PEPTIDE INFORMATION

Vol.39 No.19 December 10, 2013

Editor: M. Isoyama

Protein Research Foundation
4-1-2 Ina, Minoh, Osaka 562-0015, Japan

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E-Mail: misoyama@prf.or.jp

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TABLE OF CONTENTS

Selected articles are classified into the following categories:

A. Review, Comments, and News

B. Bioinformatics, Biophysics, and Structural Biology

C. Organic Synthetic Chemistry

D. Biochemistry and Analytical Chemistry

Q. Primary Structure Determination

E. Physiology, Medicine, and Pharmacy

I. Immunology

G. Others

A. Review, Comments, and News

3915416a: Nutr.Hosp.Suppl., 109-114(2013)
Link: [ PubMED ] [ DOI ]
Title: Influence of diabetes surgery on a gut-brain-liver axis regulating food intake and internal glucose production.
Author: Mithieux,G.
Abstract:
It has long been known that the brain, especially the hypothalamus, can modulate both insulin secretion and hepatic glucose fluxes, via the modulation of the sympathetic system (promoting glycogen breakdown) and the parasympathetic system (stimulating glycogen deposition). Central insulin signalling or hypothalamic long-chain fatty acid oxidation can also control insulin's suppression of endogenous glucose production. Interestingly, intestinal gluconeogenesis can initiate a portal glucose signal, transmitted to the hypothalamus via the gastrointestinal nervous system. This signal may modulate the sensation of hunger and satiety and insulin sensitivity of hepatic glucose fluxes as well. The rapid improvements of glucose control taking place after gastric bypass surgery in obese diabetics has long been mysterious. Actually, the specificity of gastric bypass in obese diabetic mice relates to major changes in the sensations of hunger and to rapid improvement in insulin sensitivity of endogenous glucose production. We have shown that an induction of intestinal gluconeogenesis plays a major role in these phenomena. In addition, the restoration of the secretion of glucagon like peptide 1 and consequently of insulin plays a key additional role to improve postprandial glucose tolerance. Therefore, a synergy between incretin effects and intestinal gluconeogenesis might be a key feature explaining the rapid improvement of glucose control in obese diabetics after bypass surgery.

Address: Institut National de la Sante et de la Recherche Medicale U855, Lyon, France

3915420a: Front.Oncol., 216-216(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Tumor-penetrating peptides.
Author: Teesalu,T., Sugahara,K.N., Ruoslahti,E.
Abstract:
Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular "zip code" of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the blood.

Address: Cancer Research Center, Sanford-Burnham Medical Research Institute , La Jolla, CA , USA

3915421a: Front.Microbiol., 234-234(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The MHC-II transactivator CIITA, a restriction factor against oncogenic HTLV-1 and HTLV-2 retroviruses: similarities and differences in the inhibition of Tax-1 and Tax-2 viral transactivators.
Author: Forlani,G., Abdallah,R., Accolla,R.S., Tosi,G.
Abstract:
The activation of CD4+ T helper cells is strictly dependent on the presentation of antigenic peptides by MHC class II (MHC-II) molecules. MHC-II expression is primarily regulated at the transcriptional level by the AIR-1 gene product CIITA (class II transactivator). Thus, CIITA plays a pivotal role in the triggering of the adaptive immune response against pathogens. Besides this well known function, we recently found that CIITA acts as an endogenous restriction factor against HTLV-1 (human T cell lymphotropic virus type 1) and HTLV-2 oncogenic retroviruses by targeting their viral transactivators Tax-1 and Tax-2, respectively. Here we review our findings on CIITA-mediated inhibition of viral replication and discuss similarities and differences in the molecular mechanisms by which CIITA specifically counteracts the function of Tax-1 and Tax-2 molecules. The dual function of CIITA as a key regulator of adaptive and intrinsic immunity represents a rather unique example of adaptation of host-derived factors against pathogen infections during evolution.

Address: Laboratory of General Pathology and Immunology, Department of Surgical and Morphological Sciences, University of Insubria Varese, Italy

3915431a: Adv.Clin.Exp.Med., 585-591(2013)
Link: [ PubMED ] [ OpenAccess]
Title: Hepcidin and its role in inflammatory bowel disease.
Author: Dudkowiak,R., Neubauer,K., Poniewierka,E.
Abstract:
Anemia is one of the most common extraintestinal symptoms of inflammatory bowel disease (IBD). The pathophysiology of anemia in IBD is complex. It may be developed in the course of inflammation, intestinal bleeding or disorders of iron absorption. Hepcidin, discovered in the year 2000, is an endogenous peptide responsible for iron homeostasis. Recent data suggests that hepcidin is a major mediator of anemia and plays a central role in iron homeostasis and metabolism. This paper presents information about hepcidin structure and function, mechanisms of the regulation of the synthesis and current data about the role of this hormone in IBD-related anemia. Assessment of hepcidin levels in patients with IBD may become a key element in the treatment of anemia in the near future.

Address: Division of Dietetics, Department of Gastroenterology and Hepatology, Wroclaw Medical University, Poland

3915436a: Expert Opin.Pharmacother., 2161-2169(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of canrenone in patients with metabolic syndrome.
Author: Derosa,G., Bonaventura,A., Bianchi,L., Romano,D., D'Angelo,A., Fogari,E., Maffioli,P.
Abstract:
Background: Metabolic syndrome is becoming a common disease due to a rise in obesity rates among adults. Objectives: The aim was to evaluate the effects of canrenone compared to placebo on metabolic and inflammatory parameters in patients affected by metabolic syndrome. A total of 145 patients were treated with placebo or canrenone, 50 mg/day, for 3 months and then 50 mg b.i.d. till the end of the study. Blood pressure, body weight, body mass index, fasting plasma glucose (FPG), fasting plasma insulin, HOMA-IR, lipid profile, plasma aldosterone, brain natriuretic peptide, high-sensitivity C-reactive protein (Hs-CRP), tumor necrosis factor-α (TNF-α) and M value were evaluated. Results: A decrease of blood pressure was observed in canrenone group compared to baseline; moreover, systolic blood pressure value recorded after 6 months of canrenone therapy was lower than the one recorded with placebo. Canrenone gave a significant decrease of FPI and HOMA index, and an increase of M value both compared to baseline and to placebo. Canrenone also decreased triglycerides and FPG was not observed with placebo. Canrenone also decreased plasma aldosterone, Hs-CRP and TNF-α compared to baseline and to placebo. Conclusion: Canrenone seems to be effective in reducing some factors involved in metabolic syndrome and in improving insulin-resistance and the inflammatory state observed in these patients.

Address: University of Pavia, Department of Internal Medicine and Therapeutics, Fondazione IRCCS Policlinico S. Matteo , P.le C. Golgi, 2 - 27100 Pavia , Italy +39 0382 526217

3915464a: Expert Rev.Cardiovasc.Ther., 1155-1169(2013)
Link: [ PubMED ] [ DOI ]
Title: Novel biomarkers for heart failure.
Author: Iqbal,N., Alim,K.S., Aramin,H., Iqbal,F., Green,E., Higginbotham,E., Maisel,A.S.
Abstract:
Heart failure (HF) has proven to be a major burden on the health system. The continuing prevalence of the condition and its rising associated costs and care, has amplified the need for earlier diagnosis, better risk stratification and cost-effective treatment to cut rates of hospitalization. Biomarkers seem poised to undertake such tasks, with biomarker management of patients with HF quickly evolving over the past several years. Biomarker guided diagnosis and treatment has become vital, especially during the acute setting in which the majority of patients with HF, were initially present. An adequate assessment of risk requires a multi-marker approach to a given HF patient. Established markers including brain natriuretic peptide and NT-proBNP are a significant clinical aid to physicians, though their utility is limited. In the past few years, momentous effort has been put into the discovery of new biomarkers. These endeavors have led to the emergence of several capable and promising biomarkers for HF management including troponins, mid-regional pro-adrenomedullin, GDF-15, C-reactive protein, Galectin-3, IL-6, ST-2, neutrophil gelatinase-associated lipocalin, copeptin and procalcitonin. This review will offer an insight into the novel biomarkers considered as the cutting-edge in the diagnosis and management of HF.

Address: Veterans Affairs San Diego Healthcare System, San Diego, CA, USA

3915489a: Int.J.Mol.Sci., 17410-17419(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Δ opioid receptor and its peptide: a receptor-ligand neuroprotection.
Author: Staples,M., Acosta,S., Tajiri,N., Pabon,M., Kaneko,Y., Borlongan,C.V.
Abstract:
In pursuit of neurological therapies, the opioid system, specifically δ opioid receptors and δ opioid peptides, demonstrates promising therapeutic potential for stroke, Parkinson's disease, and other degenerative neurological conditions. Recent studies offer strong evidence in support of the therapeutic use of δ opioid receptors, and provide insights into the underlying mechanisms of action. Δ opioid receptors have been shown to confer protective effects by mediating ionic homeostasis and activating endogenous neuroprotective pathways. Additionally, δ opioid agonists such as (D-Ala 2, D-Leu 5) enkephalin (DADLE) have been shown to decrease apoptosis and promote neuronal survival. In its entirety, the δ opioid system represents a promising target for neural therapies.

E-Mail: meaghans@mail.usf.edu
Address: Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL 33612, USA

3915501a: Exerc.Immunol.Rev., 86-101(2013)
Link: [ PubMED ]
Title: Influence of vitamin D status on respiratory infection incidence and immune function during 4 months of winter training in endurance sport athletes.
Author: He,C.S., Handzlik,M., Fraser,W.D., Muhamad,A., Preston,H., Richardson,A., Gleeson,M.
Abstract:
The purpose of this study was to examine the influence of vitamin D status on mucosal and systemic immunity and the incidence, severity and duration of upper respiratory tract illness (URTI) episodes in endurance athletes during a 16-week winter training period. Blood was collected from 225 subjects at the start of the study and plasma was analysed for total 25-hydroxy vitamin D (25(OH)D) and cathelicidin concentration. Blood was also collected at the end of the study and analysed for 25(OH)D and antigen-stimulated cytokine production. Unstimulated saliva samples were obtained at the start and at 4-week intervals during the study period. Saliva samples were analysed for salivary antimicrobial peptides and proteins (AMPs). Weekly training and daily illness logs were kept. At the start and end of the study 38% and 55%, respectively, of the athlete cohort had inadequate (plasma 25(OH)D 30-50 nmol/L) or deficient (plasma 25(OH)D < 30 nmol/L) vitamin D status. There was a significantly higher proportion of subjects who presented with symptoms of URTI in the vitamin D deficient status group (initial plasma 25(OH)D < 30 nmol/L) during the study period than in the optimal vitamin D group (> 120 nmol/L) and the total number of URTI symptom days and the median symptom-severity score in the vitamin D deficient group was signifi- cantly higher than in the other groups. The plasma cathelicidin concentration positively correlated with the plasma 25(OH)D concentration and the saliva secretory immunoglobulin A (SIgA) secretion rate in the optimal vitamin D status group was significantly higher than in the other groups. Low vitamin D status was associated with lower pro-inflammatory cytokine production by monocytes and lymphocytes. Low vitamin D status could be an important determinant of URTI risk in endurance athletes and mucosal as well as systemic immunity may be modified via vitamin D-dependent mechanisms.

Address: School of Sport, Exercise and Health Sciences, Loughborough University, Leucestershire, United Kingdom

3915502a: Exerc.Immunol.Rev., 72-85(2013)
Link: [ PubMED ]
Title: Eccentric exercise-induced delayed-onset muscle soreness and changes in markers of muscle damage and inflammation.
Author: Kanda,K., Sugama,K., Hayashida,H., Sakuma,J., Kawakami,Y., Miura,S., Yoshioka,H., Mori,Y., Suzuki,K.
Abstract:
The purpose of this study was to determine the relationships among delayed-onset muscle soreness (DOMS), muscle damage and inflammatory responses to eccentric exercise and investigate the underlying mechanisms. Nine healthy males performed one-leg calf-raise exercise with their right leg on a force plate. They performed 10 sets of 40 repetitions of exercise at 0.5 Hz by the load corresponding to the half of their body weight, with a rest for 3 min between sets. DOMS was evaluated by a visual analogue scale (VAS). Blood and urine samples were collected before and 2, 4, 24, 48, 72 and 96 h post-exercise. Blood samples were analyzed for leucocyte differential counts and neutrophil functions (migratory activity and oxidative burst activity). We also determined a serum marker of muscle damage, myoglobin (Mb), and plasma and urinary prostaglandin E2 as an algesic substance. As for the inflammatory mediators, plasma and urine were analyzed for cytokines (interleukin (IL)-1β, IL-1 receptor antagonist, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, tumour necrosis factor-α, interferon-γ, monocyte chemotactic protein-1, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and granulocyte macrophage colony-stimulating factor), leucocyte activation markers (calprotectin and myeloperoxidase), and neutrophil chemotactic factor complement 5a. All subjects reported muscle soreness on subsequent days and VAS peaked at 72 h after exercise. Serum Mb concentration significantly increased (p < 0.05) at 72 h after exercise as compared with the pre-exercise values which was correlated with the increases in VAS at 72 h (r = 0.73, p < 0.05). Circulating neutrophil count and migratory activity increased significantly (p < 0.01, and p < 0.05, respectively) at 4 h after exercise, whereas there were no significant changes in the other plasma and urinary inflammatory mediators. These results suggest that neutrophils can be mobilized into the circulation and migrate to the muscle tissue several hours after the eccentric exercise. There were also positive correlations between the exercise-induced increases in neutrophil migratory activity at 4 h and the increases in Mb at 48 h (r = 0.67, p < 0.05). These findings suggest that neutrophil mobilization and migration after exercise may be involved in the muscle damage and inflammatory processes.

Address: Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology/ Waseda University, Japan

3915503a: Exerc.Immunol.Rev., 29-48(2013)
Link: [ PubMED ]
Title: Urinary excretion of cytokines versus their plasma levels after endurance exercise.
Author: Sugama,K., Suzuki,K., Yoshitani,K., Shiraishi,K., Kometani,T.
Abstract:
It has been consistently shown that circulating levels of interleukin (IL)-6, IL-8, IL-1 receptor antagonist (IL-1ra) and IL-10 increase remarkably following endurance exercise longer than 2 h such as marathon and triathlon races. However, no studies have compared changes in the plasma and urinary levels of these cytokines after endurance exercise, including the recovery period. In the present study, we investigated kinetic changes in the urinary excretion of cytokines following endurance exercise up to 3 h after exercise to evaluate the magnitude of change in comparison to the plasma levels and to explore the possible biological significance and the mechanisms of cytokine dynamics following exercise. Fourteen male athletes participated in a duathlon race consisting of 5 km of running, 40 km of cycling, and 5 km of running. Venous blood and urine samples were collected before, immediately after, 1.5 h and 3 h after the race. Plasma and urine were analyzed using enzyme-linked immunosorbent assays (ELISA). Plasma concentrations of lL-1β, IL-1ra, IL-6, IL-8, IL-10 and monocyte chemotactic protein (MCP)-1 increased significantly after the race, whereas tumour necrosis factor (TNF)-α, IL-2, IL-4, IL-12 and interferon (IFN)-γ did not change significantly. Urinary concentrations of lL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-γ and MCP-1 increased significantly after the race. When the urine concentrations were adjusted by creatinine concentration, urine volume and sampling time, the increases of lL-2, IL-4, IL-8, IL-10, IFN-γ and MCP-1 were evident and these were notably present in urine of the stressed athletes suffering from renal tubular epithelial damage. The present study provides new evidence that the kinetics and magnitude of changes in urinary cytokine concentrations differ from plasma cytokine concentrations following endurance exercise, especially, in the recovery period several hours after exercise, and that the damaged kidney might be responsible at least in part for the kinetics of some cytokines. Urinary cytokines may be sensitive biomarkers of the impact of exhaustive exercise workload on renal damage and inflammation in the recovery period after endurance exercise.

Address: Tokyo University of Agriculture and Technology/Waseda University, Japan

3915519a: Front.Immunol., 336-336(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: HLA-DM Focuses on Conformational Flexibility Around P1 Pocket to Catalyze Peptide Exchange.
Author: Yin,L., Stern,L.J.
Abstract:
Peptides presented by major histocompatibility complex class II (MHCII) molecules to CD4+ T cells play a central role in the initiation of adaptive immunity. This antigen presentation process is characterized by the proteolytic cleavage of foreign and self proteins, and loading of the resultant peptides onto MHCII molecules. Loading and exchange of antigenic peptides is catalyzed by a non-classical MHCII molecule, HLA-DM. The impact of HLA-DM on epitope selection has been appreciated for a long time. However, the molecular mechanism by which HLA-DM mediates peptide exchange remains elusive. Here, we review recent efforts in elucidating how HLA-DM works, highlighted by two recently solved co-structures of HLA-DM bound to HLA-DO (a natural inhibitor of HLA-DM), or to HLA-DR1 (a common MHCII). In light of these efforts, a model for HLA-DM action in which HLA-DM utilizes conformational flexibility around the P1 pocket of the MHCII-peptide complex to catalyze peptide exchange is proposed.

Address: Department of Pathology, University of Massachusetts Medical School , Worcester, MA , USA

3915520a: Front.Endocrinol., 143-143(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Posttranslational Modifications of Proopiomelanocortin in Vertebrates and Their Biological Significance.
Author: Takahashi,A., Mizusawa,K.
Abstract:
Proopiomelanocortin (POMC) is the precursor of several peptide hormones generated in the pituitary gland. After biosynthesis, POMC undergoes several posttranslational modifications, including proteolytic cleavage, acetylation, amidation, phosphorylation, glycosylation, and disulfide linkage formation, which generate mature POMC-derived peptides. Therefore, POMC is a useful model for the investigation of posttranslational modifications. These processes have been extensively investigated in mammals, primarily in rodents. In addition, over the last decade, much information has been obtained about the posttranslational processing of POMC in non-mammalian animals such as fish, amphibians, reptiles, and birds through sequencing and peptide identification by mass spectrometry. One POMC modification, acetylation, is known to modulate the biological activities of POMC-derived α-melanocyte-stimulating hormone (α-MSH) having an acetyl group at N-terminal through potentiation or inhibition. This bidirectional regulation depends on its intrinsic roles in the tissue or cell; for example, α-MSH, as well as desacetyl (Des-Ac)-α-MSH, stimulates pigment dispersion in the xanthophores of a flounder. In contrast, α-MSH does not stimulate pigment dispersion in the melanophores of the same species, whereas Des-Ac-α-MSH does. Regulation of pigment-dispersing activities may be associated with the subtle balance in the expression of receptor genes. In this review, we consider the posttranslational modifications of POMC in vertebrates from an evolutionary aspect, with a focus on the relationship between acetylation and the biological activities of α-MSH as an important consequence of posttranslational modification.

Address: School of Marine Biosciences, Kitasato University , Sagamihara, Kanagawa , Japan

3915521a: Front.Physiol., 290-290(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Alterations of cAMP-dependent signaling in dystrophic skeletal muscle.
Author: Rudolf,R., Khan,M.M., Lustrino,D., Labeit,S., Kettelhut,I.C., Navegantes,L.C.
Abstract:
Autonomic regulation processes in striated muscles are largely mediated by cAMP/PKA-signaling. In order to achieve specificity of signaling its spatial-temporal compartmentation plays a critical role. We discuss here how specificity of cAMP/PKA-signaling can be achieved in skeletal muscle by spatio-temporal compartmentation. While a microdomain containing PKA type I in the region of the neuromuscular junction (NMJ) is important for postsynaptic, activity-dependent stabilization of the nicotinic acetylcholine receptor (AChR), PKA type I and II microdomains in the sarcomeric part of skeletal muscle are likely to play different roles, including the regulation of muscle homeostasis. These microdomains are due to specific A-kinase anchoring proteins, like rapsyn and myospryn. Importantly, recent evidence indicates that compartmentation of the cAMP/PKA-dependent signaling pathway and pharmacological activation of cAMP production are aberrant in different skeletal muscles disorders. Thus, we discuss here their potential as targets for palliative treatment of certain forms of dystrophy and myasthenia. Under physiological conditions, the neuropeptide, α-calcitonin-related peptide, as well as catecholamines are the most-mentioned natural triggers for activating cAMP/PKA signaling in skeletal muscle. While the precise domains and functions of these first messengers are still under investigation, agonists of β2-adrenoceptors clearly exhibit anabolic activity under normal conditions and reduce protein degradation during atrophic periods. Past and recent studies suggest direct sympathetic innervation of skeletal muscle fibers. In summary, the organization and roles of cAMP-dependent signaling in skeletal muscle are increasingly understood, revealing crucial functions in processes like nerve-muscle interaction and muscle trophicity.

Address: Institute of Molecular and Cell Biology, University of Applied Sciences Mannheim , Mannheim, Germany

3915546a: Mol.Brain, 44-44(2013)
Link: [ PubMED ] [ DOI ]
Title: Transcriptional regulation and its misregulation in Alzheimer's disease.
Author: Chen,X.F., Zhang,Y.W., Xu,H., Bu,G.
Abstract:
Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by loss of memory and cognitive function. A key neuropathological event in AD is the accumulation of amyloid-β (Aβ) peptide. The production and clearance of Aβ in the brain are regulated by a large group of genes. The expression levels of these genes must be fine-tuned in the brain to keep Aβ at a balanced amount under physiological condition. Misregulation of AD genes has been found to either increase AD risk or accelerate the disease progression. In recent years, important progress has been made in uncovering the regulatory elements and transcriptional factors that guide the expression of these genes. In this review, we describe the mechanisms of transcriptional regulation for the known AD genes and the misregualtion that leads to AD susceptibility.

3915547a: Vet.Clin.N.Am.Small Anim.Pract., 1261-1272(2013)
Link: [ PubMED ] [ DOI ]
Title: Using cardiac biomarkers in veterinary practice.
Author: Oyama,M.A.
Abstract:
Blood-based assays for various cardiac biomarkers can assist in the diagnosis of heart disease in dogs and cats. The two most common markers are cardiac troponin-I and N-terminal pro-B-type natriuretic peptide. Biomarker assays can assist in differentiating cardiac from noncardiac causes of respiratory signs and detection of preclinical cardiomyopathy. Increasingly, studies indicate that cardiac biomarker testing can help assess the risk of morbidity and mortality in animals with heart disease. Usage of cardiac biomarker testing in clinical practice relies on proper patient selection, correct interpretation of test results, and incorporation of biomarker testing into existing diagnostic methods.

E-Mail: maoyama@vet.upenn.edu
Address: Section of Cardiology, Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104, USA. Electronic address:

3915556a: Int.J.Nanomed., 3917-3925(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Nanoscale bacteriophage biosensors beyond phage display.
Author: Lee,J.W., Song,J., Hwang,M.P., Lee,K.H.
Abstract:
Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

Address: Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea

3915585a: Front.Physiol., 288-288(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Functional implications of insulin-like peptides in metabolism, aging, and dietary restriction.
Author: Kannan,K., Fridell,Y.W.
Abstract:
The neuroendocrine architecture and insulin/insulin-like signaling (IIS) events in Drosophila are remarkably conserved. As IIS pathway governs growth and development, metabolism, reproduction, stress response, and longevity; temporal, spatial, and nutrient regulation of dilps encoding Drosophila insulin-like peptides (DILPs) provides potential mechanisms in modulating IIS. Of eight DILPs (DILP1-8) identified, recent studies have furthered our understanding of physiological roles of DILP2, DILP3, DILP5, and DILP6 in metabolism, aging, and responses to dietary restriction (DR), which will be the focus of this review. While the DILP producing IPCs of the brain secrete DILP2, 3, and 5, fat body produces DILP6. Identification of factors that influence dilp expression and DILP secretion has provided insight into the intricate regulatory mechanisms underlying transcriptional regulation of those genes and the activity of each peptide. Studies involving loss-of-function dilp mutations have defined the roles of DILP2 and DILP6 in carbohydrate and lipid metabolism, respectively. While DILP3 has been implicated to modulate lipid metabolism, a metabolic role for DILP5 is yet to be determined. Loss of dilp2 or adult fat body specific expression of dilp6 has been shown to extend lifespan, establishing their roles in longevity regulation. The exact role of DILP3 in aging awaits further clarification. While DILP5 has been shown associated with DR-mediated lifespan extension, contradictory evidence that precludes a direct involvement of DILP5 in DR exists. This review highlights recent findings on the importance of conserved DILPs in metabolic homeostasis, DR, and aging, providing strong evidence for the use of DILPs in modeling metabolic disorders such as diabetes and hyperinsulinemia in the fly that could further our understanding of the underlying processes and identify therapeutic strategies to treat them.

Address: Department of Molecular and Cell Biology, University of Connecticut-Storrs, Storrs , CT, USA

3915586a: Front.Neurosci., 187-187(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Programming of neuroendocrine self in the thymus and its defect in the development of neuroendocrine autoimmunity.
Author: Geenen,V., Bodart,G., Henry,S., Michaux,H., Dardenne,O., Charlet-Renard,C., Martens,H., Hober,D.
Abstract:
For centuries after its first description by Galen, the thymus was considered as only a vestigial endocrine organ until the discovery in 1961 by Jacques FAP Miller of its essential role in the development of T (thymo-dependent) lymphocytes. A unique thymus first appeared in cartilaginous fishes some 500 million years ago, at the same time or shortly after the emergence of the adaptive (acquired) immune system. The thymus may be compared to a small brain or a computer highly specialized in the orchestration of central immunological self-tolerance. This was a necessity for the survival of species, given the potent evolutionary pressure imposed by the high risk of autotoxicity inherent in the stochastic generation of the diversity of immune cell receptors that characterize the adaptive immune response. A new paradigm of "neuroendocrine self-peptides" has been proposed, together with the definition of "neuroendocrine self." Neuroendocrine self-peptides are secreted by thymic epithelial cells (TECs) not according to the classic model of neuroendocrine signaling, but are processed for presentation by, or in association with, the thymic major histocompatibility complex (MHC) proteins. The autoimmune regulator (AIRE) gene/protein controls the transcription of neuroendocrine genes in TECs. The presentation of self-peptides in the thymus is responsible for the clonal deletion of self-reactive T cells, which emerge during the random recombination of gene segments that encode variable parts of the T cell receptor for the antigen (TCR). At the same time, self-antigen presentation in the thymus generates regulatory T (Treg) cells that can inhibit, in the periphery, those self-reactive T cells that escaped negative selection in the thymus. Several arguments indicate that the origin of autoimmunity directed against neuroendocrine glands results primarily from a defect in the intrathymic programming of self-tolerance to neuroendocrine functions. This defect may be genetic or acquired, for example during an enteroviral infection. This novel knowledge of normal and pathologic functions of the thymus constitutes a solid basis for the development of a novel type of tolerogenic/negative self-vaccination against type 1 diabetes (T1D).

Address: Department of Biomedical and Preclinical Sciences, Center of Immunoendocrinology, GIGA Research Institute, Fund of Scientific Research, University of Liege Liege-Sart Tilman, Belgium

3915609a: Neurology, 1184-1185(2013)
Link: [ PubMED ] [ DOI ]
Title: Is CGRP a marker for chronic migraine?
Author: Silberstein,S.D., Edvinsson,L.
Abstract:
Calcitonin gene-related peptide (CGRP) research was initiated by Poyner et al.,(1) who found that the calcitonin gene encodes the 37-amino acid neuropeptide CGRP in neuronal tissue. Within months, the Lund group produced antibodies toward α-CGRP and developed sensitive methods to study the role of this peptide in the cranial circulation. Intracranial vessels and the trigeminal ganglion harbor this potent vasodilator peptide.(2) Functionally, CGRP has a substantial role in the trigeminovascular reflex.(3) The calcitonin family is now well-characterized and contains 6 members: calcitonin, amylin, adenomedullin-2, adrenomedullin, and CGRP (2 isoforms: α-CGRP and β-CGRP).(4) The α- and β-isoforms of CGRP are similar in their biological activities but show different tissue distributions. β-CGRP is mainly found in enteric nerves and in the pituitary gland, while α-CGRP is found predominantly in sensory neurons and in the CNS.(5) CGRP causes cranial vasodilatation and facilitates nociception. During a migraine attack, trigeminal activation results in the release of CGRP from presynaptic nerve terminals and induces vasodilatation and neurogenic inflammation in leptomeningeal and extracranial vessels, which gives rise to the typical throbbing pain of migraine. Goadsby and Edvinsson(6) showed that plasma concentrations of CGRP are elevated in the external jugular venous blood during migraine headaches and that sumatriptan can abort the rise in CGRP and the headache.(4) CGRP given IV causes headache only in migraine patients and CGRP antagonists are effective for the acute treatment of migraine.(7) Until now, CGRP elevation in the plasma has not been reproduced in all studies.(8) However, technical problems have sometimes hampered the proper measurements of CGRP.(9.)

Address: From the Jefferson Headache Center (S.D.S.), Jefferson Hospital for Neuroscience, Philadelphia, PA

3915620a: Curr.Opin.Allergy Clin.Immunol., 531-536(2013)
Link: [ PubMED ] [ DOI ]
Title: Expression of antimicrobial peptides in atopic dermatitis and possible immunoregulatory functions.
Author: Kopfnagel,V., Harder,J., Werfel,T.
Abstract:
PURPOSE OF REVIEW: To summarize the findings on the expression of antimicrobial peptides in the skin in inflammatory skin diseases and particularly in atopic dermatitis. Moreover, the literature addressing the functions of antimicrobial peptides (AMPs) beyond antimicrobial activities with impact on allergic skin inflammation is summarized as well. RECENT FINDINGS: Although lower expressed than in psoriasis, most AMPs have been shown to be either constitutively expressed or upregulated in atopic dermatitis as well. A number of immunoregulatory functions which might impact on allergic skin inflammation have been described for several antimicrobial peptides, mainly for human β-defensins and the cathelicidin LL-37. SUMMARY: From the recent literature, there is considerable evidence that AMPs are induced in the skin in atopic dermatitis. However, some studies suggest that the induction, release or mobilization of some AMPs in atopic dermatitis may not reach sufficient levels to provide adequate control of cutaneous microbial colonization. Th2 cytokines appear to negatively influence the expression and induction of some AMPs. A number of immunoregulatory functions have been described for several AMPs. Most of them point to a proinflammatory function of AMPs in addition to their antimicrobial activities. Further studies are needed to clarify the role of AMPs in atopic dermatitis.

E-Mail: Kopfnagel.Verena@mh-hannover.de
Address: Division of Immunodermatology and Allergy Research, Department of Dermatology and Allergy, Hannover Medical School, Hanover, Germany

3915623a: Am.J.Med.Sci., 237-239(2013)
Link: [ PubMED ] [ DOI ]
Title: Endocarditis caused by Candida dubliniensis.
Author: Garcia,J., Soch,K., Matthew,E., Surani,S., Horseman,M.A.
Abstract:
Endocarditis caused by Candida dubliniensis is a rare event and limited to few case reports. In this report, the authors present a patient with a history of intravenous drug use and hepatitis C and endocarditis involving a prosthetic aortic valve. Also reviewed are the treatment guidelines for Candida sp. endocarditis.

Address: Pharmacy Department, Christus Spohn Hospital Corpus Christi-Memorial, Corpus Christi, TX, USA

3915643a: Biophys.J., 880-886(2013)
Link: [ PubMED ] [ DOI ]
Title: Human PIEZO1: removing inactivation.
Author: Bae,C., Gottlieb,P.A., Sachs,F.
Abstract:
PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by approximately 30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.

Address: Department of Physiology and Biophysics, State University of New York, Buffalo, NY, USA

3915645a: Semin.Oncol., 499-510(2013)
Link: [ PubMED ] [ DOI ]
Title: Selected toxicities of targeted therapies: presentation and management.
Author: Kollmannsberger,C., Mitchell,T.
Abstract:
The introduction of targeted therapy has changed the landscape of metastatic renal cell carcinoma (mRCC) but has also brought challenges. One such challenge of treating this chronic disease is long-term therapy, which produces ongoing toxicity. Knowledge of the spectrum of toxicity from targeted therapy and appropriate and timely intervention is required to maintain adequate dosing required for optimal outcome. This article addresses some of the major toxicities of newer agents in RCC and discusses management strategies.

E-Mail: ckollmannsberger@bccancer.bc.ca
Address: Division of Medical Oncology, British Columbia Cancer Agency Vancouver Cancer Centre, Vancouver BC V5Z 4E6, Canada

3915646a: Semin.Oncol., 492-498(2013)
Link: [ PubMED ] [ DOI ]
Title: Immune therapy for kidney cancer: a second dawn?
Author: McDermott,D.F., Atkins,M.B.
Abstract:
Agents targeting the immune system have been a historical standard of care in metastatic renal cell carcinoma (RCC), but have largely been supplanted by newer targeted therapy. Recent insights into the regulation of an anti-tumor immune response has led to the development of agents that can activate immune responses primarily within the tumor, enabling the possibility of achieving durable tumor response in the absence of significant systemic toxicity. In addition, a better understanding of tumor immunology has raised the potential of developing predictive biomarkers of response to immunotherapy. Novel approaches including inhibition of immune checkpoints has entered clinical testing in RCC.

Address: Biologic Therapy Program, Beth Israel Deaconess Medical Center

3915653a: Expert Rev.Clin.Pharmacol., 483-502(2013)
Link: [ PubMED ] [ DOI ]
Title: Davunetide: a review of safety and efficacy data with a focus on neurodegenerative diseases.
Author: Morimoto,B.H., Fox,A.W., Stewart,A.J., Gold,M.
Abstract:
Davunetide is the first neuroprotective peptide in its class, and has preclinical evidence for neuroprotective, neurotrophic and cognitive protective properties. Davunetide has also been shown to prevent apoptosis or programmed-cell death in a range of in vitro and in vivo models by promoting microtubule stabilization. Potential clinical uses of davunetide include neurodegenerative disorders such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD) or cognitive impairment in other diseases such as schizophrenia where microtubule structure and function is known to be impaired. The nonclinical and clinical safety of davunetide is reviewed here in detail. Pre-clinical toxicology studies in rats and dogs using the maxiμm feasible dose of davunetide provide strong evidence that davunetide is well-tolerated. Similarly, data from 10 separate clinical trials of davunetide, investigating safety and efficacy provide evidence that davunetide is generally safe and well-tolerated, and has shown some signs of clinical efficacy.

Address: Allon Therapeutics Inc., 1168 Hamilton Street, Suite 506, Vancouver, British Columbia, Canada V6B 2S2

3915654a: Expert Opin.Biol.Ther., 1495-1506(2013)
Link: [ PubMED ] [ DOI ]
Title: Berberis aristata/Silybum marianum fixed combination on lipid profile and insulin secretion in dyslipidemic patients.
Author: Derosa,G., Bonaventura,A., Bianchi,L., Romano,D., D'Angelo,A., Fogari,E., Maffioli,P.
Abstract:
Background: Relatively large number of dietary supplements and nutraceuticals have been studied for their supposed or demonstrated ability to reduce cholesterolemia in humans. Objectives: The aim of this study was to evaluate the efficacy as antihypercholesterolemic and insulin-sensitizing agent of a combination of Berberis aristata/Silybum marianum extract (Berberol(R)) in a sample of dyslipidemic patients. A total of 102 dyslipidemic subjects were enrolled. After a 6 months run-in period of diet and physical activity, the patients were randomized to placebo or Berberis aristata/Silybum marianum extract 588 mg/105 mg, twice a day for 3 months. Berberis aristata/Silybum marianum and placebo were then interrupted for 2 months (washout period), and then restarted for further 3 months. Anthropometric and metabolic parameters were assessed; moreover, all patients underwent a glucagon stimulation test. Results: Berberis aristata/Silybum marianum reduced total cholesterol, triglycerides and low-density lipoprotein cholesterol and increased high-density lipoprotein cholesterol after 3 months from randomization and compared to placebo group. When Berberis aristata/Silybum marianum was interrupted, lipid profile worsened, and it improved again when nutraceutical combination was reintroduced. During the glucagon stimulation test, a higher increase of C-peptide levels and a lower increase in glycemia after the test with Berberis aristata/Silybum marianum compared to placebo, to baseline and to randomization were recorded. No patients had serious adverse events in both groups. Conclusion: Berberis aristata/Silybum marianum is effective and safe in improving lipid profile and insulin secretion in euglycemic dyslipidemic patients.

Address: University of Pavia, Department of Internal Medicine and Therapeutics, Fondazione IRCCS Policlinico S. Matteo , P.le C. Golgi, 2 Pavia, 27100 , Italy +39 0382 526217

3915655a: S.Afr.Dent.J., 260-260(2013)
Link: [ PubMED ]
Title: The relationship between periodontitis and diabetes: a brief review.
Author: Moodley,A., Wood,N.H., Shangase,S.L.
Abstract:
It has been reported that a close association exists between inflammatory periodontal diseases and diabetes through which one exerts a reciprocal influence on the other. This two-way relationship is based on the extensively reported assumption that the one condition so modifies the systemic and local environments that the progress of the other is favoured. On the other hand, treating and eliminating inflammatory periodontal diseases results in improved glycaemic control which minimises the microvascular complications of diabetes. This paper provides a brief review in an endeavour to create a better understanding of the interaction between these two relatively common conditions.

Address: Department of Oral Medicine and Periodontology, School of Oral Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

3915658a: AANA J., 133-140(2013)
Link:
Title: Pathophysiology and management of angiotensin-converting enzyme inhibitor-associated refractory hypotension during the perioperative period.
Author: Thoma,A.
Abstract:
Hypertension is a common chronic condition in many patients requiring anesthesia. Pharmacologic therapy is a mainstay of treatment for hypertension, with angiotensin-converting enzyme (ACE) inhibitors being a frequently prescribed class of drugs. The American College of Cardiology and American Heart Association 2007 Guidelines on Perioperative Cardiovascular Evaluation and Care for Noncardiac Surgery provide information on many drug classes used in the treatment of hypertension; noticeably absent is a guideline for ACE inhibitors. Literature demonstrates that practice standards vary on whether ACE inhibitor regimens are continued or withheld during the preoperative period. When ACE inhibitor therapy is continued in patients undergoing general anesthesia, varying degrees of hypotension can be seen depending on confounding patient variables and the type of surgical procedure. In some instances, this hypotension can be refractory to traditional interventions such as administration of a fluid bolus, ephedrine, or phenylephrine. Vasopressin and methylene blue have been found to be effective treatments for ACE inhibitor-associated refractory hypotension. With the prevalence of hypertension and use of ACE inhibitors, anesthesia providers are likely to encounter refractory hypotension of this nature. The absence of guidelines regarding ACE inhibitors in the perioperative period contributes to a lack of consistency in practice.

E-Mail: athoma@lumc.edu
Address: Loyola University Medical Center, Maywood, Illinois, USA

3915662a: BioMed Res.Int., 723129-723129(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Aspergillus-associated airway disease, inflammation, and the innate immune response.
Author: Chotirmall,S.H., Al-Alawi,M., Mirkovic,B., Lavelle,G., Logan,P.M., Greene,C.M., McElvaney,N.G.
Abstract:
Aspergillus moulds exist ubiquitously as spores that are inhaled in large numbers daily. Whilst most are removed by anatomical barriers, disease may occur in certain circumstances. Depending on the underlying state of the human immune system, clinical consequences can ensue ranging from an excessive immune response during allergic bronchopulmonary aspergillosis to the formation of an aspergilloma in the immunocompetent state. The severest infections occur in those who are immunocompromised where invasive pulmonary aspergillosis results in high mortality rates. The diagnosis of Aspergillus-associated pulmonary disease is based on clinical, radiological, and immunological testing. An understanding of the innate and inflammatory consequences of exposure to Aspergillus species is critical in accounting for disease manifestations and preventing sequelae. The major components of the innate immune system involved in recognition and removal of the fungus include phagocytosis, antimicrobial peptide production, and recognition by pattern recognition receptors. The cytokine response is also critical facilitating cell-to-cell communication and promoting the initiation, maintenance, and resolution of the host response. In the following review, we discuss the above areas with a focus on the innate and inflammatory response to airway Aspergillus exposure and how these responses may be modulated for therapeutic benefit.

E-Mail: schotirmall@rcsi.ie
Address: Department of Respiratory Medicine, Beaumont Hospital, Dublin 9, Ireland

3915668a: Front.Immunol., 244-244(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies.
Author: Stone,J.D., Kranz,D.M.
Abstract:
Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional αβ T cell receptor (TCR) against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR) consisting of a single-chain antibody as an Fv fragment linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the αβ TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher-affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher-affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly.

Address: Department of Biochemistry, University of Illinois , Urbana, IL , USA

3915675a: Exp.Mol.Med., e37-e37(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Signaling mechanisms of glucose-induced F-actin remodeling in pancreatic islet β cells.
Author: Kalwat,M.A., Thurmond,D.C.
Abstract:
The maintenance of whole-body glucose homeostasis is critical for survival, and is controlled by the coordination of multiple organs and endocrine systems. Pancreatic islet β cells secrete insulin in response to nutrient stimuli, and insulin then travels through the circulation promoting glucose uptake into insulin-responsive tissues such as liver, skeletal muscle and adipose. Many of the genes identified in human genome-wide association studies of diabetic individuals are directly associated with β cell survival and function, giving credence to the idea that β-cell dysfunction is central to the development of type 2 diabetes. As such, investigations into the mechanisms by which β cells sense glucose and secrete insulin in a regulated manner are a major focus of current diabetes research. In particular, recent discoveries of the detailed role and requirements for reorganization/remodeling of filamentous actin (F-actin) in the regulation of insulin release from the β cell have appeared at the forefront of islet function research, having lapsed in prior years due to technical limitations. Recent advances in live-cell imaging and specialized reagents have revealed localized F-actin remodeling to be a requisite for the normal biphasic pattern of nutrient-stimulated insulin secretion. This review will provide an historical look at the emergent focus on the role of the actin cytoskeleton and its regulation of insulin secretion, leading up to the cutting-edge research in progress in the field today.

Address: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA

3915680a: Int.J.Mol.Med., 747-754(2013)
Link: [ PubMED ] [ DOI ]
Title: The Kiss-1/Kiss-1R complex as a negative regulator of cell motility and cancer metastasis (Review).
Author: Ji,K., Ye,L., Mason,M.D., Jiang,W.G.
Abstract:
Metastasis is a complex multistep process that involves the impairment of cell-cell adhesion in the neoplastic epithelium, invasion into adjacent tissues and the dissemination of cancer cells through the lymphatic and haematogenous routes. The inhibition of the metastatic process at an early stage has become a hot topic in cancer research. The Kiss-1 gene, initially described as a suppressor of metastasis in malignant melanoma, encodes the Kiss-1 protein which can be processed to other peptides, e.g., Kisspeptin-10, Kisspeptin-13, Kisspeptin-14 and Kisspeptin-54. These peptides are endogenous ligands of the Kiss1 receptor (Kiss-1R), a G protein-coupled receptor (GPR) also known as hOT7T175, AXOR12 or GPR54. The Kiss-1 gene has been suggested as a suppressor of metastasis in a various types of cancer, including gastric cancer, oesophageal carcinoma, pancreatic, ovarian, bladder and prostate cancer, through the regulation of cellular migration and invasion. In the current review, we summarise the current understanding of the role of Kiss1 and Kiss1R in cancer and cancer metastasis.

Address: Metastasis and Angiogenesis Research Group, Cardiff University School of Medicine, Cardiff, Wales CF14 4XN, UK

3915689a: Expert Opin.Ther.Pat., 1483-1494(2013)
Link: [ PubMED ] [ DOI ]
Title: AT1 antagonists: a patent review (2008 - 2012).
Author: Mavromoustakos,T., Agelis,G., Durdagi,S.
Abstract:
Introduction: For two decades a class of pharmaceutical molecules with proved beneficial therapeutic properties, especially in hypertension, has been introduced in the market aiming to specifically prevent the detrimental effects of the peptide hormone Angiotensin II at the AT1 receptor. The prototype of this class was losartan and based on its structure, several drugs were launched and also called 'Sartans'. New structural features on these molecules can provide multi-target properties in the RAS or other systems. New methodologies were developed for the treatment of hypertension utilizing either AT1 antagonists alone or as cocktails. Areas covered: In this review article, authors aim to cover information provided by patents of the years 2008 - 2012. The rationale of writing this review article is to cover the most important patents which can forward the field with new important discoveries. Expert opinion: From the patent investigation it is clear that new areas on the subject are still offered for new discoveries. New structural features can be still considered in the synthetic compounds that can advance the knowledge and beneficial effects on diseases related to Angiotensin II and AT1 receptor. There is era also for new formulations (i.e., cyclodextrins, polymers and liposomes). The multitarget approach can be further strengthened and more combinations can be sought in the rational drug design for seeking cocktails. Furthermore, the revealing of the complexity of the RAS offers new avenues for novel targets and this must not be overlooked.

E-Mail: tmavrom@chem.uoa.gr
Address: University of Athens, Department of Chemistry , Panepistimiopolis Zografou 15771, Athens , Greece

3915691a: Curr.Protein Pept.Sci., 459-471(2013)
Link:
Title: Pharmacological potential of exercise and RAS vasoactive peptides for prevention of diseases.
Author: Petriz,B.de A., De Almeida,J.A., Migliolo,L., Franco,O.L.
Abstract:
The Renin-Angiotensin-System (RAS) molecular network has been widely studied, especially with attention to angiotensin II, the main effector peptide among RAS. The relation of Ang II to hypertension pathogenesis has led to research being extended to other molecules from the RAS, such as angiotensin III and IV, angiotensin (1-5), and angiotensin (1-9). Moreover, great pharmacologic advances have been made in hypertension treatment by inhibiting renin and angiotensin converting enzymes and blocking the bonding of angiotensin II to its receptor AT1. Thus, RAS molecular signaling and its effect on blood pressure as well as its relationship to renal function and cardiovascular disease are still being investigated. It is a great challenge to fully cover and understand all molecules from the RAS, especially those that interfere with or have vasoactive properties. Some of these targets respond to exercise, stimulating nitric oxide synthesis and endothelial vasodilation. The activation of these specific molecules via exercise is a systematic way of controlling high blood pressure without pharmacological treatment. Angiotensin (1-7) has been focused due to its vasodilation properties and its responses to exercise, improving vascular function. Thus, stimulation of the ACE2/Ang (1-7)/Mas axis has been gaining ground as a prospective clinical means to attenuate cardiovascular diseases such as hypertension by modulating RAS activity. This review focuses on the vasoactive peptides from the RAS, their responses to exercise and possible trends for pharmacological development. In several cases where exercise training is not achievable, cardiovascular drug therapy with vasodilator peptides may possibly be an option.

E-Mail: ocfranco@pos.ucb.br
Address: Centro de Analises Proteomicas e Bioquimicas, Programa de Pos-Graduacao em Ciencias Genomicas e Biotecnologia, Universidade Catolica de Brasilia SGAN, Quadra 916, Modulo B, Av. W5 Norte, CEP 70.790-160 Brasilia-DF, Brazil

3915693a: Curr.Protein Pept.Sci., 492-503(2013)
Link:
Title: Studies on anticancer activities of lactoferrin and lactoferricin.
Author: Yin,C.M., Wong,J.H., Xia,J., Ng,T.B.
Abstract:
This review mainly summarizes results of recent studies on the anticancer activity of the multifunctional protein lactoferrin (Lf) and its derived peptide lactoferricin (Lfcin). The basic information on Lf and Lfcin, such as their sources, structures, and biological properties which favor their antitumor activity is introduced. The major anticancer mechanisms of Lf and Lfcin including cell cycle arrest, apoptosis, anti-angiogenesis, antimetastasis, immune modulation and necrosis are discussed. Other information from in vivo studies employing a mouse model is also provided. In addition, the roles of talatoferrin and δ lactoferrin, as well as improvement in drug delivery will be covered.

E-Mail: b021770@mailserv.cuhk.edu.hk
Address: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, HONG KONG

3915694a: Curr.Protein Pept.Sci., 504-514(2013)
Link:
Title: Cathelicidins: peptides with antimicrobial, immunomodulatory, anti-inflammatory, angiogenic, anticancer and procancer activities.
Author: Wong,J.H., Ye,X.J., Ng,T.B.
Abstract:
The family of peptides designated as cathelicidins was identified over a decade ago. Cathelicidins have since gained increasing recognition, both as endogenous antibiotics and as effector molecules of the innate immune system. The human cathelicidin LL-37 is widely expressed in human tissues and plays diverse biological roles. It contributes substantially to host defense and impacts multiple aspects of immunity. In view of the escalating importance of cathelicidins, the activities of LL-37 with an emphasis on antimicrobial, immunomodulatory, anti-inflammatory, angiogenic, anticancer and procancer effects are discussed in this review article.

E-Mail: jack1993@yahoo.com
Address: School of Biomedical Sciences, The Chinese University of Hong Kong

3915695a: Curr.Protein Pept.Sci., 515-531(2013)
Link:
Title: Antimicrobial activity of defensins and defensin-like peptides with special emphasis on those from fungi and invertebrate animals.
Author: Ng,T.B., Cheung,R.C., Wong,J.H., Ye,X.J.
Abstract:
Living organisms are in perpetual contact with pathogenic microbes, and in encounter with parasites and predators. In order to protect themselves, they produce a variety of antimicrobial proteins and peptides. One family of such protective or defensive proteins is known as defensins, characterized by a cationic character, a low molecular mass, and an abundance of cysteine residues. Defensins from mammals and plants have been succinctly reviewed by a number of experts in this ever-growing field. This review encompasses the defensin plectasin from the saprophytic fungus Pseudoplectania nigrella as well as defensins and defensin-like peptides from invertebrate animals such as jellyfish, sponges, nematodes, crustaceans, arachnids, insects, bivalves, snails, and sea urchins. Big defensins from mollusks are mentioned together with amphioxus big defensin. The structures and activities of these defense proteins are discussed.

E-Mail: xiujuanye2004@gmail.com
Address: Institute of Plant Virology, Fujian Province Key Laboratory of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, People's Republic of China

3915696a: Curr.Protein Pept.Sci., 532-542(2013)
Link:
Title: Recent progress towards pharmaceutical applications of disulfide-rich cyclic peptides.
Author: Schroeder,C.I., Swedberg,J.E., Craik,D.J.
Abstract:
Cyclotides and conotoxins are two classes of disulfide-rich peptides that occur in plants and animals respectively and are the major focus of study in our laboratory. In the last three years there has been significant progress in studies of these two classes of compounds and in this article we provide an overview of the findings from our laboratory in this period. Highlights include the discovery of cyclotides in the Fabaceae and Solanaceae plant families, members of which are widely used in human nutrition, and the discovery of new classes of cyclotide precursors. These discoveries confirm the widespread distribution of cyclotides in the plant kingdom and the diversity of precursor proteins involved in their biosynthesis. Other studies have delineated the mode of action of naturally occurring cyclotides and have demonstrated the versatility of synthetic cyclotides as stable protein engineering frameworks, with applications in drug design. Conotoxins continue to be a rich source of inspiration for drug design programs, and we summarize here a range of recent studies from our laboratory focusing on the development of novel synthetic strategies and the delineation of structure-activity relationships. A major highlight was the development of an orally active cyclized conotoxin derivative that is highly efficacious in a rat model of neuropathic pain. Overall the studies described herein provide much encouragement for continuing efforts to develop peptides as drugs.

E-Mail: d.craik@imb.uq.edu.au
Address: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia

3915716a: Front.Endocrinol., 103-103(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Glutamate and GABA in Appetite Regulation.
Author: Delgado,T.C.
Abstract:
Appetite is regulated by a coordinated interplay between gut, adipose tissue, and brain. A primary site for the regulation of appetite is the hypothalamus where interaction between orexigenic neurons, expressing Neuropeptide Y/Agouti-related protein, and anorexigenic neurons, expressing Pro-opiomelanocortin cocaine/Amphetamine-related transcript, controls energy homeostasis. Within the hypothalamus, several peripheral signals have been shown to modulate the activity of these neurons, including the orexigenic peptide ghrelin and the anorexigenic hormones insulin and leptin. In addition to the accumulated knowledge on neuropeptide signaling, presence and function of amino acid neurotransmitters in key hypothalamic neurons brought a new light into appetite regulation. Therefore, the principal aim of this review will be to describe the current knowledge of the role of amino acid neurotransmitters in the mechanism of neuronal activation during appetite regulation and the associated neuronal-astrocytic metabolic coupling mechanisms. Glutamate and GABA dominate synaptic transmission in the hypothalamus and administration of their receptors agonists into hypothalamic nuclei stimulates feeding. By using 13C High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy based analysis, the Cerdan group has shown that increased neuronal firing in mice hypothalamus, as triggered by appetite during the feeding-fasting paradigm, may stimulate the use of lactate as neuronal fuel leading to increased astrocytic glucose consumption and glycolysis. Moreover, fasted mice showed increased hypothalamic [2-13C]GABA content, which may be explained by the existence of GABAergic neurons in key appetite regulation hypothalamic nuclei. Interestingly, increased [2-13C]GABA concentration in the hypothalamus of fasted animals appears to result mainly from reduction in GABA metabolizing pathways, rather than increased GABA synthesis by augmented activity of the glutamate-glutamine-GABA cycle.

Address: Intermediary Metabolism Group, Center for Neurosciences and Cell Biology of Coimbra , Coimbra , Portugal

3915717a: Front.Endocrinol., 101-101(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Role of adaptor proteins in secretory granule biogenesis and maturation.
Author: Bonnemaison,M.L., Eipper,B.A., Mains,R.E.
Abstract:
In the regulated secretory pathway, secretory granules (SGs) store peptide hormones that are released on demand. SGs are formed at the trans-Golgi network and must undergo a maturation process to become responsive to secretagogues. The production of mature SGs requires concentrating newly synthesized soluble content proteins in granules whose membranes contain the appropriate integral membrane proteins. The mechanisms underlying the sorting of soluble and integral membrane proteins destined for SGs from other proteins are not yet well understood. For soluble proteins, luminal pH and divalent metals can affect aggregation and interaction with surrounding membranes. The trafficking of granule membrane proteins can be controlled by both luminal and cytosolic factors. Cytosolic adaptor proteins (APs), which recognize the cytosolic domains of proteins that span the SG membrane, have been shown to play essential roles in the assembly of functional SGs. Adaptor protein 1A (AP-1A) is known to interact with specific motifs in its cargo proteins and with the clathrin heavy chain, contributing to the formation of a clathrin coat. AP-1A is present in patches on immature SG membranes, where it removes cargo and facilitates SG maturation. AP-1A recruitment to membranes can be modulated by Phosphofurin Acidic Cluster Sorting protein 1 (PACS-1), a cytosolic protein which interacts with both AP-1A and cargo that has been phosphorylated by casein kinase II. A cargo/PACS-1/AP-1A complex is necessary to drive the appropriate transport of several cargo proteins within the regulated secretory pathway. The Golgi-localized, γ-ear containing, ADP-ribosylation factor binding (GGA) family of APs serve a similar role. We review the functions of AP-1A, PACS-1, and GGAs in facilitating the retrieval of proteins from immature SGs and review examples of cargo proteins whose trafficking within the regulated secretory pathway is governed by APs.

Address: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center , Farmington, CT , USA

3915718a: Front.Physiol., 217-217(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Insulin-like and IGF-like peptides in the silkmoth Bombyx mori: discovery, structure, secretion, and function.
Author: Mizoguchi,A., Okamoto,N.
Abstract:
A quarter of a century has passed since bombyxin, the first insulin-like peptide identified in insects, was discovered in the silkmoth Bombyx mori. During these years, bombyxin has been studied for its structure, genes, distribution, hemolymph titers, secretion control, as well as physiological functions, thereby stimulating a wide range of studies on insulin-like peptides in other insects. Moreover, recent studies have identified a new class of insulin family peptides, IGF-like peptides, in B. mori and Drosophila melanogaster, broadening the base of the research area of the insulin-related peptides in insects. In this review, we describe the achievements of the studies on insulin-like and IGF-like peptides mainly in B. mori with short histories of their discovery. Our emphasis is that bombyxins, secreted by the brain neurosecretory cells, regulate nutrient-dependent growth and metabolism, whereas the IGF-like peptides, secreted by the fat body and other peripheral tissues, regulate stage-dependent growth of tissues.

Address: Division of Biological Science, Graduate School of Science, Nagoya University Nagoya, Japan

3915719a: Front.Physiol., 202-202(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Eat to reproduce: a key role for the insulin signaling pathway in adult insects.
Author: Badisco,L., Van Wielendaele,P., Vanden Broeck,J.
Abstract:
Insects, like all heterotrophic organisms, acquire from their food the nutrients that are essential for anabolic processes that lead to growth (larval stages) or reproduction (adult stage). In adult females, this nutritional input is processed and results in a very specific output, i.e., the production of fully developed eggs ready for fertilization and deposition. An important role in this input-output transition is attributed to the insulin signaling pathway (ISP). The ISP is considered to act as a sensor of the organism's nutritional status and to stimulate the progression of anabolic events when the status is positive. In several insect species belonging to different orders, the ISP has been demonstrated to positively control vitellogenesis and oocyte growth. Whether or not ISP acts herein via a mediator action of lipophilic insect hormones (ecdysteroids and juvenile hormone) remains debatable and might be differently controlled in different insect orders. Most likely, insulin-related peptides, ecdysteroids and juvenile hormone are involved in a complex regulatory network, in which they mutually influence each other and in which the insect's nutritional status is a crucial determinant of the network's output. The current review will present an overview of the regulatory role of the ISP in female insect reproduction and its interaction with other pathways involving nutrients, lipophilic hormones and neuropeptides.

Address: Department of Animal Physiology and Neurobiology, Research Group of Molecular Developmental Physiology and Signal Transduction KU Leuven, Leuven, Belgium

3915747a: Expert Opin.Investig.Drugs, 1347-1357(2013)
Link: [ PubMED ] [ DOI ]
Title: Cat peptide antigen desensitisation for treating cat allergic rhinoconjunctivitis.
Author: Worm,M., Patel,D., Creticos,P.S.
Abstract:
INTRODUCTION: Allergic rhinoconjunctivitis is an increasingly common source of morbidity with sensitivity to cats accounting for 10-15% of the disease burden. Allergy to cats is a major risk factor for the development of asthma. AREAS COVERED: Within the present manuscript, the current data on a novel therapeutic approach to treat cat allergy is reviewed. Cat Peptide Antigen Desensitisation (Cat-PAD) is a mixture of seven small peptides developed for the treatment of cat allergy. It is designed to induce immunological tolerance via binding to MHC class II on antigen presenting cells and interacting with regulatory T cells without triggering the cross-linking of IgE on mast cells and basophils. The peptide sequences are derived from the major cat allergen Fel d 1. The peptides have been selected to ensure a similar T cell response to that generated to whole cat dander in ex-vivo PBMC derived from cat allergic individuals. The size of the peptides is insufficient to induce cross-linking of IgE. Clinical data from a series of studies shows that Cat-PAD is able to significantly reduce allergic rhinoconjunctivitis symptoms after a short course of four injections over 12 weeks, and that the treatment effect is persistent lasting 2 years after the start of treatment. EXPERT OPINION: Taken together Cat-PAD is a novel, well tolerated and promising therapeutic approach to treat cat allergic patients. Data from the current international Phase III study will unravel whether the concept is also efficient and tolerable under daily life circumstances.

Address: Charite, Allergy-Centre-Charite, Department of Dermatology and Allergology , Chariteplatz 1, 10117 Berlin , Germany +0049 30 450518105

3915752a: Dtsch.Aerztebl.Int., 477-484(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Rheumatoid arthritis--early diagnosis and disease management.
Author: Schneider,M., Kruger,K.
Abstract:
BACKGROUND: 0.5% to 0.8% of all adults suffer from rheumatoid arthritis (RA). The main considerations for persons with new-onset RA are early diagnosis, disease-modifying anti-rheumatic drugs (DMARDs), remission, and interdisciplinary treatment. METHOD: As part of the process of creating a new S3 guideline on the management of early RA and a new S1 guideline on stage-adapted pharmacotherapy for RA, the authors conducted a selective search and review of the literature and specifically updated it to 20 March 2013. RESULTS: In patients presenting with joint inflammation, the diagnosis of RA can be directly confirmed (positive predictive value, 85% to 97%), and its prognosis assessed, on the basis of the following findings: joint examination, acute phase reaction, serology (rheumatoid factor [RF], antibody against citrullinated peptides/proteins [ACPA], and duration of symptoms (ACR/Eular classification criteria, 2010). Early, remission-oriented and adapted treatment with DMARDs ("treating to target") leads to several years of normal bodily function without disability in 40% to 60% of patients. Treatment by an interdisciplinary team promotes the achievement of this goal. The risks associated with this form of treatment are low, with a dropout rate of less than 1 per 100 patient-years. Life-threatening complications are rare. CONCLUSION: Early diagnosis, intervention with DMARDs in the first three months of disease, and the achievement of a remission minimize the adverse sequelae of RA. The sequential introduction of DMARDs, including biological agents in non-responders, as part of a treat-to-target concept optimizes the long-term outcome, as has been demonstrated in clinical trials for periods of up to eight years.

E-Mail: schneider@rheumanet.org
Address: Department of Rheumatology, Dusseldorf University Hospital, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Germany

3915755a: Front.Immunol., 227-227(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Antigen and transforming growth factor β receptors contribute to long term functional and phenotypic heterogeneity of memory CD8 T cells.
Author: Hu,Y., Cauley,L.
Abstract:
Pathogen-specific CD8 T cells provide a mechanism for selectively eliminating host cells that are harboring intracellular pathogens. The pathogens are killed when lytic molecules are injected into the cytoplasm of the infected cells and begin an apoptotic cascade. Activated CD8 T cells also release large quantities of pro-inflammatory cytokines that stimulate other immune cells in the local vicinity. As the alveoli are extraordinarily sensitive to cytokine induced damage, multiple layers of immune regulation limit the activities of immune cells that enter the lungs. These mechanisms include receptor-mediated signaling pathways in CD8 T cells that respond to peptide antigens and transforming growth factor β. Both pathways influence the functional and phenotypic properties of long-lived CD8 T cells populations in peripheral and lymphoid tissues.

Address: University of Connecticut Health Center , Farmington, CT , USA

3915756a: Front.Endocrinol., 96-96(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The regulated secretory pathway and human disease: insights from gene variants and single nucleotide polymorphisms.
Author: Lin,W.J., Salton,S.R.
Abstract:
The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired.

Address: Department of Neuroscience, Icahn School of Medicine at Mount Sinai , New York, NY , USA

3915757a: Front.Aging Neurosci., 38-38(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Lessons from two prevalent amyloidoses-what amylin and Aβ have in common.
Author: Gotz,J., Lim,Y.A., Eckert,A.
Abstract:
The amyloidogenic peptide Aβ plays a key role in Alzheimer's disease (AD) forming insoluble aggregates in the brain. The peptide shares its amyloidogenic properties with amylin that forms aggregates in the pancreas of patients with Type 2 Diabetes mellitus (T2DM). While epidemiological studies establish a link between these two diseases, it is becoming increasingly clear that they also share biochemical features suggesting common pathogenic mechanisms. We discuss commonalities as to how Aβ and amylin deregulate the cellular proteome, how they impair mitochondrial functions, to which receptors they bind, aspects of their clearance and how therapeutic strategies exploit the commonalities between Aβ and amylin. We conclude that research into these two molecules is mutually beneficial for the treatment of AD and T2DM.

Address: Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland Brisbane, QLD, Australia

3915758a: Front.Integr.Neurosci., 59-59(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Inflammatory process in Alzheimer's Disease.
Author: Meraz-Rios,M.A., Toral-Rios,D., Franco-Bocanegra,D., Villeda-Hernandez,J., Campos-Pena,V.
Abstract:
Alzheimer Disease (AD) is a neurodegenerative disorder and the most common form of dementia. Histopathologically is characterized by the presence of two major hallmarks, the intracellular neurofibrillary tangles (NFTs) and extracellular neuritic plaques (NPs) surrounded by activated astrocytes and microglia. NFTs consist of paired helical filaments of truncated tau protein that is abnormally hyperphosphorylated. The main component in the NP is the amyloid-β peptide (Aβ), a small fragment of 40-42 amino acids with a molecular weight of 4 kD. It has been proposed that the amyloid aggregates and microglia activation are able to favor the neurodegenerative process observed in AD patients. However, the role of inflammation in AD is controversial, because in early stages the inflammation could have a beneficial role in the pathology, since it has been thought that the microglia and astrocytes activated could be involved in Aβ clearance. Nevertheless the chronic activation of the microglia has been related with an increase of Aβ and possibly with tau phosphorylation. Studies in AD brains have shown an upregulation of complement molecules, pro-inflammatory cytokines, acute phase reactants and other inflammatory mediators that could contribute with the neurodegenerative process. Clinical trials and animal models with non-steroidal anti-inflammatory drugs (NSAIDs) indicate that these drugs may decrease the risk of developing AD and apparently reduce Aβ deposition. Finally, further studies are needed to determine whether treatment with anti-inflammatory strategies, may decrease the neurodegenerative process that affects these patients.

Address: Departamento de Biomedicina Molecular, Centro de Investigacion y de Estudios Avanzados Mexico City, Mexico

3915773a: Curr.Microbiol., 688-695(2013)
Link: [ PubMED ] [ DOI ]
Title: How are the Non-classically Secreted Bacterial Proteins Released into the Extracellular Milieu?
Author: Wang,G., Chen,H., Xia,Y., Cui,J., Gu,Z., Song,Y., Chen,Y.Q., Zhang,H., Chen,W.
Abstract:
Most bacterial proteins that are destined to leave the cytoplasm are exported across the cell membrane to their sites of function. These proteins are generally exported via the classical secretion pathway, in which the signal peptide plays a central role. However, some bacterial proteins have been found in the extracellular milieu without any apparent signal peptide. As none of the classical secretion systems is involved in their secretion, this occurrence is termed non-classical protein secretion. The mechanism or mechanisms responsible for non-classical secretion are contentious. This review compiles evidence from the debate over whether the release of the non-classically secreted proteins is the result of cell lysis and discusses how these proteins are exported to the exterior of the cell.

Address: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, People's Republic of China

3915780a: Curr.Top.Dev.Biol., 1-36(2013)
Link: [ PubMED ] [ DOI ]
Title: How clocks and hormones act in concert to control the timing of insect development.
Author: Di Cara,F., King-Jones,K.
Abstract:
During the last century, insect model systems have provided fascinating insights into the endocrinology and developmental biology of all animals. During the insect life cycle, molts and metamorphosis delineate transitions from one developmental stage to the next. In most insects, pulses of the steroid hormone ecdysone drive these developmental transitions by activating signaling cascades in target tissues. In holometabolous insects, ecdysone triggers metamorphosis, the remarkable remodeling of an immature larva into a sexually mature adult. The input from another developmental hormone, juvenile hormone (JH), is required to repress metamorphosis by promoting juvenile fates until the larva has acquired sufficient nutrients to survive metamorphosis. Ecdysone and JH act together as key endocrine timers to precisely control the onset of developmental transitions such as the molts, pupation, or eclosion. In this review, we will focus on the role of the endocrine system and the circadian clock, both individually and together, in temporally regulating insect development. Since this is not a coherent field, we will review recent developments that serve as examples to illuminate this complex topic. First, we will consider studies conducted in Rhodnius that revealed how circadian pathways exert temporal control over the production and release of ecdysone. We will then take a look at molecular and genetic data that revealed the presence of two circadian clocks, located in the brain and the prothoracic gland, that regulate eclosion rhythms in Drosophila. In this context, we will also review recent developments that examined how the ecdysone hierarchy delays the differentiation of the crustacean cardioactive peptide (CCAP) neurons, an event that is critical for the timing of ecdysis and eclosion. Finally, we will discuss some recent findings that transformed our understanding of JH function.

Address: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

3915782a: Can.J.Cardiol., 1181-1193(2013)
Link: [ PubMED ] [ DOI ]
Title: The emerging role of biomarkers in atrial fibrillation.
Author: Kornej,J., Apostolakis,S., Bollmann,A., Lip,G.Y.
Abstract:
Improved diagnostic techniques have identified various biomarkers that might play an important role in prediction of atrial fibrillation (AF) and related outcomes (cardio- and cerebrovascular events, and mortality and rhythm outcomes). Biomarkers can include blood markers (eg, von Willebrand factor, D-dimer, natriuretic peptides, etc), urine (eg, proteinuria, estimated glomerular filtration rate, or creatinine clearance), cardiac imaging (echocardiography; transthoracic or transoesophageal), or cerebral imaging (eg, computed tomography or magnetic resonance imaging), which can provide additional refinement to clinical stroke risk stratification for identification of "high risk" subjects. Although inclusion of some blood-based biomarkers (eg, von Willebrand factor, D-dimer) in existing clinical stroke risk stratification schemes might improve their predictive value for identifying "high risk" patients, this concept might be outdated and overtaken by new developments in thromboprophylaxis (which now focus on initial identification of "low risk" patients who do not need any antithrombotic therapy, followed by patients with 1 or more stroke risk factors, to whom anticoagulation can be offered), and additional questionable practicality in "everyday" practice. Biomarkers could be applied as a "rule out" approach or as surrogates of anticoagulation efficacy in trials of new antithrombotic strategies. The present review aims to provide an update of the role of biomarkers in AF, with particular focus on AF outcomes.

Address: University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, United Kingdom

3915785a: Expert Opin.Drug Discov., 1309-1323(2013)
Link: [ PubMED ] [ DOI ]
Title: Discovery techniques for calcitonin gene-related peptide receptor antagonists for potential antimigraine therapies.
Author: Labruijere,S., Ibrahimi,K., Chan,K.Y., Maassenvandenbrink,A.
Abstract:
Introduction: Calcitonin gene-related peptide (CGRP) exerts a key function in migraine pathophysiology through the trigeminovascular system. Influencing this system via CGRP receptor antagonists seems to be an important new option in treating migraine attacks. To characterize new compounds, models are used to study the vascular effects as well as their effects on the central nervous system. Areas covered: The authors review the clinical trials and many different in vitro and in vivo experimental models that have been used to investigate the effects and side effects in animals, healthy subjects and patients. These experimental models are essential, not only in characterizing new CGRP receptor antagonists, but also in gaining more insight into the pathophysiological mechanisms behind migraines. Expert opinion: Although triptans were a major breakthrough in migraine treatment, they are not effective for every patient and contraindicated in patients with cardiovascular disease. There is still a demand for other acute antimigraine acting drugs with CGRP receptor antagonists being the most promising candidates. CGRP plays a role in protection against ischemia, but CGRP receptor antagonists do not seem to affect this protection to a harmfull extent, when used incidentally as acute antimigraine treatment. In order for drug specificity to be increased, the site of action needs to be identified; this consequently may lead to a decrease in dosing with fewer side effects.

Address: Erasmus Medical Center, Division of Pharmacology, Department of Internal Medicine , P.O. Box 2040, 3000 CA Rotterdam , The Netherlands +31 10 7043537/47

3915793a: Saudi Pharm.J., 125-141(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Prospects of pharmaceuticals and biopharmaceuticals loaded microparticles prepared by double emulsion technique for controlled delivery.
Author: Giri,T.K., Choudhary,C., Ajazuddin, Alexander,A., Badwaik,H., Tripathi,D.K.
Abstract:
Several methods and techniques are potentially useful for the preparation of microparticles in the field of controlled drug delivery. The type and the size of the microparticles, the entrapment, release characteristics and stability of drug in microparticles in the formulations are dependent on the method used. One of the most common methods of preparing microparticles is the single emulsion technique. Poorly soluble, lipophilic drugs are successfully retained within the microparticles prepared by this method. However, the encapsulation of highly water soluble compounds including protein and peptides presents formidable challenges to the researchers. The successful encapsulation of such compounds requires high drug loading in the microparticles, prevention of protein and peptide degradation by the encapsulation method involved and predictable release, both rate and extent, of the drug compound from the microparticles. The above mentioned problems can be overcome by using the double emulsion technique, alternatively called as multiple emulsion technique. Aiming to achieve this various techniques have been examined to prepare stable formulations utilizing w/o/w, s/o/w, w/o/o, and s/o/o type double emulsion methods. This article reviews the current state of the art in double emulsion based technologies for the preparation of microparticles including the investigation of various classes of substances that are pharmaceutically and biopharmaceutically active.

Address: Rungta College of Pharmaceutical Sciences and Research, Kohka Road, Kurud, Bhilai 490024, India

3915804a: Nat.Rev.Rheumatol., 595-603(2013)
Link: [ PubMED ] [ DOI ]
Title: Annexin A1: potential for glucocorticoid sparing in RA.
Author: Yang,Y.H., Morand,E., Leech,M.
Abstract:
Glucocorticoids have broad-ranging and powerful anti-inflammatory and immunomodulatory effects. Unsurprisingly, therefore, glucocorticoids are widely and persistently used to treat a large number of inflammatory diseases, including rheumatoid arthritis (RA), despite the well-described adverse effects of these drugs. Annexin A1 is a glucocorticoid-induced molecule that is known to replicate many of the described anti-inflammatory effects of glucocorticoids. In addition to the well-documented roles of this protein in neutrophil function, emerging evidence suggests that annexin A1 is involved in the modulation of T-cell function and the adaptive immune responses relevant to RA. Interest in annexin A1 was renewed after the delineation of the receptors for this protein. This breakthrough also led to advances in our understanding of anti-inflammatory annexin A1 mimetic peptides and agonistic compounds targeting these receptors, particularly those specific for the receptor N-formyl peptide receptor 2 (FPR2). Herein, we review the current knowledge of the biological activities of annexin A1 and their relevance to RA pathogenesis. We also discuss the potential of annexin A1 mimics and strategies aimed at potentiating annexin A1 signalling to become viable approaches to minimizing glucocorticoid use in RA and other inflammatory disorders.

Address: Centre for Inflammatory Diseases, Department of Medicine, Southern Clinical School, Monash University Faculty of Medicine, Nursing and Health Sciences, Monash Medical Centre, Clayton, VIC 3168, Australia

3915806a: Autoimmun.Rev., 1182-1187(2013)
Link: [ PubMED ] [ DOI ]
Title: HLA shared epitope and ACPA: Just a marker or an active player?
Author: Pratesi,F., Petit Teixeira,E., Sidney,J., Michou,L., Puxeddu,I., Sette,A., Cornelis,F., Migliorini,P.
Abstract:
Autoantibody production is genetically controlled and anti-citrullinated protein/peptide antibodies (ACPA) are not an exception to the rule. ACPA are highly specific markers of rheumatoid arthritis (RA) and are also associated with a more severe disease course. The production of ACPA is almost invariably observed in HLA-shared epitope (SE) positive patients. The DRB1 alleles sharing SE are those conferring susceptibility to RA. SE alleles behave like immune response genes, controlling both the specificity and the amount of ACPA produced. These data suggest a role of SE in the presentation of citrullinated antigens. The ability of SE alleles to bind selectively to citrullinated sequences as compared to the native counterparts has been demonstrated in the case of peptides derived from several joint associated proteins (vimentin, fibrinogen and cartilage intermediate-layer protein). On the contrary, EBV-derived citrullinated peptides do not display a biologically relevant binding to SE alleles even if the immune response to VCPs is under the genetic control of these alleles (namely *0401 and *0404). Thus, the presentation of citrullinated epitopes does not represent the only molecular mechanisms underlying the HLA-DRB1 effect on ACPA production.

Address: Clinical Immunology and Allergy Unit, Department of Internal Medicine, University of Pisa, Pisa, Italy

3915820a: Pediatr.Endocrinol.Rev., 494-502(2013)
Link:
Title: Determination of C-peptide in children: when is it useful?
Author: Besser,R.E.
Abstract:
Diabetes results from insulin deficiency but despite this endogenous insulin secretion is infrequently measured. C-peptide is not present in synthetic insulin so it's presence indicates endogenous secretion. One of the key roles for measuring C-peptide in childhood is to assist in the diagnosis of diabetes subtypes, which in turn determines appropriate management. It is also useful in Type 1 diabetes to monitor disease course, both in clinical practice and in trials following intervention with disease modifying agents. Measuring C-peptide routinely in Type 1 diabetes provides valuable information to the patient and clinician about glucose variability, risk of hypoglycemia and ketoacidosis. Newer more practical methods of C-peptide determination are now available to allow assessment of endogenous insulin secretion in routine clinical practice. We review the physiology of insulin secretion, the essential roles and methods for C-peptide determination in blood and in urine.

E-Mail: rachel.besser@nhs.net
Address: Peninsula NIHR Clinical Research Facility, Peninsula Medical School, University of Exeter, Exeter, UK

3915826a: Evid.-Based Complement.Altern.Med., 563716-563716(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: On the g-protein-coupled receptor heteromers and their allosteric receptor-receptor interactions in the central nervous system: focus on their role in pain modulation.
Author: Borroto-Escuela,D.O., Romero-Fernandez,W., Rivera,A., Van Craenenbroeck,K., Tarakanov,A.O., Agnati,L.F., Fuxe,K.
Abstract:
The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The δ opioid receptor (DOR) exerts an antagonistic allosteric influence on the μ opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β -arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/κ opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia.

Address: Department of Neuroscience, Karolinska Institutet, Retzius vag 8, 17177 Stockholm, Sweden

3915835a: Curr.Atheroscler.Rep., 357-357(2013)
Link: [ PubMED ] [ DOI ]
Title: Diabetes remission following metabolic surgery: is GLP-1 the culprit?
Author: Vidal,J., Jimenez,A.
Abstract:
The parallel occurrence of improved glucose tolerance and increased glucagon-like peptide 1 (GLP-1) response to meal intake following metabolic surgery (MS) demonstrated in several studies has led to the notion that GLP-1 is the culprit for the impressive rates of remission of type 2 diabetes mellitus (T2DM) following MS. In this article, we critically review current evidence supporting this view. Recent studies specifically designed to elucidate a causative role of GLP-1 in the antidiabetic effects of MS call into question GLP-1 as a key player for T2DM outcome following MS procedures such as Roux-en-Y gastric bypass and sleeve gastrectomy in morbidly obese subjects. Whether GLP-1 plays a more prominent role in the remission of T2DM following MS in subjects with moderate obesity warrants further studies. Appraisal of the mechanisms involved in the amelioration of hyperglycemia following MS is a priority, as it could help in the battle against the current combined epidemics of obesity and T2DM.

E-Mail: jovidal@clinic.ub.es
Address: Obesity Unit, Endocrinology and Diabetes Department, Hospital Clinic Universitari, Villarroel 170, 08036 Barcelona, Spain

3915839a: Appl.Microbiol.Biotechnol., 8439-8453(2013)
Link: [ PubMED ] [ DOI ]
Title: Tentative biosynthetic pathways of some microbial diketopiperazines.
Author: Gu,B., He,S., Yan,X., Zhang,L.
Abstract:
Cyclodipeptides and their derivatives, the diketopiperazines (DKPs), constitute a large class of natural products that exhibit various biological properties. Until recently, there are a few characterized DKP biosynthetic pathways. In all these cases, the formation of the cyclodipeptides that harbor the DKP scaffold is catalyzed either by nonribosomal peptide synthetases or by cyclodipeptide synthases. This review focuses on the DKP biosynthetic pathways and their associated molecular mechanisms.

Address: Key Laboratory of Applied Marine Biotechnology of the Ministry of Education, Ningbo University, Ningbo, 315211, China

3915849a: Br.Med.Bull., 57-68(2013)
Link: [ PubMED ] [ DOI ]
Title: Immunization to prevent congenital cytomegalovirus infection.
Author: Adler,S.P.
Abstract:
INTRODUCTION: A primary maternal cytomegalovirus (CMV) during pregnancy causes newborn disease that includes hearing deficit and/or mental retardation. SOURCES OF DATA: Relevant published literature. AREAS OF AGREEMENT: There are no biologic obstacles to immunization against fetal/placental infection with CMV. AREAS OF UNCERTAINTY: CMV vaccine trials may be difficult due to a lack of public awareness of CMV. Vaccine trials that use fetal infection as an endpoint will be prolonged, since vaccination will need to occur preconception. AREAS TIMELY FOR DEVELOPING RESEARCH: Vaccines in preclinical development include antigens of the CMV gB glycoprotein and the gH/gL UL128, 130 and 131 pentameric complex. These antigens induce antibodies that block viral entry into fibroblasts and endothelial/epithelial cells. Vaccines immunogenic in animals include an inactivated virus with a wild-type UL131 gene, a DNA vaccine using a wild-type UL130 gene and peptide vaccines using peptides from UL130 and 131. CONCLUSIONS: In spite of these potential obstacles, successful evaluation of CMV vaccines is possible.

E-Mail: sadler@vcu.edu
Address: Department of Microbiology, Medical College of Virginia Campus/Virginia Commonwealth University, PO Box 163, Richmond, VA 23298, USA

3915860a: Psychoneuroendocrinology, 2423-2438(2013)
Link: [ PubMED ] [ DOI ]
Title: The role of ghrelin signalling in second-generation antipsychotic-induced weight gain.
Author: Zhang,Q., Deng,C., Huang,X.F.
Abstract:
Based on clinical and animal studies, this review suggests a tri-phasic effect of second-generation antipsychotics (SGAs) on circulating ghrelin levels: an initial increase exerted by the acute effect of SGAs; followed by a secondary decrease possibly due to the negative feedback from the SGA-induced body weight gain or hyperphagia; and a final re-increase to reach the new equilibrium. Moreover, the results can also vary depending on individual SGAs, other hormonal states, dietary choices, and other confounding factors including medical history, co-treatments, age, gender, and ghrelin measurement techniques. Interestingly, rats treated with olanzapine, an SGA with high weight gain liabilities, are associated with increased hypothalamic ghrelin receptor (GHS-R1a) levels. In addition, expressions of downstream ghrelin signalling parameters at the hypothalamus, including neuropeptide Y (NPY)/agouti-related peptide (AgRP) and proopiomelanocortin (POMC) are also altered under SGA treatments. Thus, understanding the role of ghrelin signalling in antipsychotic drug-induced weight gain should offer potential novel pharmacological targets for tackling the obesity side-effect of SGAs and its associated metabolic syndrome.

Address: Centre for Translational Neuroscience, School of Medicine, University of Wollongong, Wollongong, NSW 2522, Australia

3915862a: Biomaterials, 8690-8707(2013)
Link: [ PubMED ] [ DOI ]
Title: Epidermal growth factor receptor targeting in cancer: a review of trends and strategies.
Author: Yewale,C., Baradia,D., Vhora,I., Patil,S., Misra,A.
Abstract:
The epidermal growth factor receptor (EGFR) is a cell-surface receptor belonging to ErbB family of tyrosine kinase and it plays a vital role in the regulation of cell proliferation, survival and differentiation. However; EGFR is aberrantly activated by various mechanisms like receptor overexpression, mutation, ligand-dependent receptor dimerization, ligand-independent activation and is associated with development of variety of tumors. Therefore, specific EGFR inhibition is one of the key targets for cancer therapy. Two major approaches have been developed and demonstrated benefits in clinical trials for targeting EGFR; monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs). EGFR inhibitors like, cetuximab, panitumumab, etc. (mAbs) and gefitinib, erlotinib, lapatinib, etc. (TKIs) are now commercially available for treatment of variety of cancers. Recently, many other agents like peptides, nanobodies, affibodies and antisense oligonucleotide have also shown better efficacy in targeting and inhibiting EGFR. Now a days, efforts are being focused to identify molecular markers that can predict patients more likely to respond to anti-EGFR therapy; to find out combinatorial approaches with EGFR inhibitors and to bring new therapeutic agents with clinical efficacy. In this review we have outlined the role of EGFR in cancer, different types of EGFR inhibitors, preclinical and clinical status of EGFR inhibitors as well as summarized the recent efforts made in the field of molecular EGFR targeting.

Address: Pharmacy Department, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara 390 001, Gujarat, India

3915865a: Semin.Nephrol., 237-247(2013)
Link: [ PubMED ] [ DOI ]
Title: Extracellular potassium homeostasis: insights from hypokalemic periodic paralysis.
Author: Cheng,C.J., Kuo,E., Huang,C.L.
Abstract:
Extracellular potassium makes up only about 2% of the total body's potassium store. The majority of the body potassium is distributed in the intracellular space, of which about 80% is in skeletal muscle. Movement of potassium in and out of skeletal muscle thus plays a pivotal role in extracellular potassium homeostasis. The exchange of potassium between the extracellular space and skeletal muscle is mediated by specific membrane transporters. These include potassium uptake by Na+, K+-adenosine triphosphatase and release by inward-rectifier K+ channels. These processes are regulated by circulating hormones, peptides, ions, and by physical activity of muscle as well as dietary potassium intake. Pharmaceutical agents, poisons, and disease conditions also affect the exchange and alter extracellular potassium concentration. Here, we review extracellular potassium homeostasis, focusing on factors and conditions that influence the balance of potassium movement in skeletal muscle. Recent findings that mutations of a skeletal muscle-specific inward-rectifier K+ channel cause hypokalemic periodic paralysis provide interesting insights into the role of skeletal muscle in extracellular potassium homeostasis. These recent findings are reviewed.

Address: Department of Medicine, Division of Nephrology, University of Texas Southwestern Medical Center, Dallas, TX 75390-8859, USA

3915866a: Biomaterials, 8533-8554(2013)
Link: [ PubMED ] [ DOI ]
Title: A review of the biomaterials technologies for infection-resistant surfaces.
Author: Campoccia,D., Montanaro,L., Arciola,C.R.
Abstract:
Anti-infective biomaterials need to be tailored according to the specific clinical application. All their properties have to be tuned to achieve the best anti-infective performance together with safe biocompatibility and appropriate tissue interactions. Innovative technologies are developing new biomaterials and surfaces endowed with anti-infective properties, relying either on antifouling, or bactericidal, or antibiofilm activities. This review aims at thoroughly surveying the numerous classes of antibacterial biomaterials and the underlying strategies behind them. Bacteria repelling and antiadhesive surfaces, materials with intrinsic antibacterial properties, antibacterial coatings, nanostructured materials, and molecules interfering with bacterial biofilm are considered. Among the new strategies, the use of phages or of antisense peptide nucleic acids are discussed, as well as the possibility to modulate the local immune response by active cytokines. Overall, there is a wealth of technical solutions to contrast the establishment of an implant infection. Many of them exhibit a great potential in preclinical models. The lack of well-structured prospective multicenter clinical trials hinders the achievement of conclusive data on the efficacy and comparative performance of anti-infective biomaterials.

Address: Research Unit on Implant Infections, Rizzoli Orthopaedic Institute, Via di Barbiano 1/10, 40136 Bologna, Italy

3915871a: Int.J.Clin.Pract., 876-887(2013)
Link: [ PubMED ] [ DOI ]
Title: Choice of early treatment regimen and impact on β-cell preservation in type 2 diabetes.
Author: Mudaliar,S.
Abstract:
The progressive deterioration of glycaemic control in individuals with type 2 diabetes mellitus (T2DM) results from insulin resistance combined with the ongoing loss of β-cell function. Although it had been suggested that most β-cell dysfunction occurs after the development of T2DM, studies have documented a substantial early loss of β-cell function, particularly during the prediabetic state. In patients diagnosed with T2DM, β-cell function continues to decline despite treatment with commonly prescribed antihyperglycaemic medications, and ultimately exogenous insulin administration is required to maintain optimal glycaemic control. Thus, interventions to address the early decline in β-cell function could potentially alter the course of T2DM, preventing or delaying its onset and decreasing the incidence of complications. Original research and review articles on this topic were identified in a PubMed search from January 2000 through August 2012. Data from prospective studies and clinical trials suggest that lifestyle modifications and certain antihyperglycaemic medications, including thiazolidinediones (TZDs), glucagon-like peptide-1 (GLP-1) agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors and insulin, may preserve or enhance β-cell function. The implication of current data is that early initiation of lifestyle modifications and antihyperglycaemic agents that preserve β-cell function might reverse or delay progression to T2DM in those with prediabetes. Moreover, improved β-cell function may confer more durable glucose control and perhaps reduce/delay the incidence of diabetic complications. Long-term studies are needed to validate this hypothesis.

E-Mail: smudaliar@vapop.ucsd.edu
Address: Center for Metabolic Research, VA San Diego Healthcare System, San Diego, CA, USA

3915924a: Immunol.Rev., 110-124(2013)
Link: [ PubMED ] [ DOI ]
Title: The great balancing act: regulation and fate of antiviral T-cell interactions.
Author: Moseman,E.A., McGavern,D.B.
Abstract:
The fate of T lymphocytes revolves around a continuous stream of interactions between the T-cell receptor (TCR) and peptide-major histocompatibility complex (MHC) molecules. Beginning in the thymus and continuing into the periphery, these interactions, refined by accessory molecules, direct the expansion, differentiation, and function of T-cell subsets. The cellular context of T-cell engagement with antigen-presenting cells, either in lymphoid or non-lymphoid tissues, plays an important role in determining how these cells respond to antigen encounters. CD8+ T cells are essential for clearance of a lymphocytic choriomeningitis virus (LCMV) infection, but the virus can present a number of unique challenges that antiviral T cells must overcome. Peripheral LCMV infection can lead to rapid cytolytic clearance or chronic viral persistence; central nervous system infection can result in T-cell-dependent fatal meningitis or an asymptomatic carrier state amenable to immunotherapeutic clearance. These diverse outcomes all depend on interactions that require TCR engagement of cognate peptide-MHC complexes. In this review, we explore the diversity in antiviral T-cell behaviors resulting from TCR engagement, beginning with an overview of the immunological synapse and progressing to regulators of TCR signaling that shape the delicate balance between immunopathology and viral clearance.

Address: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA

3915925a: Immunol.Rev., 40-56(2013)
Link: [ PubMED ] [ DOI ]
Title: The impact of macroautophagy on CD8+ T-cell-mediated antiviral immunity.
Author: Perot,B.P., Ingersoll,M.A., Albert,M.L.
Abstract:
Macroautophagy is a catabolic recycling pathway, which can be induced by various stress stimuli. Viruses are able to manipulate autophagy in the cells that they infect. The impact of autophagy on the innate immune response to viruses and its stimulatory role in antigen presentation to CD4+ T cells are well documented. Herein, we present the impact of autophagy on the activation of cytotoxic T lymphocyte (CTL)-mediated antiviral immune responses, which are required for the eradication or control of multiple viruses. We first discuss the general mechanisms by which viruses can either induce or block autophagy in cells. We then explore the cross-talk between autophagy and innate immune processes, which are both first line defenses against viruses; and constitute crucial steps for the initiation of potent adaptive immune responses. We describe the impact of autophagy on the presentation of viral peptide antigens on class I major histocompatibility complex (MHC I), a prerequisite for the priming of CTL responses. In sum, our review highlights the interplay between viruses and three integrated host response pathways - autophagy, innate and adaptive immunity - providing a framework for future mechanistic and pathogenesis-based research.

Address: Unite d'immunobiologie des cellules dendritiques, Institut Pasteur, Paris, France

3915927a: Roum.Arch.Microbiol.Immunol., 63-86(2013)
Link:
Title: Adenylate cyclases involvement in pathogenicity, a minireview.
Author: Costache,A., Bucurenci,N., Onu,A.
Abstract:
Cyclic AMP (cAMP), one of the most important secondary messengers, is produced by adenylate cyclase (AC) from adenosine triphosphate (ATP). AC is a widespread enzyme, being present both in prokaryotes and eukaryotes. Although they have the same enzymatic activity (ATP cyclization), the structure of these proteins varies, depending on their function and the producing organism. Some pathogenic bacteria utilize these enzymes as toxins which interact with calmodulin (or another eukaryote activator), causing intense cAMP synthesis and disruption of infected cell functions. In contrast, other pathogenic bacteria benefit of augmentation of AC activity for their own function. Based on sequence analysis ofAC catalytic domain from two pathogenic bacteria (Bacillus anthracis and Bordetellapertussis) with known three-dimensional structures, a possible secondary structure for 1-255 amino acid fragment from Pseudomonas aeruginosa AC (with 80TKGFSVKGKSS90 as the ATP binding site) is proposed.

E-Mail: adriana_rad@yahoo.com
Address: Laboratory of Enzymologyy and Applied Microbiology, Cantacuzino National Institute of Research-Development for Microbiology and Immunology, Bucharest, Romania

3915931a: Nanoscale, 8821-8836(2013)
Link: [ PubMED ] [ DOI ]
Title: Nanomedicine for treating spinal cord injury.
Author: Tyler,J.Y., Xu,X.M., Cheng,J.X.
Abstract:
Spinal cord injury results in significant mortality and morbidity, lifestyle changes, and difficult rehabilitation. Treatment of spinal cord injury is challenging because the spinal cord is both complex to treat acutely and difficult to regenerate. Nanomaterials can be used to provide effective treatments; their unique properties can facilitate drug delivery to the injury site, enact as neuroprotective agents, or provide platforms to stimulate regrowth of damaged tissues. We review recent uses of nanomaterials including nanowires, micelles, nanoparticles, liposomes, and carbon-based nanomaterials for neuroprotection in the acute phase. We also review the design and neural regenerative application of electrospun scaffolds, conduits, and self-assembling peptide scaffolds.

E-Mail: jcheng@purdue.edu
Address: Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA

3915932a: J.Biomol.Screen., 947-966(2013)
Link: [ PubMED ] [ DOI ]
Title: Are GPCRs Still a Source of New Targets?
Author: Garland,S.L.
Abstract:
G-protein-coupled receptors (GPCRs) still offer enormous scope for new therapeutic targets. Currently marketed agents are dominated by those with activity at aminergic receptors and yet they account for only ~10% of the family. Progress up until now with other subfamilies, notably orphans, Family A/peptide, Family A/lipid, Family B, Family C, and Family F, has been, at best, patchy. This may be attributable to the heterogeneous nature of GPCRs, their endogenous ligands, and consequently their binding sites. Our appreciation of receptor similarity has arguably been too simplistic, and screening collections have not necessarily been well suited to identifying leads in new areas. Despite the relative shortage of high-quality tool molecules in a number of cases, there is an emerging, and increasingly substantial, body of evidence associating many as yet "undrugged" receptors with a very wide range of diseases. Significant advances in our understanding of receptor pharmacology and technical advances in screening, protein X-ray crystallography, and ligand design methods are paving the way for new successes in the area. Exploitation of allosteric mechanisms; alternative signaling pathways such as G12/13, Gβγ, and β-arrestin; the discovery of "biased" ligands; and the emergence of GPCR-protein complexes as potential drug targets offer scope for new and much improved drugs.

Address: 1NQuiX Ltd, Stevenage Bioscience Catalyst, Stevenage SG1 2FX, UK

3915934a: Curr.Opin.Clin.Nutr.Metab.Care, 645-649(2013)
Link: [ PubMED ] [ DOI ]
Title: Natriuretic peptides and fat metabolism.
Author: Moro,C.
Abstract:
PURPOSE OF REVIEW: Cardiac natriuretic peptides have emerged as potent metabolic hormones during the past decade. We here discuss recent work highlighting the potential importance of these hormones in metabolic physiology and diseases. RECENT FINDINGS: Natriuretic peptides signal through a cyclic guanosine monophosphate pathway to convey their biological effects at the cell level. Similarly to cyclic adenosine monophosphate, activation of cyclic guanosine monophosphate signaling induces a browning of white fat and thermogenesis. Natriuretic peptides also enhance oxidative capacity and fat oxidation in skeletal muscle of mice and humans. The molecular mechanism involves an upregulation of mitochondrial fat oxidative capacity and respiration. This may be particularly relevant to relay the physiological adaptations of chronic exercise. Population-based studies indicate that circulating natriuretic peptides are lowered in obesity and predict type 2 diabetes. Recent work also directly link natriuretic peptides with type 2 diabetes through a gut-heart axis. SUMMARY: Natriuretic peptides exhibit a wide range of biological actions to control metabolic homeostasis. Natriuretic peptides deficiency in obesity may trigger metabolic dysfunction and lead to type 2 diabetes. Increasing circulating natriuretic peptides level and tissue signaling may help to fight against metabolic complications of obesity.

Address: aInserm, UMR1048, Obesity Research Laboratory, Institute of Metabolic and Cardiovascular Diseases (I2MC) bUMR1048, Paul Sabatier University, Toulouse, France

3915969a: Free Radic.Res., 950-957(2013)
Link: [ PubMED ] [ DOI ]
Title: Redox signaling mediated by the gut microbiota.
Author: Neish,A.S.
Abstract:
The microbiota that occupies the mammalian intestine can modulate a range of physiological functions, including control over immune responses, epithelial barrier function, and cellular proliferation. While commensal prokaryotic organisms are well known to stimulate inflammatory signaling networks, less is known about control over homeostatic pathways. Recent work has shown that gut epithelia contacted by enteric commensal bacteria rapidly generate reactive oxygen species (ROS). While the induced production of ROS in professional phagocytes via stimulation of formyl peptide receptors (FPRs) and activation of NADPH oxidase 2 (Nox2) is a well-studied process, ROS are also similarly elicited in other cell types, including intestinal epithelia, in response to microbial signals via FPRs and the epithelial NADPH oxidase 1 (Nox1). ROS generated by Nox enzymes have been shown to function as critical second messengers in multiple signal transduction pathways via the rapid and transient oxidative inactivation of a distinct class of sensor proteins bearing oxidant-sensitive thiol groups. These redox-sensitive proteins include tyrosine phosphatases that serve as regulators of MAP kinase pathways, focal adhesion kinase, as well as components involved in NF-κB activation. As microbe-elicited ROS has been shown to stimulate cellular proliferation and motility, and to modulate innate immune signaling, we hypothesize that many of the established effects of the normal microbiota on intestinal physiology may be at least partially mediated by this ROS-dependent mechanism.

Address: Department of Pathology and Laboratory Medicine, Emory University School of Medicine , Atlanta, GA , USA

3915970a: Curr.Neurovasc.Res., 356-360(2013)
Link:
Title: Vehicles for lipophilic drugs: implications for experimental design, neuroprotection, and drug discovery.
Author: Rivers-Auty,J., Ashton,J.C.
Abstract:
The delivery of some classes of drugs is challenging. Solubility, absorption, distribution, and duration of action may all be altered by combination with vehicle molecules. It has already been discovered that polyethylene glycol - which is used as a stabiliser in peptide drug formulations - has biological activity in its own right, including potential neuroprotective properties. In this article we review the evidence for confounding activity for four distinct compounds that have been used as solvents and/or carrier molecules for the delivery of lipophilic drugs under investigation for potential neuroprotective properties. We discuss the evidence that cyclodextrins, ethanol, dimethyl sulphoxide, and a castor oil derivative - Cremophor EL - have all been found to have mild to moderate neuroprotective effects. We argue that this has probably reduced the statistical power and increased the Type II error rates of neuroprotection experiments that have employed these vehicles, and suggest experimental design considerations to help correct the problem. However, we also note that the properties of these compounds may represent an opportunity for drug development, particularly for the newer compounds that have been subject to only limited experimental investigation.

E-Mail: john.ashton@otago.ac.nz
Address: Department of Pharmacology and Toxicology, School of Medical Sciences, University of Otago, P.O. Box 913, Dunedin, New Zealand

3915986a: Chem.Soc.Rev., 8517-8539(2013)
Link: [ PubMED ] [ DOI ]
Title: Functionalized magnetic nanoparticles for sample preparation in proteomics and peptidomics analysis.
Author: Li,Y., Zhang,X., Deng,C.
Abstract:
Sample preparation is a fundamental step in the proteomics and peptidomics workflow. Due to their good biocompatibility, superparamagnetic property, and high binding capacity, magnetic nanoparticles (MNPs) functionalized with different active moieties have been widely applied in recent years in various sample preparation procedures in proteomics and peptidomics analysis. The magnetic cores of the MNPs facilitate elegant handling using only magnetic devices and their small diameters are advantageous for increasing the sensitivity when using subsequent mass spectrometry (MS) analysis or gel electrophoresis. This review mainly focuses on overviewing present advances in the preparation and application of functionalized magnetic nanoparticles for sample preparation in proteomics and peptidomics analysis, including protein digestion, enrichment of low-abundance peptides/proteins and specific enrichment of peptides/proteins with post-translational modifications, such as phosphorylation and glycosylation.

Address: Department of Pharmaceutical Analysis, School of Pharmacy, Fudan University, Shanghai 201203, P. R. China

3915992a: J.Chin.Med.Assoc., 606-610(2013)
Link: [ PubMED ] [ DOI ]
Title: Amniotic fluid may act as a transporting pathway for signaling molecules and stem cells during the embryonic development of amniotes.
Author: Tong,X.
Abstract:
Amniotic fluid (AF) is formed at the very early stages of pregnancy, and is present throughout embryonic development of amniotes. It is well-known that AF provides a protective sac around the fetus that allows fetal movement and growth, and prevents mechanical and thermal shock. However, a growing body of evidence has shown that AF contains a number of proteins and peptides, including growth factors and cytokines, which potently affect cellular growth and proliferation. In addition, pluripotent stem cells have recently been identified in AF. Herein, this article reviews the biological properties of AF during embryonic development and speculates that AF may act as a transporting pathway for signaling molecules and stem cells during amniote embryonic development. Defining this novel function of AF is potentially significant for further understanding embryonic development and regenerative medicine, preventing genetic diseases, and developing therapeutic options for human malignancies.

E-Mail: xinglongtong@hotmail.com
Address: Hebei Xinglong Institute of Pharmacological and Medical Science, Shijiazhuang, China. Electronic address:

3916002a: Behav.Brain Res., 5-9(2013)
Link: [ PubMED ] [ DOI ]
Title: Lunasin-induced behavioural effects in mice: Focus on the dopaminergic system.
Author: Dzirkale,Z., Rumaks,J., Svirskis,S., Mazina,O., Allikalt,A., Rinken,A., Jekabsons,K., Muceniece,R., Klusa,V.
Abstract:
The present study for the first time is devoted to identify central effects of synthetic lunasin, a 43 amino acid peptide. A markedly expressed neuroleptic/cataleptic effect was observed at low (0.1-10nmol/mouse) centrally administered doses in male C57Bl/6 mice. Lunasin considerably reduced the amphetamine hyperlocomotion but weakly apomorphine climbing behaviour. No influence on ketamine and bicuculline effects was observed. Binding assay studies demonstrated modest affinity of lunasin for the dopamine D1 receptor (Ki=60+/-15μM). In a functional assay of cAMP accumulation on live cells lunasin antagonised apomorphine effect on D1 receptor activation (pEC50=6.1+/-0.3), but had no effect in cells expressing D2 receptors. The obtained data suggest that lunasin's action at least in part is provided via dopaminergic D1 receptor pathways. However, other non-identified mechanisms (probably intracellular) may play an important role in lunasin's central action. Nevertheless further studies of lunasin are promising, particularly taking into account a necessity for novel type of antipsychotic drugs.

Address: Department of Pharmacology, Faculty of Medicine, University of Latvia, 1A Sarlotes St, Riga, LV-1001, Latvia

3916025a: Curr.Top.Med.Chem., 1787-1807(2013)
Link: [ PubMED ]
Title: The structural evolution of β-secretase inhibitors: a focus on the development of small-molecule inhibitors.
Author: Butini,S., Brogi,S., Novellino,E., Campiani,G., Ghosh,A.K., Brindisi,M., Gemma,S.
Abstract:
Effective treatment of Alzheimer's disease (AD) remains a critical unmet need in medicine. The lack of useful treatment for AD led to an intense search for novel therapies based on the amyloid hypothesis, which states that amyloid β-42 (Aβ42) plays an early and crucial role in all cases of AD. β-Secretase (also known as BACE-1 β-site APP-cleaving enzyme, Asp-2 or memapsin-2) is an aspartyl protease representing the rate limiting step in the generation of Aβ peptide fragments, therefore it could represent an important target in the steady hunt for a disease-modifying treatment. Generally, β-secretase inhibitors are grouped into two families: peptidomimetic and nonpeptidomimetic inhibitors. However, irrespective of the class, serious challenges with respect to blood-brain barrier (BBB) penetration and selectivity still remain. Discovering a small molecule inhibitor of β-secretase represents an unnerving challenge but, due to its significant potential as a therapeutic target, growing efforts in this task are evident from both academic and industrial laboratories. In this frame, the rising availability of crystal structures of β-secretase-inhibitor complexes represents an invaluable opportunity for optimization. Nevertheless, beyond the inhibitory activity, the major issue of the current research approaches is about problems associated with BBB penetration and pharmacokinetic properties. This review follows the structural evolution of the early β-secretase inhibitors and gives a snap-shot of the hottest chemical templates in the literature of the last five years, showing research progress in this field.

Address: European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy

3916026a: Curr.Top.Med.Chem., 1820-1842(2013)
Link: [ PubMED ]
Title: Dual inhibitors of β-amyloid aggregation and acetylcholinesterase as multi-target anti-Alzheimer drug candidates.
Author: Viayna,E., Sabate,R., Munoz-Torrero,D.
Abstract:
Notwithstanding the functional role that the aggregates of some amyloidogenic proteins can play in different organisms, protein aggregation plays a pivotal role in the pathogenesis of a large number of human diseases. One of such diseases is Alzheimer's disease (AD), where the overproduction and aggregation of the β-amyloid peptide (Aβ) are regarded as early critical factors. Another protein that seems to occupy a prominent position within the complex pathological network of AD is the enzyme acetylcholinesterase (AChE), with classical and non-classical activities involved at the late (cholinergic deficit) and early (Aβ aggregation) phases of the disease. Dual inhibitors of Aβ aggregation and AChE are thus emerging as promising multi-target agents with potential to efficiently modify the natural course of AD. In the initial phases of the drug discovery process of such compounds, in vitro evaluation of the inhibition of Aβ aggregation is rather troublesome, as it is very sensitive to experimental assay conditions, and requires expensive synthetic Aβ peptides, which makes cost-prohibitive the screening of large compound libraries. Herein, we review recently developed multitarget anti-Alzheimer compounds that exhibit both Aβ aggregation and AChE inhibitory activities, and, in some cases also additional valuable activities such as BACE-1 inhibition or antioxidant properties. We also discuss the development of simplified in vivo methods for the rapid, simple, reliable, unexpensive, and high-throughput amenable screening of Aβ aggregation inhibitors that rely on the overexpression of Aβ42 alone or fused with reporter proteins in Escherichia coli.

Address: Laboratori de Quimica Farmaceutica, Facultat de Farmacia, and Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Joan XXIII, 27-31, 08028-Barcelona, Spain

3916031a: Wiley Interdiscip.Rev.Nanomed.Nanobiotechnol., 582-612(2013)
Link: [ PubMED ] [ DOI ]
Title: Self-assembly in nature: using the principles of nature to create complex nanobiomaterials.
Author: Mendes,A.C., Baran,E.T., Reis,R.L., Azevedo,H.S.
Abstract:
Self-assembly is a ubiquitous process in biology where it plays numerous important roles and underlies the formation of a wide variety of complex biological structures. Over the past two decades, materials scientists have aspired to exploit nature's assembly principles to create artificial materials, with hierarchical structures and tailored properties, for the fabrication of functional devices. Toward this goal, both biological and synthetic building blocks have been subject of extensive research in self-assembly. In fact, molecular self-assembly is becoming increasingly important for the fabrication of biomaterials because it offers a great platform for constructing materials with high level of precision and complexity, integrating order and dynamics, to achieve functions such as stimuli-responsiveness, adaptation, recognition, transport, and catalysis. The importance of peptide self-assembling building blocks has been recognized in the last years, as demonstrated by the literature available on the topic. The simple structure of peptides, as well as their facile synthesis, makes peptides an excellent family of structural units for the bottom-up fabrication of complex nanobiomaterials. Additionally, peptides offer a great diversity of biochemical (specificity, intrinsic bioactivity, biodegradability) and physical (small size, conformation) properties to form self-assembled structures with different molecular configurations. The motivation of this review is to provide an overview on the design principles for peptide self-assembly and to illustrate how these principles have been applied to manipulate their self-assembly across the scales. Applications of self-assembling peptides as nanobiomaterials, including carriers for drug delivery, hydrogels for cell culture and tissue repair are also described. WIREs Nanomed Nanobiotechnol 2013, 5:582-612. doi: 10.1002/wnan.1227 Conflict of interest: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.

Address: 3B's Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimaraes, Portugal

3916054a: Curr.Diabetes Rep., 723-732(2013)
Link: [ PubMED ] [ DOI ]
Title: Use of glucagon-like peptide-1 agonists to improve islet graft performance.
Author: Wang,Y., Qi,M., McGarrigle,J.J., Rady,B., Davis,M.E., Vaca,P., Oberholzer,J.
Abstract:
Human islet transplantation is an effective and promising therapy for type I diabetes. However, long-term insulin independence is both difficult to achieve and inconsistent. De novo or early administration of incretin-based drugs is being explored for improving islet engraftment. In addition to its glucose-dependent insulinotropic effects, incretins also lower postprandial glucose excursion by inhibiting glucagon secretion, delaying gastric emptying, and can protect β-cell function. Incretin therapy has so far proven clinically safe and tolerable with little hypoglycemic risk. The present review aims to highlight the new frontiers in research involving incretins from both in vitro and in vivo animal studies in the field of islet transplant. It also provides an overview of the current clinical status of incretin usage in islet transplantation in the management of type I diabetes.

E-Mail: wangy@uic.edu
Address: Department of Surgery/Transplant, University of Illinois at Chicago, Chicago, IL, 60612, USA,

3916064a: J.Drug Target., 787-800(2013)
Link: [ PubMED ] [ DOI ]
Title: CNS drug targeting: have we travelled in right path?
Author: Punitha,A.D., Srivastava,A.K.
Abstract:
Background: Brain disorders, their prevalence and central nervous system (CNS) targeting are now at the rise. However, complexities of blood brain barrier (BBB) have limited the success of CNS targeting. Basic criteria necessary for passive diffusion were believed to influence the CNS drug delivery. However, often, BBB transportation has differed from the dogma of basic criteria. Purpose: This communication resets the extent to which basic criteria influence the brain delivery, through commanding examples. Further, it appraises the intervention of ABC transporters in BBB transportation, with a special emphasis on P-glycoprotein; and also brings forth the successful CNS transportation of therapeutics achieved through chimeric peptide technology. As a right path to travel, it flickers light on the novel CNS molecular drug targets/biomarkers which are specially expressed by diseased cells. Conclusion: Screening for right molecular target is of great importance for cost and time effective drug discovery process. The principle of chimeric peptide technology should be applied for CNS targeting; and every diseased cell should be screened for its biomarker. Thanks to glycan/lectin arrays technique which scans expression pattern of transporter and their possible ligands, and paves way for a new dimensional research.

Address: Department of Pharmaceutics, Indian Institute of Technology (BHU), Varanasi , Uttar Pradesh , India

3916068a: Pharmazie, 484-491(2013)
Link: [ PubMED ]
Title: Advances in personalized medicine - medicinal chemistry and pharmacology of vemurafenib and ivacaftor.
Author: Pellowska,M., Merk,D., Schubert-Zsilavecz,M.
Abstract:
Pharmacogenomics offers an entrance in the field of personalized medicine. This form of adapted therapy is going to be the future concerning the reduction of side effects and efficacy of the treatment of severe diseases. Vemurafenib and Ivacaftor are the first FDA approved drugs specially addressing mutated proteins. Both substances showed promising results in all clinical trials combined with relatively mild side effects by vemurafenib and placebo-like side effects by ivacaftor. The efficacy in addressing the specific mutation of each compound was confirmed in preclinical and clinical development.

Address: Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt am Main, Germany

3916076a: Macromol.Rapid Commun., 1343-1350(2013)
Link: [ PubMED ] [ DOI ]
Title: Polydiacetylene-peptide 1D nanomaterials.
Author: Diegelmann,S.R., Tovar,J.D.
Abstract:
Polydiacetylenes have received intense attention on account of their well-established chromic alterations that are detectable often by the naked eye, making them ideal for a variety of applications such as biosensory materials. These polymers have been fabricated in a variety of materials platforms including 3D crystals, 2D monolayers, and 0D spherical vesicles; however, 1D morphologies that might be useful for directional energy migration are less common. This article describes the development and current research efforts of protein-based 1D nanowire-like supramolecular assemblies with embedded polydiacetylenes.

Address: Department of Chemistry, Department of Materials Science and Engineering, Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA

3916086a: Curr.Alzheimer Res., 797-808(2013)
Link:
Title: Multipotent, Permeable Drug ASS234 Inhibits Aβ Aggregation, Possesses Antioxidant Properties and Protects from Aβ-induced Apoptosis In Vitro.
Author: Bolea,I., Gella,A., Monjas,L., Perez,C., Rodriguez-Franco,M.I., Marco-Contelles,J., Samadi,A., Unzeta,M.
Abstract:
Amyloid β (Aβ) aggregation and deposition is a key pathological hallmark of AD. Growing evidence suggests that neurotoxicity of this peptide is related to the formation of toxic oligomeric aggregates. Therefore, a deeply investigated therapeutic strategy comes at present from blocking the formation of these species to non-toxic aggregates. Among other considered strategies, the multi-target approach has been proposed as a more suitable potential therapy, precisely due to the multifactorial nature of AD. In this context, we recently identified ASS234, a novel compound that possesses a significant multipotent profile since it is able to inhibit cholinesterase and monoamine oxidase enzymes as well as to interfere in Aβ aggregation process. In this work, we investigated more in detail the effects of ASS234 on Aβ aggregation and toxicity in vitro as well as we explored its ability to penetrate to the CNS. We report that ASS234 inhibited Aβ1-42 self-aggregation more efficiently than that of Aβ1-40, limiting the formation of fibrillar and oligomeric species. Additionally, ASS234 completely blocked the aggregation mediated by AChE of both Aβ1-42 and Aβ1-40, showing a dual binding site to AChE. Interestingly, ASS234 significantly reduced Aβ1-42-mediated toxicity in SH-SY5Y human neuroblastoma cells through the prevention of the mitochondrial apoptosis pathway activation. Also importantly, we observed a significant ability of ASS234 to capture free-radical species in vitro as well as a potent effect in preventing the Aβ1-42-induced depletion of antioxidant enzymes (catalase and SOD-1). Finally, we report the capability of ASS234 to cross the bloodbrain barrier. Overall, our in vitro results show that ASS234 may have an impact on different processes involved in AD pathogenesis and provide evidences that it has encouraging attributes as a therapeutic lead compound.

E-Mail: Mercedes.unzeta@uab.cat
Address: Departament de Bioquimica i Biologia Molecular, Facultat de Medicina, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

3916087a: Curr.Alzheimer Res., 809-817(2013)
Link:
Title: Impact of apolipoprotein e on Alzheimer's disease.
Author: Hauser,P.S., Ryan,R.O.
Abstract:
A key feature of Alzheimer's disease (AD) is deposition of extracellular amyloid plaque comprised chiefly of the amyloid β (Aβ) peptide. Studies of Aβ have shown that it may be catabolized by proteolysis or cleared from brain via members of the low-density lipoprotein receptor family. Alternatively, Aβ can undergo a conformational transition from α-helix to β-sheet, a conformer that displays a propensity to self-associate, oligomerize and form fibrils. Furthermore, β- sheet conformers catalyze conversion of other α-helical Aβ peptides to β-sheet, feeding the oligomer and fibril assembly process. A factor that influences the fate of Aβ in the extracellular space is apolipoprotein (apo) E. Polymorphism at position 112 or 158 in apoE give rise to three major isoforms. One isoform in particular, apoE4 (Arg at 112 and 158), has generated considerable interest since the discovery that it is the major genetic risk factor for development of late onset AD. Despite this striking correlation, the molecular mechanism underlying apoE4's association with AD remains unclear. A tertiary structural feature distinguishing apoE4 from apoE2 and apoE3, termed domain interaction, is postulated to affect the conformation and orientation of its' two independently folded domains. This feature has the potential to influence apoE4's interaction with Aβ, its sensitivity to proteolysis or its lipid accrual and receptor binding activities. Thus, domain interaction may constitute the principal molecular feature of apoE4 that predisposes carriers to late onset AD. By understanding the contribution of apoE4 to AD at the molecular level new therapeutic or prevention strategies will emerge.

E-Mail: rryan@chori.org
Address: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609 USA

3916090a: Expert Opin.Pharmacother., 2047-2058(2013)
Link: [ PubMED ] [ DOI ]
Title: Dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes: comparison, efficacy and safety.
Author: Deacon,C.F., Holst,J.J.
Abstract:
INTRODUCTION: Dipeptidyl peptidase (DPP)-4 inhibitors belong to one class of drugs that have been approved for treatment of type 2 diabetes (T2D) based on the glucose-lowering actions of the gastrointestinal hormone glucagon-like peptide (GLP)-1. Several different compounds are now available, and although their mechanism of action (inhibition of the catalytic activity of DPP-4) is the same, there are fundamental differences between them. AREAS COVERED: The authors discuss the differences between different DPP-4 inhibitors and review their therapeutic efficacy and key safety data. The literature covered includes original studies and meta-analyses identified in PubMed, recent abstracts presented at major diabetes scientific conferences, and clinical trials registered at ClinicalTrials.gov. EXPERT OPINION: Although there are some differences in the pharmacokinetic and pharmacodynamic profiles of the different DPP-4 inhibitors, all are small orally active compounds with broadly similar HbA1c-lowering efficacy. They improve glycaemic control in T2D, without increasing the risk of hypoglycaemia or causing weight gain. They can be used as monotherapy or in combination with other anti-diabetic therapies, including insulin, regardless of renal or hepatic function, and are efficacious across the spectrum of patients with T2D, including those with long-standing disease duration. DPP-4 inhibitors may also have beneficial effects beyond glycaemic control, although this remains to be demonstrated in purpose-designed clinical trials.

Address: University of Copenhagen, Panum Institute 12.2.23, Department of Biomedical Sciences , Blegdamsvej 2, DK-2200 Copenhagen N , Denmark +45 3532 7523

3916092a: BioDrugs, 509-523(2013)
Link: [ PubMED ] [ DOI ]
Title: Lixisenatide: a review of its use in patients with type 2 diabetes mellitus.
Author: Scott,L.J.
Abstract:
Lixisenatide (Lyxumia((R))) is a glucagon-like peptide-1 receptor agonist that acts in a glucose-dependent manner to improve glycemic control in adult patients with type 2 diabetes mellitus. Subcutaneous once-daily prandial lixisenatide is indicated for the treatment of adult patients with type 2 diabetes to achieve glycemic control in combination with oral antihyperglycemic drugs (OADs) and/or basal insulin when these antihyperglycemic drugs do not provide adequate glycemic control. In an extensive phase III clinical trial program, lixisenatide once daily in combination with OADs and/or basal insulin for 24 weeks improved glycemic control, had beneficial effects on bodyweight, and was generally well tolerated in adult patients with inadequately controlled type 2 diabetes despite treatment with OADs and/or basal insulin. At 24 weeks, in terms of the primary efficacy endpoint of each trial, combination therapy with lixisenatide was associated with better efficacy than placebo in patients inadequately controlled on OADs and/or basal insulin, was shown to be noninferior to exenatide in patients inadequately controlled on background metformin therapy, and showed similar efficacy to sitagliptin in patients inadequately controlled on background metformin therapy. Further clinical experience/post-marketing surveillance studies and long-term safety data, along with pharmacoeconomic analyses, are required to fully define the position of lixisenatide in relation to other antihyperglycemics. In the meantime, once-daily prandial lixisenatide in combination with OADs and/or basal insulin (plus diet and exercise) is an effective option for improving glycemic control in adult patients with type 2 diabetes, including in patients where bodyweight loss is an essential component in their management.

E-Mail: demail@springer.com
Address: Adis, 41 Centorian Drive, Private Bag 65901, Mairangi Bay, North Shore, 0754, Auckland, New Zealand,

3916137a: Int.J.Biochem.Cell Biol., 2179-2185(2013)
Link: [ PubMED ] [ DOI ]
Title: Control of food intake and muscle wasting in cachexia.
Author: Amitani,M., Asakawa,A., Amitani,H., Inui,A.
Abstract:
Cachexia is characterized by anorexia, weakness, weight loss, and muscle wasting. Anorexia and muscle wasting are the key features of cachexia and they affect mortality, morbidity, and quality of life. Consistent studies have found that feeding-regulating peptides such as melanocortin, ghrelin, and leptin are related to muscle metabolism, and the balance of catabolism and anabolism in muscle is regulated in the hypothalamus, which also regulates appetite and energy expenditure. In cachexia, proinflammatory cytokines, such as TNF-α, IL-1, IL-6 and Angiotensin II induce muscle atrophy. The mechanism is suggested via upregulation of MuRF1 and MAFbx. In contrast, the orexigenic peptide, AgRP and ghrelin have the effect to decrease proinflammatory cytokines and increase body weight, food intake, and muscle mass. The understandings of the pathological mechanism of anorexia and muscle metabolism in view of the crosstalk between brain and muscle will open the new way for the management of cachexia. In this review, we describe recent experimental and clinical studies that have examined the regulation of food intake and muscle wasting in cachexia. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.

Address: Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

3916184a: Expert Rev.Gastroenterol.Hepatol., 683-687(2013)
Link: [ PubMED ] [ DOI ]
Title: Study of teduglutide effectiveness in parenteral nutrition-dependent short-bowel syndrome subjects.
Author: Vipperla,K., O'Keefe,S.J.
Abstract:
Loss of intestinal absorptive capacity from congenital defect, surgical resection or mucosal disease results in short bowel syndrome (SBS)-associated intestinal failure. In the past, few medical management options were available besides dietary modification, controlling diarrhea or high stomal output, and providing parenteral fluid, electrolyte and nutrient support (parenteral support). Recent research on strategies to enhance the intestinal absorptive capacity focused on glucagon-like peptide-2, an intestinotrophic hormone that has been shown to increase the villus height and crypt depth, and decrease gastric motility and intestinal secretory losses. STEPS is a Phase III randomized double-blinded controlled trial in which teduglutide, a recombinant analog of glucagon-like peptide-2, or placebo was given subcutaneously to SBS patients for 24 weeks. A clinically meaningful response, defined as a 20-100% reduction in parenteral support volume, was achieved in 63% of the treatment group compared with 30% in the placebo group (p = 0.002) without an increase in serious side effects. Teduglutide offers a new targeted approach to SBS-associated intestinal failure management. Its specific role in clinical practice remains to be evaluated.

Address: Clinical Instructor of Medicine, Division of General Internal Medicine, University of Pittsburgh Medical Center, 200 Lothrop Street, 933W MUH, Pittsburgh, PA 15213, USA

3916190a: Front.Physiol., 264-264(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Targeting cardiac β-adrenergic signaling via GRK2 inhibition for heart failure therapy.
Author: Cannavo,A., Liccardo,D., Koch,W.J.
Abstract:
Cardiac cells, like those of the other tissues, undergo regulation through membrane-bound proteins known as G protein-coupled receptors (GPCRs). β-adrenergic receptors (βARs) are key GPCRs expressed on cardiomyocytes and their role is crucial in cardiac physiology since they regulate inotropic and chronotropic responses of the sympathetic nervous system (SNS). In compromised conditions such as heart failure (HF), chronic βAR hyperstimulation occurs via SNS activation resulting in receptor dysregulation and down-regulation and consequently there is a marked reduction of myocardial inotropic reserve and continued loss of pump function. Data accumulated over the last two decades indicates that a primary culprit in initiating and maintain βAR dysfunction in the injured and stressed heart is GPCR kinase 2 (GRK2), which was originally known as βARK1 (for βAR kinase). GRK2 is up-regulated in the failing heart due to chronic SNS activity and targeting this kinase has emerged as a novel therapeutic strategy in HF. Indeed, its inhibition or genetic deletion in several disparate animal models of HF including a pre-clinical pig model has shown that GRK2 targeting improves functional and morphological parameters of the failing heart. Moreover, non-βAR properties of GRK2 appear to also contribute to its pathological effects and thus, its inhibition will likely complement existing therapies such as βAR blockade. This review will explore recent research regarding GRK2 inhibition; in particular it will focus on the GRK2 inhibitor peptide known as βARKct, which represents new hope in the treatment against HF progression.

Address: Center for Translational Medicine, Department of Pharmacology, Temple University Philadelphia, PA, USA

3916192a: Front.Neurosci., 185-185(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Circadian adaptations to meal timing: neuroendocrine mechanisms.
Author: Patton,D.F., Mistlberger,R.E.
Abstract:
Circadian rhythms of behavior and physiology are generated by central and peripheral circadian oscillators entrained by periodic environmental or physiological stimuli. A master circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) is directly entrained by daily light-dark (LD) cycles, and coordinates the timing of other oscillators by direct and indirect neural, hormonal and behavioral outputs. The daily rhythm of food intake provides stimuli that entrain most peripheral and central oscillators, some of which can drive a daily rhythm of food anticipatory activity if food is restricted to one daily mealtime. The location of food-entrainable oscillators (FEOs) that drive food anticipatory rhythms, and the food-related stimuli that entrain these oscillators, remain to be clarified. Here, we critically examine the role of peripheral metabolic hormones as potential internal entrainment stimuli or outputs for FEOs controlling food anticipatory rhythms in rats and mice. Hormones for which data are available include corticosterone, ghrelin, leptin, insulin, glucagon, and glucagon-like peptide 1. All of these hormones exhibit daily rhythms of synthesis and secretion that are synchronized by meal timing. There is some evidence that ghrelin and leptin modulate the expression of food anticipatory rhythms, but none of the hormones examined so far are necessary for entrainment. Ghrelin and leptin likely modulate food-entrained rhythms by actions in hypothalamic circuits utilizing melanocortin and orexin signaling, although again food-entrained behavioral rhythms can persist in lesion and gene knockout models in which these systems are disabled. Actions of these hormones on circadian oscillators in central reward circuits remain to be evaluated. Food-entrained activity rhythms are likely mediated by a distributed system of circadian oscillators sensitive to multiple feeding related inputs. Metabolic hormones appear to play a modulatory role within this system.

Address: Department of Psychology, Simon Fraser University Burnaby, BC, Canada

3916212a: Front.Immunol., 335-335(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Recent Advances in Defining the Immunoproteome of
Author: Kunnath-Velayudhan,S., Porcelli,S.A.
Abstract:
Immunity conferred by antigen-specific CD4+ T cells is critical for controlling infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. However, despite research that spans more than a century, many of the characteristics of protective immune responses to Mtb remain elusive. Defining the repertoire of antigenic targets is central to understanding the immune response against this pathogen. Although traditional methods of antigen discovery have identified many immunodominant antigens, they afford limited proteome coverage. Recent advances in proteomic techniques that are based on peptide library and protein microarray technology have enabled interrogation of the entire proteome of Mtb for antigens. Though these techniques have limitations and are still evolving, early studies using these techniques provide an unbiased view of the immune response to Mtb. Here we review proteome-wide approaches to antigen discovery and summarize what these have revealed so far on the composition of the Mtb immunoproteome.

Address: Department of Microbiology and Immunology, Albert Einstein College of Medicine of Yeshiva University , Bronx, NY , USA

3916213a: Front.Immunol., 318-318(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Immunostimulation by Synthetic Lipopeptide-Based Vaccine Candidates: Structure-Activity Relationships.
Author: Zaman,M., Toth,I.
Abstract:
Peptide-based vaccines offer several advantages over conventional whole organism or protein approaches by offering improved purity and specificity in inducing immune response. However, peptides alone are generally non-immunogenic. Concerns remain about the toxicity of adjuvants which are critical for immunogenicity of synthetic peptides. The use of lipopeptides in peptide vaccines is currently under intensive investigation because potent immune responses can be generated without the use of adjuvant (thus are self-adjuvanting). Several lipopeptides derived from microbial origin, and their synthetic versions or simpler fatty acid moieties impart this self-adjuvanting activity by signaling via Toll-like receptor 2 (TLR2). Engagement of this innate immune receptor on antigen-presenting cell leads to the initiation and development of potent immune responses. Therefore optimization of lipopeptides to enhance TLR2-mediated activation is a promising strategy for vaccine development. Considerable structure-activity relationships that determine TLR2 binding and consequent stimulation of innate immune responses have been investigated for a range of lipopeptides. In this mini review we address the development of lipopeptide vaccines, mechanism of TLR2 recognition, and immune activation. An overview is provided of the best studied lipopeptide vaccine systems.

Address: School of Chemistry and Molecular Biosciences, The University of Queensland , St Lucia, QLD , Australia

3916219a: Sci.Signal., pe33-pe33(2013)
Link: [ PubMED ] [ DOI ]
Title: Too fast to die.
Author: Brzostek,J., Gascoigne,N.R.
Abstract:
Signals generated by the T cell receptor (TCR) result in the death or survival of developing thymocytes (T cell precursors), depending on the affinity of the TCR for peptide ligands bound to the major histocompatibility complex (MHC) that are encountered in the thymus. A new study by Ellen Robey's group in this issue of Science Signaling presents a systematic evaluation of signaling and motility changes in thymocytes that encounter ligands of different affinities in the thymic environment. In contrast to previous in vitro studies, the authors found that low-affinity ligands stimulated infrequent transient mobilization of intracellular Ca2+, whereas high-affinity ligands triggered sustained Ca2+ signaling and periods of migratory arrest. For ligands of intermediate affinity, changes in thymocyte motility, rather than in Ca2+ signaling patterns, provided the best correlation with functional outcomes. These findings suggest that transient signaling events in the absence of strong stop signals are required for thymocyte survival and functional maturation in the thymus.

Address: 1Department of Microbiology, Yong Loo Lin School of Medicine, and Immunology Programme, National University of Singapore, 5 Science Drive 2, Singapore 117545

3916241a: Expert Rev.Vaccines, 1173-1193(2013)
Link: [ PubMED ] [ DOI ]
Title: VLPs and particle strategies for cancer vaccines.
Author: Ungaro,F., Conte,C., Quaglia,F., Tornesello,M.L., Buonaguro,F.M., Buonaguro,L.
Abstract:
Effective delivery of tumor antigens to APCs is one of the key steps for eliciting a strong and durable immune response to tumors. Several cancer vaccines have been evaluated in clinical trials, based on soluble peptides, but results have not been fully satisfactory. To improve immunogenicity particles provide a valid strategy to display and/or incorporate epitopes which can be efficiently targeted to APCs for effective induction of adaptive immunity. In the present review, we report some leading technologies for developing particulate vaccines employed in cancer immunotherapy, highlighting the key parameters for a rational design to elicit both humoral and cellular responses.

Address: Department of Pharmacy, University of Napoli Federico II, Via Domenico Montesano 49, 80131, Napoli, Italy

3916242a: Expert Rev.Anti-Infect.Ther., 1097-1113(2013)
Link: [ PubMED ] [ DOI ]
Title: Oral inflammation, a role for antimicrobial peptide modulation of cytokine and chemokine responses.
Author: Brogden,K.A., Johnson,G.K., Vincent,S.D., Abbasi,T., Vali,S.
Abstract:
Acute and chronic inflammation commonly occurs throughout the oral cavity. The most common causes are physical damage and microbial infections, and less frequently immune reactions and malignant changes. All of these processes result in the induction of antimicrobial peptides, chemokines and cytokines that lead to cellular infiltrates, a vascular response, tissue destruction and cellular proliferation. A fascinating concept developing in the current literature suggests that antimicrobial peptides modulate the production of chemokines, cytokines and other cellular mediators and that this may have a larger ramification as an underlying mechanism mediating inflammation. Here, we propose that the ability of antimicrobial peptides to induce chemokines and anti-inflammatory or proinflammatory cytokines plays an important role in the early events of oral inflammation and may be a target for the prevention or treatment of oral inflammatory conditions.

Address: Dows Institute for Dental Research, College of Dentistry, University of Iowa, N423 DSB, 801 Newton Road, Iowa City, IA 52242, USA

3916269a: Anat.Rec., 1346-1363(2013)
Link: [ PubMED ] [ DOI ]
Title: Of pheromones and kairomones: what receptors mediate innate emotional responses?
Author: Fortes-Marco,L., Lanuza,E., Martinez-Garcia,F.
Abstract:
Some chemicals elicit innate emotionally laden behavioral responses. Pheromones mediate sexual attraction, parental care or agonistic confrontation, whereas predators' kairomones elicit defensive behaviors in their preys. This essay explores the hypothesis that the detection of these semiochemicals relies on highly specific olfactory and/or vomeronasal receptors. The V1R, V2R, and formyl-peptide vomeronasal receptors bind their ligands in highly specific and sensitive way, thus being good candidates for pheromone- or kairomone-detectors (e.g., secreted and excreted proteins, peptides and lipophilic volatiles). The olfactory epithelium also expresses specific receptors, for example trace amine-associated receptors (TAAR) and guanylyl cyclase receptors (GC-D and other types), some of which bind kairomones and putative pheromones. However, most of the olfactory neurons express canonical olfactory receptors (ORs) that bind many ligands with different affinity, being not suitable for mediating responses to pheromones and kairomones. In this respect, trimethylthiazoline (TMT) is considered a fox-derived kairomone for mice and rats, but it seems to be detected by canonical ORs. Therefore, we have reassessed the kairomonal nature of TMT by analyzing the behavioral responses of outbred (CD1) and inbred mice (C57BL/J6) to TMT. Our results confirm that both mouse strains avoid TMT, which increases immobility in C57BL/J6, but not CD1 mice. However, mice of both strains sniff at TMT throughout the test and show no trace of TMT-induced contextual conditioning (immobility or avoidance). This suggests that TMT is not a kairomone but, similar to a loud noise, in high concentrations it induces aversion and stress as unspecific responses to a strong olfactory stimulation.

Address: Laboratori de Neuroanatomia Funcional Comparada, Department of Functional Biology, University of Valencia, C. Dr. Moliner, 50, 46100, Burjassot, Spain

3916297a: Expert Opin.Investig.Drugs, 1329-1336(2013)
Link: [ PubMED ] [ DOI ]
Title: IMA901: a peptide vaccine in renal cell carcinoma.
Author: Bedke,J., Stenzl,A.
Abstract:
INTRODUCTION: Immunotherapy has always been a promising therapeutic approach in metastatic renal cell carcinoma (mRCC) with frequently observed long-term responders. Since then, immunotherapy emerged from rather unspecific approaches to a specific stimulation of the immune system by tumor-associated antigens (TAAs) in therapeutic vaccination trials. Current vaccine trials are mainly based on the unspecific stimulation of antigen-presenting cells (APCs) by tumor cell lysates with not clearly defined TAAs. AREAS COVERED: IMA901 is a novel synthetic off-the-shelf vaccine consisting of 10 different tumor-associated peptides (TUMAPs), which has entered a Phase III trial. The preceding Phase I and II trials demonstrated a clear association of a clinical benefit in mRCC patients with an immunological response to the administered TUMAPs. EXPERT OPINION: IMA901 is a first-in-class drug, which is administered together with GM-CSF and single-dose cyclophosphamide. This triumvirate of vaccine, a local and a systemic immunomodulator showed an improved clinical benefit in mRCC patients. This interplay effectively activated cytotoxic T cells. Future strategies will lead to improved local immunomodulators to boost the activation of APCs, systemic immunomodulators to suppress Tregs and myeloid-derived suppressor cells (MDSCs) and antigens of higher cancer specificity and immunogenicity, together with an optimal schedule and dosage of the vaccine.

E-Mail: bedke@live.com
Address: University of Tubingen, Department of Urology , Hoppe-Seyler-Str. 3, Tubingen, 72076 , Germany

B. Bioinformatics, Biophysics, and Structural Biology

3915432b: Phys.Chem.Chem.Phys., 16693-16703(2013)
Link: [ PubMED ] [ DOI ]
Title: Hydration water and peptide dynamics--two sides of a coin. A neutron scattering and adiabatic calorimetry study at low hydration and cryogenic temperatures.
Author: Bastos,M., Alves,N., Maia,S., Gomes,P., Inaba,A., Miyazaki,Y., Zanotti,J.M.
Abstract:
In the present work we bridge neutron scattering and calorimetry in the study of a low-hydration sample of a 15-residue hybrid peptide from cecropin and mellitin CA(1-7)M(2-9) of proven antimicrobial activity. Quasielastic and low-frequency inelastic neutron spectra were measured at defined hydration levels - a nominally 'dry' sample (specific residual hydration h = 0.060 g/g), a H2O-hydrated (h = 0.49) and a D2O-hydrated one (h = 0.51). Averaged mean square proton mobilities were derived over a large temperature range (50-300 K) and the vibrational density of states (VDOS) were evaluated for the hydrated samples. The heat capacity of the H2O-hydrated CA(1-7)M(2-9) peptide was measured by adiabatic calorimetry in the temperature range 5-300 K, for different hydration levels. The glass transition and water crystallization temperatures were derived in each case. The existence of different types of water was inferred and their amounts calculated. The heat capacities as obtained from direct calorimetric measurements were compared to the values derived from the neutron spectroscopy by way of integrating appropriately normalized VDOS functions. While there is remarkable agreement with respect to both temperature dependence and glass transition temperatures, the results also show that the VDOS derived part represents only a fraction of the total heat capacity obtained from calorimetry. Finally our results indicate that both hydration water and the peptide are involved in the experimentally observed transitions.

E-Mail: mbastos@fc.up.pt
Address: CIQ (UP), Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, R. Campo Alegre 687, P-4169-007 Porto, Portugal

3915445b: Adv.Appl.Bioinform.Chem., 37-46(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV) serotype Asia 1.
Author: Alam,S.M., Amin,R., Rahman,M.Z., Hossain,M.A., Sultana,M.
Abstract:
Foot and mouth disease virus (FMDV), with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.

Address: Department of Microbiology, University of Dhaka, Dhaka, Bangladesh

3915463b: Biochemistry, 6662-6671(2013)
Link: [ PubMED ] [ DOI ]
Title: Structure of a Dinuclear Iron Cluster-Containing β-Hydroxylase Active in Antibiotic Biosynthesis.
Author: Makris,T.M., Knoot,C.J., Wilmot,C.M., Lipscomb,J.D.
Abstract:
A family of dinuclear iron cluster-containing oxygenases that catalyze β-hydroxylation tailoring reactions in natural product biosynthesis by nonribosomal peptide synthetase (NRPS) systems was recently described [Makris, T. M., Chakrabarti, M., Munck, E., and Lipscomb, J. D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 15391-15396]. Here, the 2.17 A X-ray crystal structure of the archetypal enzyme from the family, CmlA, is reported. CmlA catalyzes β-hydroxylation of l-p-aminophenylalanine during chloramphenicol biosynthesis. The fold of the N-terminal domain of CmlA is unlike any previously reported, but the C-terminal domain has the αββα fold of the metallo-β-lactamase (MBL) superfamily. The diiron cluster bound in the C-terminal domain is coordinated by an acetate, three His residues, two Asp residues, one Glu residue, and a bridging oxo moiety. One of the Asp ligands forms an unusual monodentate bridge. No other oxygen-activating diiron enzyme utilizes this ligation or the MBL protein fold. The N-terminal domain facilitates dimerization, but using computational docking and a sequence-based structural comparison to homologues, we hypothesize that it likely serves additional roles in NRPS recognition and the regulation of O2 activation.

Address: Department of Biochemistry, Molecular Biology and Biophysics and Center for Metals in Biocatalysis, University of Minnesota , Minneapolis, Minnesota 55455, United States

3915497b: Langmuir, 11982-11990(2013)
Link: [ PubMED ] [ DOI ]
Title: Enhancing On-Surface Mismatch Discrimination Capability of PNA Probes by AuNP Modification of Gold(111) Surface.
Author: Ghosh,S., Mishra,S., Mukhopadhyay,R.
Abstract:
Unambiguous identification of single base mismatches in nucleic acid sequences is of great importance in nucleic acid detection assays. However, ambiguities are often encountered with, and therefore, a strategy for attaining substantially large enhancement of mismatch discrimination has been worked upon in this study. Short single-stranded peptide nucleic acid (PNA) and deoxyribonucleic acid (DNA) sensor probes that are immobilized onto gold nanoparticle (AuNP) modified Au(111) surface have been applied for target DNA detection. It will be shown that while both PNA and the analogous DNA probes exhibit generally better target detection abilities on the AuNP-modified Au(111) surface (elicited from fluorescence-based measurement of on-surface Tm values), compared to the bare Au(111) surface, PNA supersedes DNA, for all sizes of AuNPs (10, 50, and 90 nm) applied, with the difference being quite drastic in the case of the smallest 10 nm AuNP. It is found that while the AuNP curvature plays a pivotal role in target detection abilities of the PNA probes, the changes in the surface roughness caused by AuNP treatment do not exert any significant influence. This study also presents a means for preparing PNA-AuNP hybrids without altering PNA functionality and without AuNP aggregation by working with the surface-affixed AuNPs.

Address: Department of Biological Chemistry, Indian Association for the Cultivation of Science , Jadavpur, Kolkata-700 032, India

3915506b: PLoS One, e72575-e72575(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Molecular dynamics simulations to provide insights into epitopes coupled to the soluble and membrane-bound MHC-II complexes.
Author: Bello,M., Correa-Basurto,J.
Abstract:
Epitope recognition by major histocompatibility complex II (MHC-II) is essential for the activation of immunological responses to infectious diseases. Several studies have demonstrated that this molecular event takes place in the MHC-II peptide-binding groove constituted by the α and β light chains of the heterodimer. This MHC-II peptide-binding groove has several pockets (P1-P11) involved in peptide recognition and complex stabilization that have been probed through crystallographic experiments and in silico calculations. However, most of these theoretical calculations have been performed without taking into consideration the heavy chains, which could generate misleading information about conformational mobility both in water and in the membrane environment. Therefore, in absence of structural information about the difference in the conformational changes between the peptide-free and peptide-bound states (pMHC-II) when the system is soluble in an aqueous environment or non-covalently bound to a cell membrane, as the physiological environment for MHC-II is. In this study, we explored the mechanistic basis of these MHC-II components using molecular dynamics (MD) simulations in which MHC-II was previously co-crystallized with a small epitope (P7) or coupled by docking procedures to a large (P22) epitope. These MD simulations were performed at 310 K over 100 ns for the water-soluble (MHC-IIw, MHC-II-P(7w), and MHC-II-P(22w)) and 150 ns for the membrane-bound species (MHC-IIm, MHC-II-P(7m), and MHC-II-P(22m)). Our results reveal that despite the different epitope sizes and MD simulation environments, both peptides are stabilized primarily by residues lining P1, P4, and P6-7, and similar noncovalent intermolecular energies were observed for the soluble and membrane-bound complexes. However, there were remarkably differences in the conformational mobility and intramolecular energies upon complex formation, causing some differences with respect to how the two peptides are stabilized in the peptide-binding groove.

E-Mail: bellomartini@gmail.com
Address: Laboratorio de Modelado Molecular y Bioinformatica de la Escuela Superior de Medicina, Instituto Politecnico Nacional, Mexico, Plan de San Luis Y Diaz Miron S/N, Col. Casco de Santo Tomas, Mexico City, Mexico

3915507b: PLoS One, e72424-e72424(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Theoretical insights into catalytic mechanism of protein arginine methyltransferase 1.
Author: Zhang,R., Li,X., Liang,Z., Zhu,K., Lu,J., Kong,X., Ouyang,S., Li,L., Zheng,Y.G., Luo,C.
Abstract:
Protein arginine methyltransferase 1 (PRMT1), the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet) as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical SN2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.

Address: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

3915513b: PLoS One, e76956-e76956(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Combinatorial Optimization of Cystine-Knot Peptides towards High-Affinity Inhibitors of Human Matriptase-1.
Author: Glotzbach,B., Reinwarth,M., Weber,N., Fabritz,S., Tomaszowski,M., Fittler,H., Christmann,A., Avrutina,O., Kolmar,H.
Abstract:
Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.

Address: Clemens-Schopf-Institut fur Organische Chemie und Biochemie, Technische Universitat Darmstadt, Darmstadt, Germany

3915514b: PLoS One, e76876-e76876(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Inhibition of influenza a virus infection in vitro by peptides designed in silico.
Author: Lopez-Martinez,R., Ramirez-Salinas,G.L., Correa-Basurto,J., Barron,B.L.
Abstract:
Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H+ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.

Address: Laboratorio de Virologia, Departamento de Microbiologia, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Carpio y Plan de Ayala S/N, Casco de Santo Tomas, Mexico D.F., Mexico

3915545b: Comput.Biol.Chem., 207-214(2013)
Link: [ PubMED ] [ DOI ]
Title: Bioinformatics analysis of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica.
Author: Li,Z.H., Tang,Z.X., Fang,X.J., Zhang,Z.L., Shi,L.E.
Abstract:
In this paper, the physical and chemical characteristics, biological structure and function of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica (Y. NSN) found in our group were studied using multiple bioinformatics approaches. The results showed that Y. NSN had 283 amino acids, a weight of 30,692.5ku and a certain hydrophilic property. Y. NSN had a signal peptide, no transmembrane domains and disulphide bonds. Cleavage site in Y. NSN was between pos. 23 and 24. The prediction result of the secondary structure showed Y. NSN was a coil structure-based protein. The ratio of α-helix, β-folded and random coil were 18.73%, 16.96% and 64.31%, respectively. Active sites were pos. 124, 125, 127, 157, 165 and 169. Mg2+ binding site was pos. 157. Substrate binding sites were pos. 124, 125 and 169. The analysis of multisequencing alignment and phylogenetic tree indicated that Y. NSN shared high similarity with the nuclease from Y. enterocolitica subsp. enterocolitica 8081. The enzyme activity results showed that Y. NSN was a nuclease with good thermostability.

Address: College of Life and Environmental Sciences, Hangzhou Normal University, 310016 Hangzhou, Zhejiang, China

3915554b: PLoS One, e77074-e77074(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: β-Propeller Blades as Ancestral Peptides in Protein Evolution.
Author: Kopec,K.O., Lupas,A.N.
Abstract:
Proteins of the β-propeller fold are ubiquitous in nature and widely used as structural scaffolds for ligand binding and enzymatic activity. This fold comprises between four and twelve four-stranded β-meanders, the so called blades that are arranged circularly around a central funnel-shaped pore. Despite the large size range of β-propellers, their blades frequently show sequence similarity indicative of a common ancestry and it has been proposed that the majority of β-propellers arose divergently by amplification and diversification of an ancestral blade. Given the structural versatility of β-propellers and the hypothesis that the first folded proteins evolved from a simpler set of peptides, we investigated whether this blade may have given rise to other folds as well. Using sequence comparisons, we identified proteins of four other folds as potential homologs of β-propellers: the luminal domain of inositol-requiring enzyme 1 (IRE1-LD), type II β-prisms, β-pinwheels, and WW domains. Because, with increasing evolutionary distance and decreasing sequence length, the statistical significance of sequence comparisons becomes progressively harder to distinguish from the background of convergent similarities, we complemented our analyses with a new method that evaluates possible homology based on the correlation between sequence and structure similarity. Our results indicate a homologous relationship of IRE1-LD and type II β-prisms with β-propellers, and an analogous one for β-pinwheels and WW domains. Whereas IRE1-LD most likely originated by fold-changing mutations from a fully formed PQQ motif β-propeller, type II β-prisms originated by amplification and differentiation of a single blade, possibly also of the PQQ type. We conclude that both β-propellers and type II β-prisms arose by independent amplification of a blade-sized fragment, which represents a remnant of an ancient peptide world.

Address: Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, Tubingen, Baden-Wurttemberg, Germany

3915566b: Biophys.J., 1829-1837(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular Dynamics Simulations of Scorpion Toxin Recognition by the Ca2+-Activated Potassium Channel KCa3.1.
Author: Chen,R., Chung,S.H.
Abstract:
The Ca2+-activated channel of intermediate-conductance (KCa3.1) is a target for antisickling and immunosuppressant agents. Many small peptides isolated from animal venoms inhibit KCa3.1 with nanomolar affinities and are promising drug scaffolds. Although the inhibitory effect of peptide toxins on KCa3.1 has been examined extensively, the structural basis of toxin-channel recognition has not been understood in detail. Here, the binding modes of two selected scorpion toxins, charybdotoxin (ChTx) and OSK1, to human KCa3.1 are examined in atomic detail using molecular dynamics (MD) simulations. Employing a homology model of KCa3.1, we first determine conduction properties of the channel using Brownian dynamics and ascertain that the simulated results are in accord with experiment. The model structures of ChTx-KCa3.1 and OSK1-KCa3.1 complexes are then constructed using MD simulations biased with distance restraints. The ChTx-KCa3.1 complex predicted from biased MD is consistent with the crystal structure of ChTx bound to a voltage-gated K+ channel. The dissociation constants (Kd) for the binding of both ChTx and OSK1 to KCa3.1 determined experimentally are reproduced within fivefold using potential of mean force calculations. Making use of the knowledge we gained by studying the ChTx-KCa3.1 complex, we attempt to enhance the binding affinity of the toxin by carrying out a theoretical mutagenesis. A mutant toxin, in which the positions of two amino acid residues are interchanged, exhibits a 35-fold lower Kd value for KCa3.1 than that of the wild-type. This study provides insight into the key molecular determinants for the high-affinity binding of peptide toxins to KCa3.1, and demonstrates the power of computational methods in the design of novel toxins.

E-Mail: rong.chen@anu.edu.au
Address: Research School of Biology, Australian National University, Canberra, Australia. Electronic address:

3915596b: PLoS One, e71733-e71733(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Conformational changes in the lower palm domain of ASIC1a contribute to desensitization and RFamide modulation.
Author: Frey,E.N., Pavlovicz,R.E., Wegman,C.J., Li,C., Askwith,C.C.
Abstract:
Acid-sensing ion channel 1a (ASIC1a) is a proton-gated cation channel that contributes to fear and pain as well as neuronal damage following persistent cerebral acidosis. Neuropeptides can affect acid-induced neuronal injury by altering ASIC1a inactivation and/or steady-state desensitization. Yet, exactly how ASIC1a inactivation and desensitization occur or are modulated by peptides is not completely understood. We found that regions of the extracellular palm domain and the β(11-12) linker are important for inactivation and steady-state desensitization of ASIC1a. The single amino acid substitutions L280C and L415C dramatically enhanced the rate of inactivation and altered the pH-dependence of steady-state desensitization. Further, the use of methanethiosulfonate (MTS) reagents suggests that the lower palm region (L280C) undergoes a conformational change when ASIC1a transitions from closed to desensitized. We determined that L280C also displays an altered response to the RFamide peptide, FRRFamide. Further, the presence of FRRFamide limited MTS modification of L280C. Together, these results indicate a potential role of the lower palm domain in peptide modulation and suggest RFamide-related peptides promote conformational changes within this region. These data provide empirical support for the idea that L280, and likely this region of the central vestibule, is intimately involved in channel inactivation and desensitization.

Address: Department of Neuroscience, Wexner Medical Center, The Ohio State University, Columbus, Ohio, United States of America

3915629b: Peptides, 32-40(2013)
Link: [ PubMED ] [ DOI ]
Title: NMR solution structure and SRP54M predicted interaction of the N-terminal sequence (1-30) of the ovine Doppel protein.
Author: Pimenta,J., Viegas,A., Sardinha,J., Martins,I.C., Cabrita,E.J., Fontes,C.M., Prates,J.A., Pereira,R.M.
Abstract:
Prion protein (PrPC) biosynthesis involves a multi-step process that includes translation and post-translational modifications. While PrP has been widely investigated, for the homolog Doppel (Dpl), limited knowledge is available. In this study, we focused on a vital step of eukaryotic protein biosynthesis: targeting by the signal recognition particle (SRP). Taking the ovine Dpl (OvDpl(1-30)) peptide as a template, we studied its behavior in two different hydrophobic environments using CD and NMR spectroscopy. In both trifluoroethanol (TFE) and dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC), the OvDpl(1-30) peptide revealed to fold in an α-helical conformation with a well-defined central region extending from residue Cys8 until Ser22. The NMR structure was subsequently included in a computational docking complex with the conserved M-domain of SRP54 protein (SRP54M), and further compared with the N-terminal structures of mouse Dpl and bovine PrPC proteins. This allowed the determination of (i) common predicted N-terminal/SRP54M polar contacts (Asp331, Gln335, Glu365 and Lys432) and (ii) different N-C orientations between prion and Dpl peptides at the SRP54M hydrophobic groove, that are in agreement with each peptide electrostatic potential. Together, these findings provide new insights into the biosynthesis of prion-like proteins. Besides they also show the role of protein conformational switches in signalization toward the endoplasmic membrane, a key event of major significance in the cell cycle. They are thus of general applicability to the study of the biological function of prion-like as well as other proteins.

Address: Unidade de Biotecnologia e Recursos Geneticos, Instituto Nacional de Investigacao Agraria e Veterinaria, Quinta da Fonte Boa, Vale de Santarem, Portugal

3915635b: Infect.Genet.Evol., 83-95(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of a vaccine against schistosomiasis using bioinformatics and molecular modeling tools.
Author: De Oliveira Lopes,D., De Oliveira,F.M., Do Vale Coelho,I.E., De Oliveira Santana,K.T., Mendonca,F.C., Taranto,A.G., Dos Santos,L.L., Miyoshi,A., De Carvalho Azevedo,V.A., Comar,M.Jr.
Abstract:
Schistosomiasis is a serious public health problem in Brazil and worldwide. Although the drugs used to treatment schistosomiasis are effective, the disease continues to expand in all endemic countries due to constant reinfection, poor sanitation, and the lack of effective programs for disease control. However, advances generated through genome projects have provided important information that has improved the understanding of the biology of this parasite. These advances, associated with the advent of bioinformatic analysis, are becoming an important tool in reverse vaccinology. Through database access to the DNA and protein sequences of Schistosoma mansoni and the use of bioinformatics programs, fourteen epitopes were identified. Five epitopes were obtained from proteins whose immunogenic potential had already been assessed in other studies (KP), and nine whose immunogenic potential is unknown (UP). To improve stimulation of the host immune system, the selected epitopes were modeled with a sugar moiety. After this addition, all of the epitopes showed structures similar to those observed in the native proteins, but only eleven of the peptides presented thermodynamically stable structures. Prediction analysis and molecular modeling showed that the glycopeptides presented here are important targets in the search for a vaccine against schistosomiasis. Additionally, they suggest that these molecules may be used in immunological assays to evaluate the level of protection, the effect on pathology reduction and the profile of cytokines and antibodies induced by them.

Address: Laboratory of Molecular Biology of Federal University of Sao Joao del-Rei, Av Sebastiao Goncalves Coelho, 400 Chanadour, Divinopolis MG CEP 35.501.296, Brazil

3915642b: Biophys.J., 993-1003(2013)
Link: [ PubMED ] [ DOI ]
Title: Characterizing a histidine switch controlling pH-dependent conformational changes of the influenza virus hemagglutinin.
Author: Kalani,M.R., Moradi,A., Moradi,M., Tajkhorshid,E.
Abstract:
During the fusion of the influenza virus to the host cell, bending of the HA2 chain of hemagglutinin into a hairpin-shaped structure in a pH-dependent manner facilitates the fusion of the viral envelope and the endosomal membrane. To characterize the structural and dynamical responses of the hinge region of HA2 to pH changes and examine the role of a conserved histidine in this region (the hinge histidine), we have performed an extensive set of molecular dynamics (MD) simulations of 26-residue peptides encompassing the hinge regions of several hemagglutinin subtypes under both neutral and low pH conditions, modeled by the change of the protonation state of the hinge histidine. More than 70 sets of MD simulations (collectively amounting to 25.1 mus) were performed in both implicit and explicit solvents to study the effect of histidine protonation on structural dynamics of the hinge region. In both explicit and implicit solvent simulations, hinge bending was consistently observed upon the protonation of the histidine in all the simulations starting with an initial straight helical conformation, whereas the systems with a neutral histidine retained their primarily straight conformation throughout the simulations. Conversely, the MD simulations starting from an initially bent conformation resulted in the formation of a straight helical structure upon the neutralization of the hinge histidine, whereas the bent structure was maintained when the hinge histidine remained protonated. Finally, mutation of the hinge histidine to alanine abolishes the bending response of the peptide altogether. A molecular mechanism based on the interaction of the hinge histidine with neighboring acidic residues is proposed to be responsible for its role in controlling the conformation of the hinge. We propose that this might present a common mechanism for pH-controlled structural changes in helical structures when histidines act as the pH sensor.

Address: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA

3915669b: Front.Pharmacol., 103-103(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Development of antibody arrays for monoclonal antibody Higher Order Structure analysis.
Author: Wang,X., Li,Q., Davies,M.
Abstract:
Antibody arrays were developed to probe a monoclonal antibody's three-dimensional structure (3-D structure). Peptides with overlapping regions were designed to cover the whole mAb light chain and heavy chain, respectively, and used to generate polyclonal antibodies after the conjugation of the peptides to a carrier protein, KLH. It was shown that good peptide specificity was achieved from the antibodies generated. Using more than 30 different polyclonal antibodies to measure the surface epitope distribution, it was shown that the mAb antibody array can detect epitope exposure as low as 0.1% of defined mAb populations. This ELISA-based analysis of mAb epitope exposure can be considered as a measurement of "conformational impurity" in biologics development, similar to the analysis of other product-related impurities such as different forms of glycosylation, deamidation, and oxidation. This analysis of "conformational impurity" could provide valuable information on the mAb conformational comparability for biosimilar mAbs as well as novel mAbs, especially in the area of protein immunogenicity. Furthermore, stability studies indicated that there are several conformational "hot spots" in many mAbs tested, especially in the hinge region. This antibody array technology can be used for novel mAb Higher Order Structure (HOS) analysis during process and formulation development. Another important area of application is for biosimilar mAb development where the innovator molecule and biosimilar molecule could be compared based on their systemic "fingerprint" from the 30 plus antibodies.

Address: Array Bridge Inc. St. Louis, MO, USA

3915709b: PLoS One, e70847-e70847(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: A hierarchical coarse-grained (all-atom-to-all-residue) computer simulation approach: self-assembly of peptides.
Author: Pandey,R.B., Kuang,Z., Farmer,B.L.
Abstract:
A hierarchical computational approach (all-atom residue to all-residue peptide) is introduced to study self-organizing structures of peptides as a function of temperature. A simulated residue-residue interaction involving all-atom description, analogous to knowledge-based analysis (with different input), is used as an input to a phenomenological coarse-grained interaction for large scales computer simulations. A set of short peptides P1 (1H (2)S (3)S (4)Y (5)W (6)Y (7)A (8)F (9)N (10)N (11)K (12)T) is considered as an example to illustrate the utility. We find that peptides assemble rather fast into globular aggregates at low temperatures and disperse as random-coil at high temperatures. The specificity of the mass distribution of the self-assembly depends on the temperature and spatial lengths which are identified from the scaling of the structure factor. Analysis of energy and mobility profiles, gyration radius of peptide, and radial distribution function of the assembly provide insight into the multi-scale (intra- and inter-chain) characteristics. Thermal response of the global assembly with the simulated residue-residue interaction is consistent with that of the knowledge-based analysis despite expected quantitative differences.

E-Mail: ras.pandey@usm.edu
Address: Department of Physics and Astronomy, University of Southern Mississippi, Hattiesburg, Mississippi, USA

3915727b: Molecules, 10056-10080(2013)
Link: [ PubMED ] [ DOI ]
Title: Modeling the interaction of dodecylphosphocholine micelles with the anticoccidial peptide PW2 guided by NMR data.
Author: Gomes-Neto,F., Valente,A.P., Almeida,F.C.
Abstract:
Antimicrobial peptides are highly dynamic entities that acquire structure upon binding to a membrane interface. To better understand the structure and the mechanism for the molecular recognition of dodecylphosphocholine (DPC) micelles by the anticoccidial peptide PW2, we performed molecular dynamics (MD) simulations guided by NMR experimental data, focusing on strategies to explore the transient nature of micelles, which rearrange on a millisecond to second timescale. We simulated the association of PW2 with a pre-built DPC micelle and with free-DPC molecules that spontaneously forms micelles in the presence of the peptide along the simulation. The simulation with spontaneous micelle formation provided the adequate environment which replicated the experimental data. The unrestrained MD simulations reproduced the NMR structure for the entire 100 ns MD simulation time. Hidden discrete conformational states could be described. Coulomb interactions are important for initial approximation and hydrogen bonds for anchoring the aromatic region at the interface, being essential for the stabilization of the interaction. Arg9 is strongly attached with phosphate. We observed a helix elongation process stabilized by the intermolecular peptide-micelle association. Full association that mimics the experimental data only happens after complete micelle re-association. Fast micelle dynamics without dissociation of surfactants leads to only superficial binding.

E-Mail: francisco.neto@ioc.fiocruz.br
Address: Laboratory of Toxinology, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro 21045-900, Brazil

3915729b: Molecules, 9567-9581(2013)
Link: [ PubMED ] [ DOI ]
Title: Accidental interaction between PDZ domains and diclofenac revealed by NMR-assisted virtual screening.
Author: Tenno,T., Goda,N., Umetsu,Y., Ota,M., Kinoshita,K., Hiroaki,H.
Abstract:
In silico approaches have become indispensable for drug discovery as well as drug repositioning and adverse effect prediction. We have developed the eF-seek program to predict protein-ligand interactions based on the surface structure of proteins using a clique search algorithm. We have also developed a special protein structure prediction pipeline and accumulated predicted 3D models in the Structural Atlas of the Human Genome (SAHG) database. Using this database, genome-wide prediction of non-peptide ligands for proteins in the human genome was performed, and a subset of predicted interactions including 14 PDZ domains was then confirmed by NMR titration. Surprisingly, diclofenac, a non-steroidal anti-inflammatory drug, was found to be a non-peptide PDZ domain ligand, which bound to 5 of 15 tested PDZ domains. The critical residues for the PDZ-diclofenac interaction were also determined. Pharmacological implications of the accidental PDZ-diclofenac interaction are further discussed.

E-Mail: tenno.takeshi@e.mbox.nagoya-u.ac.jp
Address: Laboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Japan

3915731b: Mar.Drugs, 3015-3024(2013)
Link: [ PubMED ] [ DOI ]
Title: Bouillonamide: a mixed polyketide-peptide cytotoxin from the marine cyanobacterium Moorea bouillonii.
Author: Tan,L.T., Okino,T., Gerwick,W.H.
Abstract:
The tropical marine cyanobacterium, Moorea bouillonii, has gained recent attention as a rich source of bioactive natural products. Continued chemical investigation of this cyanobacterium, collected from New Britain, Papua New Guinea, yielded a novel cytotoxic cyclic depsipeptide, bouillonamide (1), along with previously reported molecules, ulongamide A and apratoxin A. Planar structure of bouillonamide was established by extensive 1D and 2D NMR experiments, including multi-edited HSQC, TOCSY, HBMC, and ROESY experiments. In addition to the presence of α-amino acid residues, compound 1 contained two unique polyketide-derived moieties, namely a 2-methyl-6-methylamino-hex-5-enoic acid (Mmaha) residue and a unit of 3-methyl-5-hydroxy-heptanoic acid (Mhha). Absolute stereochemistry of the α-amino acid units in bouillonamide was determined mainly by Marfey's analysis. Compound 1 exhibited mild toxicity with IC50's of 6.0 μM against the neuron 2a mouse neuroblastoma cells.

E-Mail: liktong.tan@nie.edu.sg
Address: Natural Sciences and Science Education, National Institute of Education, Nanyang Technological University, 1 Nanyang Walk, 637616, Singapore

3915776b: Dalton Trans., 15953-15966(2013)
Link: [ PubMED ] [ DOI ]
Title: Metal array fabrication based on ultrasound-induced self-assembly of metalated dipeptides.
Author: Isozaki,K., Haga,Y., Ogata,K., Naota,T., Takaya,H.
Abstract:
Pd- and Pt-bound bis-metalated peptides were synthesised by the condensation of Pd- or Pt-aldimine-complex-bound glutamic acids to afford the four possible metal isomers of bis-Pd and bis-Pt-homometalated dipeptides and PdPt- and PtPd-heterometalated dipeptides without metal disproportionation. Ultrasound-induced self-assembly of these bis-metalated peptides proceeded effectively to afford supramolecular gels that displayed well-ordered metal arrays. The formation of parallel β-sheet type aggregates through interpeptide amide-amide hydrogen bonding was confirmed by IR, scanning electron microscopy (SEM), and synchrotron X-ray diffraction analyses (WAXS and SAXS). The mechanism of the ultrasound-induced self-assembly of the metalated dipeptides was elucidated via kinetic and association experiments by 1H NMR, in which ultrasound-triggered dissociation of intramolecular hydrogen bonds between the chloride ligands of the Pd- and Pt-complexes and amides initially occurred. This was followed by the formation of intermolecular amide-amide hydrogen bonds, which afforded the corresponding oligomeric peptide self-assembly as the nucleus for supramolecular aggregation. The observed first-order relationship of the gelation rate versus the sonication frequency suggested that the microcavitation generated under sonication conditions acted as a crucial trigger and provided a reaction field for efficient self-assembly.

Address: Department of Chemistry, Graduate School of Engineering Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-8531, Japan

3915784b: Bioinformatics, 2799-2800(2013)
Link: [ PubMED ] [ DOI ]
Title: ProteoStats--a library for estimating false discovery rates in proteomics pipelines.
Author: Yadav,A.K., Kadimi,P.K., Kumar,D., Dash,D.
Abstract:
SUMMARY: Statistical validation of peptide assignments from a large-scale shotgun proteomics experiment is a critical step, and various methods for evaluating significance based on decoy database search are in practice. False discovery rate (FDR) estimation of peptide assignments assesses global significance and corrects for multiple comparisons. Various approaches have been proposed for FDR estimation but unavailability of standard tools or libraries leads to development of many in-house scripts followed by manual steps that are error-prone and low-throughput. The ProteoStats library provides an open-source framework for developers with many FDR estimation and visualization features for several popular search algorithms. It also provides accurate q-values, which can be easily integrated in any proteomics pipeline to provide automated, accurate, high-throughput statistical validation and minimize manual errors. AVAILABILITY: https://sourceforge.net/projects/mssuite/files/ProteoStats/. CONTACT: ddash@igib.res.in or aky.compbio@gmail.com or amit.yadav@igib.in SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Address: G.N.R. Knowledge Center for Genome Informatics, CSIR-Institute of Genomics and Integrative Biology, New Delhi-110020, India

3915803b: Dalton Trans., 13817-13820(2013)
Link: [ PubMED ] [ DOI ]
Title: Effect of peptide conformation on TiO2 biomineralization.
Author: Park,S., Lee,H., Lee,S.Y.
Abstract:
The conformational effects of lysine-rich peptides on the biomineralization of TiO2 were investigated. The biomineralization activity of the peptide was dependent on the spatial proximity of the amino groups, which is determined by the secondary structure.

E-Mail: leessy@yonsei.ac.kr
Address: Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea

3915805b: Bioinformatics, 2808-2809(2013)
Link: [ PubMED ] [ DOI ]
Title: A fast Peptide Match service for UniProt Knowledgebase.
Author: Chen,C., Li,Z., Huang,H., Suzek,B.E., Wu,C.H.
Abstract:
SUMMARY: We have developed a new web application for peptide matching using Apache Lucene-based search engine. The Peptide Match service is designed to quickly retrieve all occurrences of a given query peptide from UniProt Knowledgebase (UniProtKB) with isoforms. The matched proteins are shown in summary tables with rich annotations, including matched sequence region(s) and links to corresponding proteins in a number of proteomic/peptide spectral databases. The results are grouped by taxonomy and can be browsed by organism, taxonomic group or taxonomy tree. The service supports queries where isobaric leucine and isoleucine are treated equivalent, and an option for searching UniRef100 representative sequences, as well as dynamic queries to major proteomic databases. In addition to the web interface, we also provide RESTful web services. The underlying data are updated every 4 weeks in accordance with the UniProt releases. AVAILABILITY: http://proteininformationresource.org/peptide.shtml CONTACT: chenc@udel.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Address: Washington, DC 20007, USA, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK and Swiss Institute of Bioinformatics, Centre Medical Universitaire, 1211 Geneva 4, Switzerland

3915817b: Langmuir, 11667-11680(2013)
Link: [ PubMED ] [ DOI ]
Title: Application of peptide gemini surfactants as novel solubilization surfactants for photosystems I and II of cyanobacteria.
Author: Koeda,S., Umezaki,K., Noji,T., Ikeda,A., Kawakami,K., Kondo,M., Yamamoto,Y., Shen,J.R., Taga,K., Dewa,T., Ito,S., Nango,M., Tanaka,T., Mizuno,T.
Abstract:
We designed novel peptide gemini surfactants (PG-surfactants), DKDKC12K and DKDKC12D, which can solubilize Photosystem I (PSI) of Thermosynecoccus elongatus and Photosystem II (PSII) of Thermosynecoccus vulcanus in an aqueous buffer solution. To assess the detailed effects of PG-surfactants on the original supramolecular membrane protein complexes and functions of PSI and PSII, we applied the surfactant exchange method to the isolated PSI and PSII. Spectroscopic properties, light-induced electron transfer activity, and dynamic light scattering measurements showed that PSI and PSII could be solubilized not only with retention of the original supramolecular protein complexes and functions but also without forming aggregates. Furthermore, measurement of the lifetime of light-induced charge-separation state in PSI revealed that both surfactants, especially DKDKC12D, displayed slight improvement against thermal denaturation below 60 degrees C compared with that using β-DDM. This degree of improvement in thermal resistance still seems low, implying that the peptide moieties did not interact directly with membrane protein surfaces. By conjugating an electron mediator such as methyl viologen (MV2+) to DKDKC12K (denoted MV-DKDKC12K), we obtained derivatives that can trap the generated reductive electrons from the light-irradiated PSI. After immobilization onto an indium tin oxide electrode, a cathodic photocurrent from the electrode to the PSI/MV-DKDKC12K conjugate was observed in response to the interval of light irradiation. These findings indicate that the PG-surfactants DKDKC12K and DKDKC12D provide not only a new class of solubilization surfactants but also insights into designing other derivatives that confer new functions on PSI and PSII.

Address: Graduate School of Engineering, Nagoya Institute of Technology , Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555, Japan

3915824b: J.Phys.Chem.Lett., 1913-1917(2013)
Link: [ PubMED ] [ DOI ]
Title: Two-Dimensional Infrared (2DIR) Spectroscopy of the Peptide Beta3s Folding.
Author: Lai,Z., Preketes,N.K., Jiang,J., Mukamel,S., Wang,J.
Abstract:
Probing underlying free energy landscape, pathways, and mechanism is the key for understanding protein folding in theory and experiment. Recently time-resolved two-dimensional infrared (2DIR) with femtosecond laser pulses, has emerged as a promising tool for investigating the protein folding dynamics on faster timescales than possible by NMR. We have employed molecular dynamics simulations to compute 2DIR spectra of the folding process of a peptide, Beta3s. Simulated non-chiral and chiral 2DIR signals illustrate the variation of the spectra as the peptide conformation evolves along the free energy landscape. Chiral spectra show stronger changes than the non-chiral signals because cross peaks caused by the formation of the β-sheet are clearly resolved. Chirality-induced 2DIR may be used to detect the folding of β-sheet proteins with high spectral and temporal resolution.

Address: Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY11794

3915837b: Protein Cell, 761-770(2013)
Link: [ PubMED ] [ DOI ]
Title: Crystal structures of the two membrane-proximal Ig-like domains (D3D4) of LILRB1/B2: alternative models for their involvement in peptide-HLA binding.
Author: Nam,G., Shi,Y., Ryu,M., Wang,Q., Song,H., Liu,J., Yan,J., Qi,J., Gao,G.F.
Abstract:
Leukocyte immunoglobulin-like receptors (LILRs), also called CD85s, ILTs, or LIRs, are important mediators of immune activation and tolerance that contain tandem immunoglobulin (Ig)-like folds. There are 11 (in addition to two pseudogenes) LILRs in total, two with two Ig-like domains (D1D2) and the remaining nine with four Ig-like domains (D1D2D3D4). Thus far, the structural features of the D1D2 domains of LILR proteins are well defined, but no structures for the D3D4 domains have been reported. This is a very important field to be studied as it relates to the unknown functions of the D3D4 domains, as well as their relative orientation to the D1D2 domains on the cell surface. Here, we report the crystal structures of the D3D4 domains of both LILRB1 and LILRB2. The two Iglike domains of both LILRB1-D3D4 and LILRB2-D3D4 are arranged at an acute angle ( approximately 60 degrees ) to form a bent structure, resembling the structures of natural killer inhibitory receptors. Based on these two D3D4 domain structures and previously reported D1D2/HLA I complex structures, two alternative models of full-length (four Ig-like domains) LILR molecules bound to HLA I are proposed.

Address: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

3915844b: Int.J.Mol.Sci., 16836-16850(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Trastuzumab-peptide interactions: mechanism and application in structure-based ligand design.
Author: Sun,T.Y., Wang,Q., Zhang,J., Wu,T., Zhang,F.
Abstract:
Understanding of protein-ligand interactions and its influences on protein stability is necessary in the research on all biological processes and correlative applications, for instance, the appropriate affinity ligand design for the purification of bio-drugs. In this study, computational methods were applied to identify binding site interaction details between trastuzumab and its natural receptor. Trastuzumab is an approved antibody used in the treatment of human breast cancer for patients whose tumors overexpress the HER2 (human epidermal growth factor receptor 2) protein. However, rational design of affinity ligands to keep the stability of protein during the binding process is still a challenge. Herein, molecular simulations and quantum mechanics were used on protein-ligand interaction analysis and protein ligand design. We analyzed the structure of the HER2-trastuzumab complex by molecular dynamics (MD) simulations. The interaction energies of the mutated peptides indicate that trastuzumab binds to ligand through electrostatic and hydrophobic interactions. Quantitative investigation of interactions shows that electrostatic interactions play the most important role in the binding of the peptide ligand. Prime/MM-GBSA calculations were carried out to predict the binding affinity of the designed peptide ligands. A high binding affinity and specificity peptide ligand is designed rationally with equivalent interaction energy to the wild-type octadecapeptide. The results offer new insights into affinity ligand design.

E-Mail: suntyy@gmail.com
Address: Soft Matter Research Center, Department of Chemistry, Zhejiang University, Hangzhou 310027, China

3915845b: Sci.Rep., 2461-2461(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Visualizing cyclic peptide hydration at the single-molecule level.
Author: Chen,Y., Deng,K., Qiu,X., Wang,C.
Abstract:
The role of water molecules in the selective transport of potassium ions across cell membranes is important. Experimental investigations of valinomycin-water interactions remain huge challenge due to the poor solubility of valinomycin in water. Herein, we removed this experimental obstacle by introducing gaseous water and valinomycin onto a Cu(111) surface to investigate the hydration of valinomycin. By combining scanning tunneling microscopy (STM) with density functional theory (DFT) calculations, we revealed that water could affect the adsorption structure of valinomycin. Hydrogen bond interactions occurred primarily at the carbonyl oxygen of valinomycin and resulted in the formation of valinomycin hydrates. The single-molecule perspective revealed in our investigation could provide new insight into the role of water on the conformation transition of valinomycin, which might provide a new molecular basis for the ion transport mechanism at the water/membrane interface.

Address: National Center for Nanoscience and Technology, China, Beijing 100190, PR China

3915870b: Omics, 502-509(2013)
Link: [ PubMED ] [ DOI ]
Title: A Database for Mycobacterium Secretome Analysis: 'MycoSec' to Accelerate Global Health Research.
Author: Roy,A., Bhattacharya,S., Bothra,A.K., Sen,A.
Abstract:
Members of the genus Mycobacterium are notorious for their pathogenesis. Investigations from various perspectives have identified the pathogenic strategies employed by these lethal pathogens. Secretomes are believed to play crucial roles in host cell recognition and cross-talks, in cellular attachment, and in triggering other functions related to host pathogen interactions. However, a proper idea of the mycobacterial secretomes and their mechanism of functionality still remains elusive. In the present study, we have developed a comprehensive database of potential mycobacterial secretomes (MycoSec) using pre-existing algorithms for secretome prediction for researchers interested in this particular field. The database provides a platform for retrieval and analysis of identified secretomes in all finished genomes of the family Mycobacteriaceae. The database contains valuable information regarding secretory signal peptides (Sec type), lipoprotein signal peptides (Lipo type), and Twin arginine (RR/KR) signal peptides (TAT type), prevalent in mycobacteria. Information pertaining to COG analysis, codon usage, and gene expression of the predicted secretomes has also been incorporated in the database. MycoSec promises to be a useful repertoire providing a plethora of information regarding mycobacterial secretomes and may well be a platform to speed global health research. MycoSec is freely accessible at http://www.bicnbu.in/mycosec .

Address: 1 Bioinformatics Facility, Department of Botany, University of North Bengal , Siliguri, India

3915884b: PLoS One, e71752-e71752(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Chemical-functional diversity in cell-penetrating peptides.
Author: Stalmans,S., Wynendaele,E., Bracke,N., Gevaert,B., D'Hondt,M., Peremans,K., Burvenich,C., De Spiegeleer,B.
Abstract:
Cell-penetrating peptides (CPPs) are a promising tool to overcome cell membrane barriers. They have already been successfully applied as carriers for several problematic cargoes, like e.g. plasmid DNA and (si)RNA, opening doors for new therapeutics. Although several hundreds of CPPs are already described in the literature, only a few commercial applications of CPPs are currently available. Cellular uptake studies of these peptides suffer from inconsistencies in used techniques and other experimental conditions, leading to uncertainties about their uptake mechanisms and structural properties. To clarify the structural characteristics influencing the cell-penetrating properties of peptides, the chemical-functional space of peptides, already investigated for cellular uptake, was explored. For 186 peptides, a new cell-penetrating (CP)-response was proposed, based upon the scattered quantitative results for cellular influx available in the literature. Principal component analysis (PCA) and a quantitative structure-property relationship study (QSPR), using chemo-molecular descriptors and our newly defined CP-response, learned that besides typical well-known properties of CPPs, i.e. positive charge and amphipathicity, the shape, structure complexity and the 3D-pattern of constituting atoms influence the cellular uptake capacity of peptides.

Address: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium

3915885b: PLoS One, e71687-e71687(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: De-novo design of antimicrobial peptides for plant protection.
Author: Zeitler,B., Herrera Diaz,A., Dangel,A., Thellmann,M., Meyer,H., Sattler,M., Lindermayr,C.
Abstract:
This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 μg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 μg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of "healthy" food, these peptides might serve as templates for novel antibacterial and antifungal agents.

E-Mail: lindermayr@helmholtz-muenchen.de
Address: Department of Environmental Science, Institute of Biochemical Plant Pathology, Helmholtz Zentrum Munchen, German Research Center for Environmental Health, Munchen-Neuherberg, Germany

3915890b: PLoS One, e71340-e71340(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: An in silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-peptide interactions.
Author: Tiwari,G., Mohanty,D.
Abstract:
Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known Ki or Kd values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optiμm binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD) simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high Ki or Kd moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors of PDZ-peptide complexes.

Address: Bioinformatics Center, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India

3915892b: PLoS One, e71308-e71308(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Binding, conformational transition and dimerization of amyloid-β peptide on GM1-containing ternary membrane: insights from molecular dynamics simulation.
Author: Manna,M., Mukhopadhyay,C.
Abstract:
Interactions of amyloid-β (Aβ) with neuronal membrane are associated with the progression of Alzheimer's disease (AD). Ganglioside GM1 has been shown to promote the structural conversion of Aβ and increase the rate of peptide aggregation; but the exact nature of interaction driving theses processes remains to be explored. In this work, we have carried out atomistic-scale computer simulations (totaling 2.65 micros) to investigate the behavior of Aβ monomer and dimers in GM1-containing raft-like membrane. The oligosaccharide head-group of GM1 was observed to act as scaffold for Aβ-binding through sugar-specific interactions. Starting from the initial helical peptide conformation, a β-hairpin motif was formed at the C-terminus of the GM1-bound Aβ-monomer; that didn't appear in absence of GM1 (both in fluid POPC and liquid-ordered cholesterol/POPC bilayers and also in aqueous medium) within the simulation time span. For Aβ-dimers, the β-structure was further enhanced by peptide-peptide interactions, which might influence the propensity of Aβ to aggregate into higher-ordered structures. The salt-bridges and inter-peptide hydrogen bonds were found to account for dimer stability. We observed spontaneous formation of intra-peptide D(23)-K(28) salt-bridge and a turn at V(24)GSN(27) region - long been accepted as characteristic structural-motifs for amyloid self-assembly. Altogether, our results provide atomistic details of Aβ-GM1 and Aβ-Aβ interactions and demonstrate their importance in the early-stages of GM1-mediated Aβ-oligomerisation on membrane surface.

Address: Department of Chemistry, University of Calcutta, Kolkata, India

3915937b: Langmuir, 11695-11704(2013)
Link: [ PubMED ] [ DOI ]
Title: Creation of cross-linked bilayer membranes that can incorporate membrane proteins from oligo-Asp-based peptide gemini surfactants.
Author: Koeda,S., Umezaki,K., Sumino,A., Noji,T., Ikeda,A., Yamamoto,Y., Dewa,T., Taga,K., Nango,M., Tanaka,T., Mizuno,T.
Abstract:
We designed novel bilayer-forming amphiphiles based on the cyclic oligo-Asp-based peptide gemini (PG) surfactants cr-D2C12 and cr-D3C12, which consist of -Cys(Asp)nCys- (n = 2 or 3) as a core peptide and two Cys residues containing a dodecylamidomethyl group. Dynamic light scattering and transmission electron microscopy measurements revealed the formation of spherical bilayer membranes that could incorporate the light-harvesting antenna complex 2 (LH2) from Rhodopseudomonas acidophila . Furthermore, this proteoliposome-like conjugate could be assembled onto cationized glass and mica to form planar bilayer membranes incorporating LH2. Using atomic force microscopy, we observed LH2 protruding (ca. 1.2-1.5 nm) from flat terraces of the planar bilayer membranes formed from cr-D2C12 or cr-D3C12. Thus, our designed PG surfactants are a new class of bilayer-forming amphiphiles that may be applied to the study of various membrane proteins.

Address: Graduate School of Engineering, Nagoya Institute of Technology , Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555, Japan

3915945b: Phys.Chem.Chem.Phys., 15321-15324(2013)
Link: [ PubMED ] [ DOI ]
Title: Stabilizing effect of electrostatic vs. aromatic interactions in diproline nucleated peptide β-hairpins.
Author: Makwana,K.M., Raghothama,S., Mahalakshmi,R.
Abstract:
The contribution of Tyr-His vs. Cys-His interacting pairs to the scaffold stability of (D)Pro-(L)Pro nucleated peptide β-hairpins has been examined. We present direct evidence for the superiority of the Cys-His pairs, mediated by sulphur-imidazole interactions, as added stabilizing agents of the β-hairpin scaffold.

E-Mail: maha@iiserb.ac.in
Address: Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India

3915946b: Phys.Chem.Chem.Phys., 15869-15875(2013)
Link: [ PubMED ] [ DOI ]
Title: Attenuated second-order Moller-Plesset perturbation theory: performance within the aug-cc-pVTZ basis.
Author: Goldey,M., Dutoi,A., Head-Gordon,M.
Abstract:
Attenuated second-order Moller-Plesset perturbation theory (MP2) within the finite aug-cc-pVTZ (aTZ) basis set is developed for inter- and intra-molecular non-bonded interactions. A single attenuation parameter is optimized on the S66 database of 66 intermolecular interactions, leading to a very large RMS error reduction by a factor of greater than 5 relative to standard MP2/aTZ. Attenuation introduces an error of opposite sign to basis set superposition error (BSSE) and overestimation of dispersion interactions in finite basis MP2. A variety of tests including the S22 set, conformer energies of peptides, alkanes, sugars, sulfate-water clusters, and the coronene dimer establish the transferability of the MP2(terfc, aTZ) model to other inter and intra-molecular interactions. Direct comparisons against attenuation in the smaller aug-cc-pVDZ basis shows that MP2(terfc, aTZ) often significantly outperforms MP2(terfc, aDZ), although at higher computational cost. MP2(terfc, aDZ) and MP2(terfc, aTZ) often out-perform MP2 at the complete basis set limit. Comparison of the two attenuated MP2 models against each other and against attenuation using non-augmented basis sets gives insight into the error cancellation responsible for their remarkable success.

E-Mail: mhg@cchem.berkeley.edu
Address: Department of Chemistry, University of California, Berkeley, California 94720, USA

3915947b: Mol.BioSyst., 2597-2603(2013)
Link: [ PubMED ] [ DOI ]
Title: BH3 helix-derived biophotonic nanoswitches regulate cytochrome c release in permeabilised cells.
Author: Mart,R.J., Errington,R.J., Watkins,C.L., Chappell,S.C., Wiltshire,M., Jones,A.T., Smith,P.J., Allemann,R.K.
Abstract:
Dynamic physical interactions between proteins underpin all key cellular processes and are a highly attractive area for the development of research tools and medicines. Protein-protein interactions frequently involve α-helical structures, but peptides matching the sequences of these structures usually do not fold correctly in isolation. Therefore, much research has focused on the creation of small peptides that adopt stable α-helical structures even in the absence of their intended protein targets. We show that short peptides alkylated with azobenzene crosslinkers can be used to photo-stimulate mitochondrial membrane depolarization and cytochrome c release in permeabilised cells, the initial events of the intrinsic apoptosis pathway.

E-Mail: allemannrk@cardiff.ac.uk
Address: School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK

3915948b: Naturwissenschaften, 853-859(2013)
Link: [ PubMED ] [ DOI ]
Title: The preferred conformation of dipeptides in the context of biosynthesis.
Author: Bywater,R.P., Veryazov,V.
Abstract:
Globular proteins are folded polypeptide structures comprising stretches of secondary structures (helical (α- or 310 helix type), polyproline helix or β-strands) interspersed by regions of less well-ordered structure ("random coil"). Protein fold prediction is a very active field impacting inte alia on protein engineering and misfolding studies. Apart from the many studies of protein refolding from the denatured state, there has been considerable interest in studying the initial formation of peptides during biosynthesis, when there are at the outset only a few residues in the emerging polypeptide. Although there have been many studies employing quantum chemical methods of the conformation of dipeptides, these have mostly been carried out in the gas phase or simulated water. None of these conditions really apply in the interior confines of the ribosome. In the present work, we are concerned with the conformation of dipeptides in this low dielectric environment. Furthermore, only the residue types glycine and alanine have been studied by previous authors, but we extend this repertoire to include leucine and isoleucine, position isomers which have very different structural propensities.

E-Mail: romeobravo@adelard.org.uk
Address: Magdalen College, High Street, Oxford, OX1 4AU, UK

3915974b: Nucleic Acids Res., 9020-9032(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction.
Author: Subramanyam,S., Jones,W.T., Spies,M., Spies,M.A.
Abstract:
RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction.

Address: University of Iowa, Iowa City, IA 52242, USA and Division of Medicinal and Natural Products Chemistry, Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA

3916005b: J.Mol.Biol., 4134-4144(2013)
Link: [ PubMED ] [ DOI ]
Title: Crystal Structures of the Free and Ligand-Bound FK1-FK2 Domain Segment of FKBP52 Reveal a Flexible Inter-Domain Hinge.
Author: Bracher,A., Kozany,C., Hahle,A., Wild,P., Zacharias,M., Hausch,F.
Abstract:
The human Hsp90 co-chaperone FKBP52 belongs to the family of FK506-binding proteins, which act as peptidyl-prolyl isomerases. FKBP52 specifically enhances the signaling of steroid hormone receptors, modulates ion channels and regulates neuronal outgrowth dynamics. In turn, small-molecule ligands of FKBP52 have been suggested as potential neurotrophic or anti-prostate cancer agents. The usefulness of available ligands is however limited by a lack of selectivity. The immunophilin FKBP52 is composed of three domains, an FK506-binding domain with peptidyl-prolyl isomerase activity, an FKBP-like domain of unknown function and a TPR-clamp domain, which recognizes the C-terminal peptide of Hsp90 with high affinity. The herein reported crystal structures of FKBP52 reveal that the short linker connecting the FK506-binding domain and the FKBP-like domain acts as a flexible hinge. This enhanced flexibility and its modulation by phosphorylation might explain some of the functional antagonism between the closely related homologs FKBP51 and FKBP52. We further present two co-crystal structures of FKBP52 in complex with the prototypic ligand FK506 and a synthetic analog thereof. These structures revealed the molecular interactions in great detail, which enabled in-depth comparison with the corresponding complexes of the other cytosolic FKBPs, FKBP51 and FKBP12. The observed subtle differences provide crucial insights for the rational design of ligands with improved selectivity for FKBP52.

E-Mail: bracher@biochem.mpg.de
Address: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address:

3916011b: Structure, 1698-1706(2013)
Link: [ PubMED ] [ DOI ]
Title: Structural basis for the interaction of the Golgi-Associated Retrograde Protein Complex with the t-SNARE Syntaxin 6.
Author: Abascal-Palacios,G., Schindler,C., Rojas,A.L., Bonifacino,J.S., Hierro,A.
Abstract:
The Golgi-Associated Retrograde Protein (GARP) complex is a tethering factor involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein, we report the structural basis for the interaction of the human Ang2 subunit of GARP with the Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of the Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a binding mode in which a dityrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction.

Address: Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain

3916016b: Biophys.Chem., 86-93(2013)
Link: [ PubMED ] [ DOI ]
Title: A micro-environmental study of the Zn(+2)-Aβ1-16 structural properties.
Author: Maiorana,A., Marino,T., Minicozzi,V., Morante,S., Russo,N.
Abstract:
Relying on a combination of classical molecular dynamics and hybrid QM/MM computational methods, we study the influence of the nature of the local physico-chemical environment on the structural features of β-amyloid peptides complexed with Zn(+2) ions. The analysis is carried out by comparing among themselves different Zn(+2)-ligand force fields and studying their influence on metal coordination and long-range peptide folding. The system in the non-physiological so-called "gas phase" (no solvent) was also simulated with the purpose of identifying to what extent, if at all, the solvent can affect the Zn coordination mode, besides its long-range structural properties. There are two main results of this investigation. The first is that the Zn(+2) coordination mode in classical molecular dynamics simulations markedly depends on the partial charge attributed to the ion and the atoms surrounding it. Comparing with experiments, it is possible to identify the most appropriate Zn(+2) force field for the Zn(+2)-Aβ1-16 complex in study. Secondly, although the presence of water naturally influences the peptide folding propensity, it does not affect the structure of the Zn(+2) inner coordination shell. A useful way to validate classical results and in particular those referring to the structural differences visible when different force fields are employed, was to use a hybrid QM/MM optimization step. When the classical system configurations are submitted to such a quantum minimization step, the geometries of the resulting Zn(+2) site turn out to be all very similar and structurally in good agreement with what is experimentally known.

Address: Universita Cattolica del Sacro Cuore, Largo Agostino Gemelli, 8, 00168 Rome, Italy

3916019b: Acc.Chem.Res., 2136-2144(2013)
Link: [ PubMED ] [ DOI ]
Title: Solid-state NMR studies of biomineralization peptides and proteins.
Author: Roehrich,A., Drobny,G.
Abstract:
Nature has evolved sophisticated strategies for engineering hard tissues through the interaction of proteins, and ultimately cells, with inorganic mineral phases. This process, called biomineralization, is how living organisms transform inorganic materials such as hydroxyapatite, calcite, and silica into highly intricate and organized structures. The remarkable material properties of shell, bone, and teeth come from the activities of proteins that function at the organic-inorganic interface. A better understanding of the biomolecular mechanisms used to promote or retard the formation of mineral-based structures could provide important design principles for the development of calcification inhibitors and promoters in orthopedics, cardiology, urology, and dentistry. With the knowledge of the structural basis for control of hard tissue growth by proteins, scientists could potentially develop materials using biomimetic principles with applications in catalysis, biosensors, electronic devices, and chromatographic separations, to name a few. Additionally, biomineralization also has potential applications in electronics, catalysis, magnetism, sensory devices, and mechanical design. Where man-made hard materials require the use of extreme temperatures, high pressure, and pH, biological organisms can accomplish these feats at ambient temperature and at physiological pH. Despite the fact that many researchers want to identify and control the structure of proteins at material and biomineral interfaces, there is a decided lack of molecular-level structure information available for proteins at biomaterial interfaces in general. In particular, this holds for mammalian proteins that directly control calcification processes in hard tissue. The most fundamental questions regarding the secondary and tertiary structures of proteins adsorbed to material surfaces, how proteins catalyze the formation of biomineral composites, or how proteins interact at biomaterial interfaces remain unanswered. This is largely due to a lack of methods capable of providing high-resolution structural information for proteins adsorbed to material surfaces under physiologically relevant conditions. In this Account, we highlight recent work that is providing insight into the structure and crystal recognition mechanisms of a salivary protein model system, as well as the structure and interactions of a peptide that catalyzes the formation of biosilica composites. To develop a better understanding of the structure and interactions of proteins in biomaterials, we have used solid-state NMR techniques to determine the molecular structure and dynamics of proteins and peptides adsorbed onto inorganic crystal surfaces and embedded within biomineral composites. This work adds to the understanding of the structure and crystal recognition mechanisms of an acidic human salivary phosphoprotein, statherin.

Address: Department of Chemistry, University of Washington , Box 351700, Seattle, Washington 98195, United States

3916049b: J.Comput.Chem., 2493-2497(2013)
Link: [ PubMED ] [ DOI ]
Title: Hamiltonian replica-permutation method and its applications to an alanine dipeptide and amyloid-β(29-42) peptides.
Author: Itoh,S.G., Okumura,H.
Abstract:
We propose the Hamiltonian replica-permutation method (RPM) (or multidimensional RPM) for molecular dynamics and Monte Carlo simulations, in which parameters in the Hamiltonian are permuted among more than two replicas with the Suwa-Todo algorithm. We apply the Coulomb RPM, which is one of realization of the Hamiltonian RPM, to an alanine dipeptide and to two amyloid-β(29-42) molecules. The Hamiltonian RPM realizes more efficient sampling than the Hamiltonian replica-exchange method. We illustrate the protein misfolding funnel of amyloid-β(29-42) and reveal its dimerization pathways.

Address: Department of Theoretical and Computational Molecular Science, Institute for Molecular Science, Okazaki, Aichi, 444-8585, Japan

3916051b: Phys.Chem.Chem.Phys., 15062-15077(2013)
Link: [ PubMED ] [ DOI ]
Title: Exploring the conformational and reactive dynamics of biomolecules in solution using an extended version of the glycine reactive force field.
Author: Monti,S., Corozzi,A., Fristrup,P., Joshi,K.L., Shin,Y.K., Oelschlaeger,P., Van Duin,A.C., Barone,V.
Abstract:
In order to describe possible reaction mechanisms involving amino acids, and the evolution of the protonation state of amino acid side chains in solution, a reactive force field (ReaxFF-based description) for peptide and protein simulations has been developed as an expansion of the previously reported glycine parameters. This expansion consists of adding to the training set more than five hundred molecular systems, including all the amino acids and some short peptide structures, which have been investigated by means of quantum mechanical calculations. The performance of this ReaxFF protein force field on a relatively short time scale (500 ps) is validated by comparison with classical non-reactive simulations and experimental data of well characterized test cases, comprising capped amino acids, peptides, and small proteins, and reaction mechanisms connected to the pharmaceutical sector. A good agreement of ReaxFF predicted conformations and kinetics with reference data is obtained.

E-Mail: sapeptides@gmail.com
Address: CNR-Institute of Chemistry of Organometallic Compounds-UOS Pisa, Pisa, Italy

3916073b: Proc.Natl.Acad.Sci.U.S.A., 13833-13838(2013)
Link: [ PubMED ] [ DOI ]
Title: Structure of the processive rubber oxygenase RoxA from Xanthomonas sp.
Author: Seidel,J., Schmitt,G., Hoffmann,M., Jendrossek,D., Einsle,O.
Abstract:
Rubber oxygenase A (RoxA) is one of only two known enzymes able to catalyze the oxidative cleavage of latex for biodegradation. RoxA acts as a processive dioxygenase to yield the predominant product 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD), a tri-isoprene unit. Here we present a structural analysis of RoxA from Xanthomonas sp. strain 35Y at a resolution of 1.8 A. The enzyme is a 75-kDa diheme c-type cytochrome with an unusually low degree of secondary structure. Analysis of the heme group arrangement and peptide chain topology of RoxA confirmed a distant kinship with diheme peroxidases of the CcpA family, but the proteins are functionally distinct, and the extracellular RoxA has evolved to have twice the molecular mass by successively accumulating extensions of peripheral loops. RoxA incorporates both oxygen atoms of its cosubstrate dioxygen into the rubber cleavage product ODTD, and we show that RoxA is isolated with O2 stably bound to the active site heme iron. Activation and cleavage of O2 require binding of polyisoprene, and thus the substrate needs to use hydrophobic access channels to reach the deeply buried active site of RoxA. The location and nature of these channels support a processive mechanism of latex cleavage.

Address: Lehrstuhl fur Biochemie, Institut fur Biochemie, Albert-Ludwigs-Universitat Freiburg, 79104 Freiburg, Germany

3916085b: Langmuir, 11705-11712(2013)
Link: [ PubMED ] [ DOI ]
Title: Different interfacial behaviors of N- and C-terminus cysteine-modified cecropin P1 chemically immobilized onto polymer surface.
Author: Han,X., Uzarski,J.R., Mello,C.M., Chen,Z.
Abstract:
Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) were used to investigate the orientation of N-terminus cysteine-modified cecropin P1 (cCP1) at the polystyrene maleimide (PS-MA)/peptide phosphate buffer solution interface. The cCP1 cysteine group reacts with the maleimide group on the PS-MA surface to chemically immobilize cCP1. Previously, we found that the C-terminus cysteine-modified cecropin P1 (CP1c) molecules exhibit a multiple-orientation distribution at the PS-MA/peptide phosphate buffer solution interface, due to simultaneous physical adsorption and chemical immobilization of CP1c on the PS-MA surface. Differently, in this research, it was found that the interfacial orientation of cCP1 molecules varied from a horizontal orientation to the "tilting" orientation to the "standing up" orientation and then to the "multiple-orientation" distribution as the peptide concentration increased from 0.19 to 3.74 μM. This research shows the different interaction mechanisms between CP1c and PS-MA and between cCP1 and PS-MA.

Address: Department of Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States

3916089b: J.Proteome Res., 3831-3842(2013)
Link: [ PubMED ] [ DOI ]
Title: ISPTM: an iterative search algorithm for systematic identification of post-translational modifications from complex proteome mixtures.
Author: Huang,X., Huang,L., Peng,H., Guru,A., Xue,W., Hong,S.Y., Liu,M., Sharma,S., Fu,K., Caprez,A.P., Swanson,D.R., Zhang,Z., Ding,S.J.
Abstract:
Identifying protein post-translational modifications (PTMs) from tandem mass spectrometry data of complex proteome mixtures is a highly challenging task. Here we present a new strategy, named iterative search for identifying PTMs (ISPTM), for tackling this challenge. The ISPTM approach consists of a basic search with no variable modification, followed by iterative searches of many PTMs using a small number of them (usually two) in each search. The performance of the ISPTM approach was evaluated on mixtures of 70 synthetic peptides with known modifications, on an 18-protein standard mixture with unknown modifications and on real, complex biological samples of mouse nuclear matrix proteins with unknown modifications. ISPTM revealed that many chemical PTMs were introduced by urea and iodoacetamide during sample preparation and many biological PTMs, including dimethylation of arginine and lysine, were significantly activated by Adriamycin treatment in nuclear matrix associated proteins. ISPTM increased the MS/MS spectral identification rate substantially, displayed significantly better sensitivity for systematic PTM identification compared with that of the conventional all-in-one search approach, and offered PTM identification results that were complementary to InsPecT and MODa, both of which are established PTM identification algorithms. In summary, ISPTM is a new and powerful tool for unbiased identification of many different PTMs with high confidence from complex proteome mixtures.

Address: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States

3916125b: Nat.Struct.Mol.Biol., 1098-1105(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.
Author: Liszczak,G., Goldberg,J.M., Foyn,H., Petersson,E.J., Arnesen,T., Marmorstein,R.
Abstract:
N-terminal acetylation is ubiquitous among eukaryotic proteins and controls a myriad of biological processes. Of the N-terminal acetyltransferases (NATs) that facilitate this cotranslational modification, the heterodimeric NatA complex has the most diversity for substrate selection and modifies the majority of all N-terminally acetylated proteins. Here, we report the X-ray crystal structure of the 100-kDa holo-NatA complex from Schizosaccharomyces pombe, in the absence and presence of a bisubstrate peptide-CoA-conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit. The NatA-Naa15p auxiliary subunit contains 13 tetratricopeptide motifs and adopts a ring-like topology that wraps around the NatA-Naa10p subunit, an interaction that alters the Naa10p active site for substrate-specific acetylation. These studies have implications for understanding the mechanistic details of other NAT complexes and how regulatory subunits modulate the activity of the broader family of protein acetyltransferases.

Address: 1] Program in Gene Expression and Regulation, Wistar Institute, Philadelphia, Pennsylvania, USA. [2] Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA

3916126b: Nat.Struct.Mol.Biol., 1069-1076(2013)
Link: [ PubMED ] [ DOI ]
Title: Dimeric WH2 domains in Vibrio VopF promote actin filament barbed-end uncapping and assisted elongation.
Author: Pernier,J., Orban,J., Avvaru,B.S., Jegou,A., Romet-Lemonne,G., Guichard,B., Carlier,M.F.
Abstract:
Proteins containing repeats of the WASP homology 2 (WH2) actin-binding module are multifunctional regulators of actin nucleation and assembly. The bacterial effector VopF in Vibrio cholerae, like VopL in Vibrio parahaemolyticus, is a unique homodimer of three WH2 motifs linked by a C-terminal dimerization domain. We show that only the first and third WH2 domains of VopF bind G-actin in a non-nucleating, sequestered conformation. Moreover, dimeric WH2 domains in VopF give rise to unprecedented regulation of actin assembly. Specifically, two WH2 domains on opposite protomers of VopF direct filament assembly from actin or profilin-actin by binding terminal subunits and uncapping capping protein from barbed ends by a new mechanism. Thus, VopF does not nucleate filaments by capping a pointed-end F-actin hexamer. These properties may contribute to VopF pathogenicity, and they show how dimeric WH2 peptides may mediate processive filament growth.

Address: Cytoskeleton Dynamics and Motility Group, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France

3916173b: Sci.Rep., 2984-2984(2013)
Link: [ PubMED ] [ DOI ]
Title: In silico models for designing and discovering novel anticancer peptides.
Author: Tyagi,A., Kapoor,P., Kumar,R., Chaudhary,K., Gautam,A., Raghava,G.P.
Abstract:
Use of therapeutic peptides in cancer therapy has been receiving considerable attention in the recent years. Present study describes the development of computational models for predicting and discovering novel anticancer peptides. Preliminary analysis revealed that Cys, Gly, Ile, Lys, and Trp are dominated at various positions in anticancer peptides. Support vector machine models were developed using amino acid composition and binary profiles as input features on main dataset that contains experimentally validated anticancer peptides and random peptides derived from SwissProt database. In addition, models were developed on alternate dataset that contains antimicrobial peptides instead of random peptides. Binary profiles-based model achieved maxiμm accuracy 91.44% with MCC 0.83. We have developed a webserver, which would be helpful in: (i) predicting miniμm mutations required for improving anticancer potency; (ii) virtual screening of peptides for discovering novel anticancer peptides, and (iii) scanning natural proteins for identification of anticancer peptides (http://crdd.osdd.net/raghava/anticp/).

Address: 1] Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh-160036, India [2]

3916205b: PLoS One, e77020-e77020(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Solution NMR Structure and Histone Binding of the PHD Domain of Human MLL5.
Author: Lemak,A., Yee,A., Wu,H., Yap,D., Zeng,H., Dombrovski,L., Houliston,S., Aparicio,S., Arrowsmith,C.H.
Abstract:
Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal α-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3), the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide 'reader' domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.

Address: Northeast Structural Genomics Consortium and Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada

3916216b: Sci.Rep., 2956-2956(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Detecting secondary structure and surface orientation of helical peptide monolayers from resonant hybridization signals.
Author: Alici,K.B., Gallardo,I.F.
Abstract:
Hybridization of dominant vibrational modes with meta-surface resonance allows detection of both structural changes and surface orientations of bound helical peptides. Depending on the resonance frequency of meta-molecules, a red- or blue- shift in peptide Amide-I frequency is observed. The underlying coupling mechanism is described by using a temporal coupled mode theory that is in very good agreement with the experimental results. This hybridization phenomenon constitutes the basis of many nanophotonic systems such as tunable coupled mode bio-sensors and dynamic peptide systems driven by infrared signals.

Address: Nanotechnology Research Center, Bilkent University, Ankara 06800, Turkey

3916237b: Phys.Chem.Chem.Phys., 19457-19464(2013)
Link: [ PubMED ] [ DOI ]
Title: Two-dimensional stimulated resonance Raman spectroscopy study of the Trp-cage peptide folding.
Author: Ren,H., Lai,Z., Biggs,J.D., Wang,J., Mukamel,S.
Abstract:
We report a combined molecular dynamics (MD) and ab initio simulation study of the ultrafast broadband ultraviolet (UV) stimulated resonance Raman (SRR) spectra of the Trp-cage mini protein. Characteristic two dimensional (2D) SRR features of various folding states are identified. Structural fluctuations erode the cross peaks and the correlation between diagonal peaks is a good indicator of the α-helix formation.

E-Mail: smukamel@uci.edu
Address: Department of Chemistry, University of California, Irvine, California 92697, USA

3916243b: J.Phys.Chem.C, 17039-17049(2013)
Link: [ PubMED ] [ DOI ]
Title: Lipid Fluid-Gel Phase Transition Induced Alamethicin Orientational Change Probed by Sum Frequency Generation Vibrational Spectroscopy.
Author: Yang,P., Wu,F.G., Chen,Z.
Abstract:
Alamethicin has been extensively studied as an antimicrobial peptide (AMP) and is widely used as a simple model for ion channel proteins. It has been shown that the antimicrobial activity of AMPs is related to their cell membrane orientation, which may be influenced by the phase of the lipid molecules in the cell membrane. The "healthy" cell membranes contain fluid phase lipids, while gel phase lipids can be found in injured or aged cells or in some phase separated membrane regions. Thus, investigations on how the phase of the lipids influences the membrane orientation of AMPs are important to understand more details regarding the AMP's action on cell membranes. In this study, we determined the orientational changes of alamethicin molecules associated with planar substrate supported single lipid bilayers (serving as model cell membranes) with different phases (fluid or gel) as a function of peptide concentration using sum frequency generation (SFG) vibrational spectroscopy. The phase changes of the lipid bilayers were realized by varying the sample temperature. Our SFG results indicated that alamethicin lies down on the surface of fluid and gel phase 1,2-dimyristoyl(d54)-sn-glycero-3-phosphocholine (d-DMPC) lipid bilayers when the lipid bilayers are in contact with a peptide solution with a low concentration of 0.84 μM. However, at a medium peptide concentration of 10.80 μM, alamethicin inserts into the fluid phase lipid bilayer. Its orientation switches from a transmembrane to an in-plane (or lying down) orientation when the phase of the lipid bilayer changes from a fluid state to a gel state. At a high peptide concentration of 21.60 μM, alamethicin adopts a transmembrane orientation while associated with both fluid and gel phase lipid bilayers. We also studied the structural changes of the fluid and gel phase lipid bilayers upon their interactions with alamethicin molecules at different peptide concentrations.

Address: Department of Chemistry, 930 North University Avenue, University of Michigan, Ann Arbor, Michigan 48109

3916268b: Bioinformatics, 2469-2476(2013)
Link: [ PubMED ] [ DOI ]
Title: Graph-based peak alignment algorithms for multiple liquid chromatography-mass spectrometry datasets.
Author: Wang,J., Lam,H.
Abstract:
Liquid chromatography coupled to mass spectrometry (LC-MS) is the dominant technological platform for proteomics. An LC-MS analysis of a complex biological sample can be visualized as a 'map' of which the positional coordinates are the mass-to-charge ratio (m/z) and chromatographic retention time (RT) of the chemical species profiled. Label-free quantitative proteomics requires the alignment and comparison of multiple LC-MS maps to ascertain the reproducibility of experiments or reveal proteome changes under different conditions. The main challenge in this task lies in correcting inevitable RT shifts. Similar, but not identical, LC instruments and settings can cause peptides to elute at very different times and sometimes in a different order, violating the assumptions of many state-of-the-art alignment tools. To meet this challenge, we developed LWBMatch, a new algorithm based on weighted bipartite matching. Unlike existing tools, which search for accurate warping functions to correct RT shifts, we directly seek a peak-to-peak mapping by maximizing a global similarity function between two LC-MS maps. For alignment tasks with large RT shifts (>500 s), an approximate warping function is determined by locally weighted scatterplot smoothing of potential matched features, detected using a novel voting scheme based on co-elution. For validation, we defined the ground truth for alignment success based on tandem mass spectrometry identifications from sequence searching. We showed that our method outperforms several existing tools in terms of precision and recall, and is capable of aligning maps from different instruments and settings. AVAILABILITY: Available at https://sourceforge.net/projects/rt-alignment/. CONTACT: kehlam@ust.hk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Address: Division of Biomedical Engineering and Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong

3916270b: Protein Sci., 1349-1357(2013)
Link: [ PubMED ] [ DOI ]
Title: Structure of an early native-like intermediate of β2-microglobulin amyloidogenesis.
Author: Vanderhaegen,S., Fislage,M., Domanska,K., Versees,W., Pardon,E., Bellotti,V., Steyaert,J.
Abstract:
To investigate early intermediates of β2-microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X-ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co-crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild-type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.

Address: Structural Biology Research Centre, VIB, Pleinlaan 2, 1050, Brussel, Belgium

3916284b: J.Mol.Model., 4305-4318(2013)
Link: [ PubMED ] [ DOI ]
Title: Structure-based approach to the design of BakBH3 mimetic peptides with increased helical propensity.
Author: Delgado-Soler,L., Del Mar Orzaez,M., Rubio-Martinez,J.
Abstract:
The Bcl-2 family of proteins are well-characterized regulators of the intrinsic apoptotic pathway. Proteins within this family can be classified as either prosurvival or prodeath members and the balance between them present at the mitochondrial membrane is what determines if the cell lives or dies. Specific interactions among Bcl-2 family proteins play a crucial role in regulating programmed cell death. Structural studies have established a conserved interaction pattern among Bcl-2 family members. This interaction is mediated by the binding of the hydrophobic face of the amphipathic α-helical BH3 domain into a conserved hydrophobic groove on the prosurvival partners. It has been reported that an increase in the helical content of BH3 mimetic peptides considerably improves the binding affinity. In this context, this work states for designing peptides derived from the BH3 domain of the proapoptotic protein Bak by substitution of some non-interacting residues by the helical inducing residue Aib. Different synthetic peptides preserving BakBH3 relevant interactions were proposed and simulated presenting a better predicted binding energy and higher helical content than the wild type Bak peptide.

Address: Department of Physical Chemistry, University of Barcelona (UB) and Institut de Quimica Teorica i Computacional (IQTCUB), Universitat de Barcelona, C/Marti i Franques 1, 08028, Barcelona, Spain

3916285b: J.Mol.Model., 4271-4282(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular dynamics study of Na+ transportation in a cyclic peptide nanotube and its influences on water behaviors in the tube.
Author: Song,X., Fan,J., Liu,D., Li,H., Li,R.
Abstract:
The dynamics of Na+ transportation in a transmembrane cyclic peptide nanotube of 8 x (WL)4/POPE has been simulated. The curve of PMF (potential of mean force) for Na+ moving through the tube, based on ABF (adaptive biasing force) method, indicates that Na+ possesses lower free energy in an α-plane region than in a mid-plane one. It was found that Na+ would desorb one or two water molecules in the first solvation shell when entering the tube and later maintain in a solvation state. The average numbers of water molecules around Na+ are 4.50, 4.09 in the first solvation shell, and 3.10, 4.08 in the second one for Na+ locating in an α-plane zone and a mid-plane region, respectively. However, water molecules far away from Na+ location still nearly arrange in a form of 1-2-1-2 file. The dipole orientations of water molecules in the regions of gaps 1 and 7 display "D-defects", resulted from the simultaneous electrostatic potentials generated by Na+ and the bare carbonyls at the tube mouths. Such "D-defects" accommodate the energetically favorable water orientations thereby.

Address: Faculty of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, People's Republic of China

3916289b: Phys.Chem.Chem.Phys., 14931-14935(2013)
Link: [ PubMED ] [ DOI ]
Title: A mechanistic study supports a two-step mechanism for peptide bond formation on the ribosome.
Author: Byun,B.J., Kang,Y.K.
Abstract:
We report the feasible pathways of the quaternary model system for the ribosome-catalyzed PT reaction obtained by density functional calculations. Our results indicate that the step from the reactant complex to the first six-membered TS involving a proton shuttle via the 2'-OH of the P-site A76 in the stepwise pathway is the most favored rate-limiting step in solution. It is found that the C-O3' bond-breaking of A76 is not significant but the C-N bond formation with a tetrahedral intermediate occurs in the rate-limiting step and that the fast breakdown of the C-O3' bond is followed in the second transition state. These are consistent with recent kinetic experiments.

Address: Department of Chemistry, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea

C. Organic Synthetic Chemistry

3915450c: J.Biol.Inorg.Chem., 767-778(2013)
Link: [ PubMED ] [ DOI ]
Title: Gold complexes inhibit the aggregation of prion neuropeptides.
Author: Wang,X., He,L., Zhao,C., Du,W., Lin,J.
Abstract:
Prion diseases are characterized by conformational conversion of prion protein from a normal cellular form to an abnormal scrapie isoform (PrP(Sc)). PrP106-126 is a prion neuropeptide and an accepted model used to study the characteristics of PrP(Sc) because such a model has biological and physiochemical properties similar to those of PrP(Sc). Some metal complexes have a strong binding affinity for PrP106-126 and a good inhibitory effect against amyloid fibril formation. However, the effects of the metal ligand configuration on peptide binding and aggregation are not well known. To investigate interaction and peptide aggregation between prion neuropeptides and two gold complexes with different ligand configurations ([Au(bpy)Cl2]PF6 and [Au(dien)Cl]Cl2, where bpy is 2,2'-bipyridine and dien is diethylenetriamine), six prion peptides with either a His111-mutated or a Met109/112-mutated residue were used in this study. The selection of the mutant was based on the corresponding neuropeptide from other species. The results showed that the aromatic gold complex [Au(bpy)Cl2]PF6 exhibits better binding affinity and a better inhibitory effect against peptide aggregation than the tridentate complex [Au(dien)Cl]Cl2. For the sequence-specific PrP106-126 and its mutants, His111 plays the most important role in peptide aggregation and binding affinity. Furthermore, Met112 has a greater effect on the binding affinity than Met109. Compared with the mutated short 14 amino acid peptides, the hydrophobic region of PrP106-126 contributes to both binding affinity and self-aggregation behavior. This work will help to understand and develop potential metallodrugs against amyloid disorder.

Address: Department of Chemistry, Renmin University of China, Beijing, 100872, China

3915467c: Nat.Prod.Commun., 897-901(2013)
Link: [ PubMED ]
Title: Formal synthesis of +-madindoline A, a potent IL-6 inhibitor, utilizing enzymatic discrimination of quaternary carbon.
Author: Shimizu,K., Tomita,M., Fuhshuku,K., Sugai,T., Shoji,M.
Abstract:
A formal synthesis of +-madindoline A was achieved. The Sunazuka's key intermediate, (4R,5S)---3-butyl-4-(tert-butyldimethylsiloxy)-5-methoxycarbonyl-2,5-dimethyl-2 -cyclopentenone, was synthesized from easily available (2S,3S)-3-acetoxy-2-ethenyl-2-methylcyclopentanone. The starting material was reliably supplied by a chemo-enzymatic procedure in enantiomerically pure form. The synthesis was performed by straightforward transformations involving enone formation and regioselective introduction of the two alkyl side chains.

Address: Department of Pharmaceutical Science, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan

3915604c: Soft Matter, 1589-1599(2013)
Link: [ PubMED ] [ DOI ]
Title: Nanoparticle Formation from Hybrid, Multiblock Copolymers of Poly(Acrylic Acid) and VPGVG Peptide.
Author: Grieshaber,S.E., Paik,B.A., Bai,S., Kiick,K.L., Jia,X.
Abstract:
Elastin-mimetic hybrid copolymers with an alternating molecular architecture were synthesized via the step growth polymerization of azide-functionalized, telechelic poly(tert-butyl acrylate) (PtBA) and an alkyne-terminated, valine and glycine-rich peptide with a sequence of (VPGVG)2 (VG2). The resultant hybrid copolymer, [PtBA-VG2]n, contains up to six constituent building blocks and has a polydispersity index (PDI) of ~1.9. Trifluoroacetic acid (TFA) treatment of [PtBA-VG2]n gave rise to an alternating copolymer of poly(acrylic acid) (PAA) and VG2 ([PAA-VG2]n). The modular design permits facile adjustment of the copolymer composition by varying the molecular weight of PAA (22 and 63 repeat units). Characterization by dynamic light scattering indicated that the multiblock copolymers formed discrete nanoparticles at room temperature in aqueous solution at pH 3.8, with an average diameter of 250-270 nm and a particle size distribution of 0.34 for multiblock copolymers containing PAA22 and 0.17 for those containing PAA63. Upon increasing the pH to 7.4, both types of particles were able to swell without being disintegrated, reaching an average diameter of 285-300 nm for [PAA22-VG2]n and 330-350 nm for [PAA63-VG2]n, respectively. The nanoparticles were not dissociated upon the addition of urea, further confirming their unusual stability. The nanoparticles were capable of sequestering a hydrophobic fluorescent dye (pyrene), and the critical aggregation concentration (CAC) was determined to be 1.09 x 10-2 or 1.05 x 10-2 mg/mL for [PAA22-VG2]n and [PAA63-VG2]n, respectively. We suggest that the multiblock copolymers form through collective H-bonding and hydrophobic interactions between the PAA and VG2 peptide units, and that the unusual stability of the multiblock nanoparticles is conferred by the multiblock architecture. These hybrid multiblock copolymers are potentially useful as pH-responsive drug delivery vehicles, with the possibility of drug loading through concerted H-bonds and hydrophobic interactions.

Address: Department of Materials Science and Engineering, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19716, USA

3915605c: Adv.Funct.Mater., 3239-3246(2012)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Ellipsoidal Polyaspartamide Polymersomes with Enhanced Cell-Targeting Ability.
Author: Lai,M.H., Jeong,J.H., Devolder,R.J., Brockman,C., Schroeder,C., Kong,H.
Abstract:
Nano-sized polymersomes functionalized with peptides or proteins are being increasingly studied for targeted delivery of diagnostic and therapeutic molecules. Earlier computational studies have suggested that ellipsoidal nanoparticles, compared to spherical ones, display enhanced binding efficiency with target cells, but this has not yet been experimentally validated. We hypothesize that hydrophilic polymer chains coupled to vesicle-forming polymers would result in ellipsoidal polymersomes. In addition, ellipsoidal polymersomes modified with cell adhesion peptides bind with target cells more actively than spherical ones. We examine this hypothesis by substituting polyaspartamide with octadecyl chains and varying numbers of poly(ethylene glycol) (PEG) chains. Increasing the degree of substitution of PEG from 0.5 to 1.0 mol% drives the polymer to self-assemble into an ellipsoidal polymersome with an aspect ratio of 2.1. Further modification of these ellipsoidal polymersomes with peptides containing an Arg-Gly-Asp sequence (RGD peptides) lead to a significant increase in the rate of association and decrease in the rate of dissociation with a substrate coated with αvβ3 integrins. In addition, in a circulation-mimicking flow, the ellipsoidal polymersomes linked with RGD peptides adhere to target tissues more favorably than their spherical equivalents do. Overall, the results of this study will greatly serve to improve the efficiency of targeted delivery of a wide array of polymersomes loaded with various biomedical modalities.

Address: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Matthews Avenue, Urbana IL, 61801, USA

3915624c: Nanotechnology, 375603-375603(2013)
Link: [ PubMED ] [ DOI ]
Title: Lighting up silica nanotubes transcribed from the submicron structure of a metal-peptide hybrid.
Author: Pu,F., Ren,J.
Abstract:
Fluorescent silica nanotubes are attracting increasing interest because of their versatile applicability in a range of diverse fields. By using sol-gel transcription of tetraethylorthosilicate (TEOS) from various soft templates, silica nanotubes can be conveniently prepared. Metal-peptide hybrids with well-defined nanostructures and outstanding functionalities are very interesting candidates to serve as templates. Herein, we demonstrate that glutathione (GSH) can act as a building block for a bioinspired structure with dimensions down to the nanoscale, based on specific interactions between metal ions and the peptide. Congo red is able to selectively stain the nanofibres obtained, and appears apple-green in colour, implying that Congo red is promising to serve as an effective and convenient probe for determining the self-assembly of GSH and copper ions. Furthermore, silica nanotubes are synthesized using the nanofibres as a template in a very simple way. The silica nanotubes can be lit up by biomolecule-templated metal nanoparticles or nanoclusters and emit bright fluorescence. This work will certainly open up new opportunities in fabricating a broad range of nanostructured materials with versatile functionalities.

Address: State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, People's Republic of China

3915632c: J.Control.Release, 169-178(2013)
Link: [ PubMED ] [ DOI ]
Title: Chemically and biologically synthesized CPP-modified gelonin for enhanced anti-tumor activity.
Author: Shin,M.C., Zhang,J., David,A.E., Trommer,W.E., Kwon,Y.M., Min,K.A., Kim,J.H., Yang,V.C.
Abstract:
The ineffectiveness of small molecule drugs against cancer has generated significant interest in more potent macromolecular agents. Gelonin, a plant-derived toxin that inhibits protein translation, has attracted much attention in this regard. Due to its inability to internalize into cells, however, gelonin exerts only limited tumoricidal effect. To overcome this cell membrane barrier, we modified gelonin, via both chemical conjugation and genetic recombination methods, with low molecular weight protamine (LMWP), a cell-penetrating peptide (CPP) which was shown to efficiently ferry various cargos into cells. Results confirmed that gelonin-LMWP chemical conjugate (cG-L) and recombinant gelonin-LMWP chimera (rG-L) possessed N-glycosidase activity equivalent to that of unmodified recombinant gelonin (rGel); however, unlike rGel, both gelonin-LMWPs were able to internalize into cells. Cytotoxicity studies further demonstrated that cG-L and rG-L exhibited significantly improved tumoricidal effects, with IC50 values being 120-fold lower than that of rGel. Moreover, when tested against a CT26 s.c. xenograft tumor mouse model, significant inhibition of tumor growth was observed with rG-L doses as low as 2μg/tumor, while no detectable therapeutic effects were seen with rGel at 10-fold higher doses. Overall, this study demonstrated the potential of utilizing CPP-modified gelonin as a highly potent anticancer drug to overcome limitations of current chemotherapeutic agents.

Address: Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China

3915683c: Nucl.Med.Biol., 1025-1034(2013)
Link: [ PubMED ] [ DOI ]
Title: Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer.
Author: Richter,S., Wuest,M., Krieger,S.S., Rogers,B.E., Friebe,M., Bergmann,R., Wuest,F.
Abstract:
INTRODUCTION: Bombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3). METHODS: Three fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against (125)I-[Tyr(4)]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC50=8.7+/-2.2nM) and was subsequently selected for radiolabeling with fluorine-18 (18F) through acylation with N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). The radiopharmacological profile of 18F-labeled bombesin [18F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies. RESULTS: All fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC50=8.7-16.7nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The 18F-labeled BBN analogue [18F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. [18F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [18F]BBN-2 in PC3 xenografts (2.75+/-1.82 %ID/g after 5min and 2.45+/-1.25 %ID/g after 60min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments. CONCLUSION: The present study demonstrates that 18F-labeled BBN analogue [18F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.

Address: Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Canada T6G 1Z2

3915699c: Chimia, 279-282(2013)
Link: [ PubMED ]
Title: Peptide-catalyzed 1,4-addition reactions of aldehydes to nitroolefins.
Author: Kastl,R., Arakawa,Y., Duschmale,J., Wiesner,M., Wennemers,H.
Abstract:
Conjugate addition reactions of aldehydes to nitroolefins provide synthetically useful γ-nitroaldehydes. Here we summarize our research on peptide-catalyzed conjugate addition reactions of aldehydes to differently substituted nitroolefins. We show that peptides of the general type Pro-Pro-Xaa (Xaa = acidic amino acid) are not only highly active, robust and stereoselective catalysts but have also remarkable chemoselectivities.

Address: ETH Zurich, Laboratorium fur Organische Chemie, Wolfgang-Pauli-Str. 10, CH-8093 Zurich

3915700c: Chimia, 262-266(2013)
Link: [ PubMED ]
Title: 1,2,3-Triazoles as amide-bond surrogates in peptidomimetics.
Author: Valverde,I.E., Mindt,T.L.
Abstract:
1,2,3-Triazoles represent a class of heterocycles with interesting properties for application in peptide sciences since they closely resemble amide bonds while being stable to enzymatic degradation. These characteristics make 1,2,3-triazoles promising candidates as amide-bond surrogates for the development of novel peptidomimetics with potentially improved biological characteristics. Despite the potential of the heterocycle as an amide-bond isoster, only few examples of triazole-based peptidomimetics can be found in the literature. With the intention to promote this new and promising strategy for peptide modification, this review summarizes synthetic methods available for the facile preparation of α-amino acid and α-amino alkyne building blocks and their use for the incorporation of 1,4-disubstituted 1,2,3 triazoles into the backbone of peptides mediated by the Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC). In addition, examples of the successful amide-to-triazole substitution in biologically active peptides are presented.

Address: University of Basel Hospital, Department of Radiology and Nuclear Medicine, Division of Radiopharmaceutical Chemistry, Petersgraben 4, CH-4031 Basel

3915701c: Chimia, 244-249(2013)
Link: [ PubMED ]
Title: Practical considerations for improving the productivity of mass spectrometry-based proteomics.
Author: Laskay,U.A., Srzentic,K., Fornelli,L., Upir,O., Kozhinov,A.N., Monod,M., Tsybin,Y.O.
Abstract:
Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/ MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.

Address: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Federale de Lausanne, EPFL LSMB BCH 4307, CH-1015 Lausanne

3915800c: Molecules, 9833-9849(2013)
Link: [ PubMED ] [ DOI ]
Title: ADIBO-based "click" chemistry for diagnostic peptide micro-array fabrication: physicochemical and assay characteristics.
Author: Prim,D., Rebeaud,F., Cosandey,V., Marti,R., Passeraub,P., Pfeifer,M.E.
Abstract:
Several azide-derivatized and fluorescently-labeled peptides were immobilized on azadibenzocyclooctyne (ADIBO)-activated slide surfaces via a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction revealing excellent immobilization kinetics, good spot homogeneities and reproducible fluorescence signal intensities. A myc-peptide micro-array immunoassay showed an antibody limit-of-detection (LOD) superior to a microtiter plate-based ELISA. Bovine serum albumin (BSA) and dextran covalently attached via "click" chemistry more efficiently reduced non-specific binding (NSB) of fluorescently-labeled IgG to the microarray surface in comparison to immobilized hexanoic acid and various types of polyethylene glycol (PEG) derivatives. Confirmation of these findings via further studies with other proteins and serum components could open up new possibilities for human sample and microarray platform-based molecular diagnostic tests.

E-Mail: denis.prim@hevs.ch
Address: Institute of Life Technologies, University of Applied Sciences Western Switzerland, Sion 1950, Switzerland

3915801c: Molecules, 9797-9817(2013)
Link: [ PubMED ] [ DOI ]
Title: Click chemistry in peptide-based drug design.
Author: Li,H., Aneja,R., Chaiken,I.
Abstract:
Click chemistry is an efficient and chemoselective synthetic method for coupling molecular fragments under mild reaction conditions. Since the advent in 2001 of methods to improve stereochemical conservation, the click chemistry approach has been broadly used to construct diverse chemotypes in both chemical and biological fields. In this review, we discuss the application of click chemistry in peptide-based drug design. We highlight how triazoles formed by click reactions have been used for mimicking peptide and disulfide bonds, building secondary structural components of peptides, linking functional groups together, and bioconjugation. The progress made in this field opens the way for synthetic approaches to convert peptides with promising functional leads into structure-minimized and more stable forms.

E-Mail: lindalihuiyuan@gmail.com
Address: Department of Biochemistry and Molecular Biology, College of Medicine, Drexel University, 245 N 15th Street, New College Building, Room 11102, Philadelphia, PA 19102, USA

3915807c: Chem.Commun., 8788-8790(2013)
Link: [ PubMED ] [ DOI ]
Title: Sensitive label-free monitoring of protein kinase activity and inhibition using ferric ions coordinated to phosphorylated sites as electrocatalysts.
Author: Wang,M., Wang,G.X., Xiao,F.N., Zhao,Y., Wang,K., Xia,X.H.
Abstract:
Ferric ions show high affinity to the kinase-generated phosphorylated sites of peptides. Thus, the high electrocatalytic activity of the coordinated ferric ions toward the reduction of hydrogen peroxide can be used for sensitive label-free monitoring of protein kinase activity and inhibition.

E-Mail: xhxia@nju.edu.cn
Address: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China

3915828c: Eur.J.Inorg.Chem., 1265-1268(2013)
Link: [ PubMED ] [ DOI ]
Title: Synthesis of -Symmetric Dimeric Re(I) Peptide Complexes.
Author: Herrick,R.S., Ziegler,C.J., Kennedy,K.L., Luu,C., Engle,J.T.
Abstract:
The reactions of [Re(CO)3(H2O)3]Br or Re(CO)5Cl with two peptides, glycylglycine or glycylalanine, were investigated. Each reaction produced a unique, well-defined product. Structural elucidation showed the formation of chiral compounds with the formula [Re(CO)3(Gly-Xxx-O)]2, Xxx = Gly 1, Ala 2. Each dimer displays C2 -symmetry and a nearly rectangular shape. The ligands are bound via the amine and amide carbonyl at a rhenium center and via the pendant carboxylate to the adjacent rhenium center. Both products are fully characterized, via X-ray structure elucidation.

Address: Department of Chemistry, College of the Holy Cross, Worcester, MA 01610

3915873c: J.Org.Chem., 9450-9456(2013)
Link: [ PubMED ] [ DOI ]
Title: Catalytic sp3 C-H oxidation of peptides and their analogues by radical cation salts: from glycine amides to quinolines.
Author: Jia,X., Wang,Y., Peng,F., Huo,C., Yu,L., Liu,J., Wang,X.
Abstract:
A catalytic α-sp(3) C-H oxidation of peptides and glycine amides was achieved under radical cation salt catalysis in the presence of O2, producing a series of substituted quinolines. The scope of this reaction shows good functional group tolerance and high efficiency of the oxidative functionalization.

Address: Key Laboratory of Eco-Environment-Related Polymer Materials, Ministry of Education, China and Gansu Key Laboratory of Polymer Materials, College of Chemistry and Chemical Engineering, Northwest Normal University , Lanzhou, Gansu 730070, China

3915905c: Glycoconj.J., 857-870(2013)
Link: [ PubMED ] [ DOI ]
Title: Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates.
Author: McCarthy,P.C., Saksena,R., Peterson,D.C., Lee,C.H., An,Y., Cipollo,J.F., Vann,W.F.
Abstract:
Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP < 47 (median DP 25) and DP < 100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MS(E) analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP < 47 and DP < 100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates.

Address: Laboratory of Bacterial Polysaccharides, CBER-FDA, 8800 Rockville Pike, Bethesda, MD, 20892, USA

3915923c: Biomaterials, 9190-9200(2013)
Link: [ PubMED ] [ DOI ]
Title: Multifunctional receptor-targeted nanocomplexes for the delivery of therapeutic nucleic acids to the Brain.
Author: Kenny,G.D., Bienemann,A.S., Tagalakis,A.D., Pugh,J.A., Welser,K., Campbell,F., Tabor,A.B., Hailes,H.C., Gill,S.S., Lythgoe,M.F., McLeod,C.W., White,E.A., Hart,S.L.
Abstract:
Convection enhanced delivery (CED) is a method of direct injection to the brain that can achieve widespread dispersal of therapeutics, including gene therapies, from a single dose. Non-viral, nanocomplexes are of interest as vectors for gene therapy in the brain, but it is essential that administration should achieve maximal dispersal to minimise the number of injections required. We hypothesised that anionic nanocomplexes administered by CED should disperse more widely in rat brains than cationics of similar size, which bind electrostatically to cell-surface anionic moieties such as proteoglycans, limiting their spread. Anionic, receptor-targeted nanocomplexes (RTN) containing a neurotensin-targeting peptide were prepared with plasmid DNA and compared with cationic RTNs for dispersal and transfection efficiency. Both RTNs were labelled with gadolinium for localisation in the brain by MRI and in brain sections by LA-ICP-MS, as well as with rhodamine fluorophore for detection by fluorescence microscopy. MRI distribution studies confirmed that the anionic RTNs dispersed more widely than cationic RTNs, particularly in the corpus callosum. Gene expression levels from anionic formulations were similar to those of cationic RTNs. Thus, anionic RTN formulations can achieve both widespread dispersal and effective gene expression in brains after administration of a single dose by CED.

Address: Molecular Immunology Unit, UCL Institute of Child Health, London WC1N 1EH, UK

3915929c: Proc.Natl.Acad.Sci.U.S.A., E3445-E3454(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.
Author: Chang,Y.S., Graves,B., Guerlavais,V., Tovar,C., Packman,K., To,K.H., Olson,K.A., Kesavan,K., Gangurde,P., Mukherjee,A., Baker,T., Darlak,K., Elkin,C., Filipovic,Z., Qureshi,F.Z., Cai,H., Berry,P., Feyfant,E., Shi,X.E., Horstick,J., Annis,D.A., Manning,A.M., Fotouhi,N., Nash,H., Vassilev,L.T., Sawyer,T.K.
Abstract:
Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-A) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

E-Mail: ychang@aileronrx.com
Address: Aileron Therapeutics, Inc., Cambridge, MA 02139, USA

3915959c: Angew.Chem.Int.Ed., 10515-10518(2013)
Link: [ PubMED ] [ DOI ]
Title: Vinylsulfonamide and Acrylamide Modification of DNA for Cross-linking with Proteins.
Author: Dadova,J., Orsag,P., Pohl,R., Brazdova,M., Fojta,M., Hocek,M.
Abstract:
Bioorthogonal covalent cross-linking of DNA-binding proteins (p53) to DNA was achieved through novel DNA probes bearing a reactive vinylsulfonamide (VS) group. The VS-modified dCTP served as building block for polymerase synthesis of modified DNA, which was readily conjugated with cysteine-containing peptides and proteins by Michael addition.

Address: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences & IOCB Research Center, Flemingovo nam. 2, 16610 Prague 6 (Czech Republic) http://www.uochb.cas.cz/hocekgroup

3915999c: Anal.Biochem., 223-230(2013)
Link: [ PubMED ] [ DOI ]
Title: Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A.
Author: Hardes,K., Hammamy,M.Z., Steinmetzer,T.
Abstract:
Further development of our recently published Glu(pNA)-containing peptides (Anal. Biochem. 428 (2012) 73-80) provided new fluorogenic substrates for the activated blood coagulation factor XIII. A first series was designed by incorporation of Glu(AMC) at the penultimate position from the N terminus. For the best derivative H-Tyr-Glu(AMC)-Val-Lys-Val-Ile-NH2, a moderate kcat/Km value of 34s(-1)M(-1) was determined, which is more than 100-fold reduced compared with the previously reported Glu(pNA) substrates. Furthermore, two fluorescence resonance energy transfer (FRET) substrates were prepared by incorporation of an N-methyl-anthraniloyl fluorophore and a 2,4-dinitrophenyl quencher. Both substrates were excellently cleaved by FXIII-A2( *), which is generated from its zymogen by activation of thrombin in the presence of calcium ions. In the absence and presence of H-Gly-ethyl ester, kcat/Km values of 8010 and 8660s-(1)M-(1), respectively, were found for the conversion of H-Lys(N(Me)Abz)-Glu(NH-(CH2)4-NH-Dnp)-Val-Lys-Val-Ile-Gly-NH2 (substrate 8). These values are more than 200-fold improved compared with the Glu(AMC) substrates. Substrate 8 is suitable for the measurement of FXIII-A2( *) activities in plasma samples as well as for in vitro measurements. Furthermore, it was used for the determination of the inhibitory potency of a newly synthesized chloromethyl ketone derivative, Cbz-Phe-Glu(CMK)-Val-Lys-Val-Ile-Gly-NH2, which was found to be a potent irreversible inhibitor of FXIII-A2( *).

Address: Institute of Pharmaceutical Chemistry, Philipps University Marburg, D-35032 Marburg, Germany

3916010c: Nucl.Med.Biol., 1006-1012(2013)
Link: [ PubMED ] [ DOI ]
Title: Synthesis and preclinical characterization of [(64)Cu]NODAGA-MAL-exendin-4 with a N(ε)-maleoyl-l-lysyl-glycine linkage.
Author: Yim,C.B., Mikkola,K., Fagerholm,V., Elomaa,V.V., Ishizu,T., Rajander,J., Schlesinger,J., Roivainen,A., Nuutila,P., Solin,O.
Abstract:
INTRODUCTION: Renal localization of high radioactivity levels during targeted imaging compromises tissue visualization in the kidney region and limits diagnostic accuracy. Radioiodinated antibody fragments with a renal enzyme-cleavable N(ε)-maleoyl-l-lysyl-glycine (MAL) linkage demonstrated low renal radioactivity levels in mice, from early postinjection times. This study tested the hypothesis whether a (64)Cu-labeled NODAGA-exendin-4 peptide with a MAL linkage ([(64)Cu]NODAGA-MAL-exendin-4) could decrease kidney radioactivity levels in rats, compared to a [(64)Cu]NODAGA-exendin-4 reference, without impairing the radioactivity levels in the target tissue. METHODS: NODAGA-MAL-exendin-4 was synthesized in a two-phase approach using solid support to prepare maleoyl-derivatized NODAGA followed by Michael addition to cysteine-derivatized exendin-4 in solution. Radiolabeling was performed in buffered aqua with [(64)Cu]CuCl2, which was produced via the (64)Ni(p,n)(64)Cu nuclear reaction. The in vitro and in vivo stability, lipophilicity, and distribution kinetics in major rat organs for [(64)Cu]NODAGA-MAL-exendin-4 were studied and compared to [(64)Cu]NODAGA-exendin-4. Labeling of pancreatic islets was assessed using autoradiography. RESULTS: NODAGA-MAL-exendin-4 was synthesized, with an overall yield of 9%, and radiolabeled with (64)Cu with high specific radioactivity. Serum incubation studies showed high stability for [(64)Cu]NODAGA-MAL-exendin-4. Similar tissue distribution kinetics was observed for [(64)Cu]NODAGA-MAL-exendin-4 and [(64)Cu]NODAGA-exendin-4, with high kidney radioactivity levels. CONCLUSIONS: The incorporated MAL linkage in [(64)Cu]NODAGA-MAL-exendin-4 was unable to reduce kidney radioactivity levels, compared to [(64)Cu]NODAGA-exendin-4. The applicability of metabolizable linkages in the design of kidney-saving exendin-4 analogs requires further investigation.

Address: Turku PET Centre, University of Turku and Turku University Central Hospital, FI-20520 Turku, Finland

3916028c: J.Am.Chem.Soc., 12604-12607(2013)
Link: [ PubMED ] [ DOI ]
Title: Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa.
Author: Lee,M., Artola-Recolons,C., Carrasco-Lopez,C., Martinez-Caballero,S., Hesek,D., Spink,E., Lastochkin,E., Zhang,W., Hellman,L.M., Boggess,B., Hermoso,J.A., Mobashery,S.
Abstract:
Bacterial cell wall is a polymer of considerable complexity that is in constant equilibrium between synthesis and recycling. AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa , which is intimately involved in cell-wall remodeling. We document the hydrolytic reactions that this enzyme performs on the cell wall. The process removes the peptide stems from the peptidoglycan, the major constituent of the cell wall. We document that the majority of the reactions of this enzyme takes place on the polymeric insoluble portion of the cell wall, as opposed to the fraction that is released from it. We show that AmpDh3 is tetrameric both in crystals and in solution. Based on the X-ray structures of the enzyme in complex with two synthetic cell-wall-based ligands, we present for the first time a model for a multivalent anchoring of AmpDh3 onto the cell wall, which lends itself to its processive remodeling.

Address: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA

3916069c: Z.Naturforsch.C, 223-230(2013)
Link: [ PubMED ]
Title: Optimization of enniatin production by solid-phase fermentation of Fusarium tricinctum.
Author: Wang,J.P., Debbab,A., Hemphill,C.F., Proksch,P.
Abstract:
Enniatins are cyclic depsipeptides produced by fungi of the genus Fusarium that are of interest due to their pronounced biological activities; especially enniatins A, A1, B, and B1 possess anticarcinogenic and anti-HIV properties. In the present study, F. tricinctum was grown on seven solid media and in one liquid growth medium with or without addition of peptone or of amino acid precursors in order to identify favourable media with simple cultivation conditions for maxiμm enniatin production. Additionally, the optimal duration of growth was investigated for the highest yields of enniatins. From the different media analysed, white beans (Phaseolus vulgaris, solid medium) induced the highest accumulation of enniatins A, A1, B, and B1, that reached a maxiμm of 1,365 mg total enniatins in 1 L growth medium after 18 days of fermentation. Fermentation of F. tricinctum on white beans gave the highest yield of enniatins compared to all other media analysed in this study.

Address: Institut fur Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universitat Dusseldorf, Universitatsstr. 1, Geb.26.23, D-40225 Dusseldorf, Germany

3916088c: Inorg.Chem., 9297-9308(2013)
Link: [ PubMED ] [ DOI ]
Title: Synthesis, spectroscopic properties, and photoinduced CO-release studies of functionalized ruthenium(II) polypyridyl complexes: versatile building blocks for development of CORM-peptide nucleic acid bioconjugates.
Author: Bischof,C., Joshi,T., Dimri,A., Spiccia,L., Schatzschneider,U.
Abstract:
A series of ruthenium(II) dicarbonyl complexes of formula [RuCl2(L)(CO)2] (L = bpy(CH3,CH3) = 4,4'-dimethyl-2,2'-bipyridine, bpy(CH3,CHO) = 4'-methyl-2,2'-bipyridine-4-carboxyaldehyde, bpy(CH3,COOH) = 4'-methyl-2,2'-bipyridine-4-carboxylic acid, CppH = 2-(pyridin-2-yl)pyrimidine-4-carboxylic acid, dppzcH = dipyrido[3,2-a:2',3'-c]phenazine-11-carboxylic acid), and [RuCl(L)(CO)2]+ (L = tpy(COOH) = 6-(2,2':6',2''-terpyridine-4'-yloxy)hexanoic acid) has been synthesized. In addition, a high-yield synthesis of a peptide nucleic acid (PNA) monomer containing the 2-(pyridin-2-yl)pyrimidine ligand was also developed, and this compound was used to prepare the first Ru(II) dicarbonyl complex, [RuCl2(Cpp-L-PNA)(CO)2],(Cpp-L-PNA = tert-butyl-N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2-yl)p yrimidine-4-carboxamido)hexanoyl]glycinate) attached to a PNA monomer backbone. Such metal-complex PNA-bioconjugates are attracting profound interest for biosensing and biomedical applications. Characterization of all complexes has been undertaken by IR and NMR spectroscopy, mass spectrometry, elemental analysis, and UV-vis spectroscopy. Investigation of the CO-release properties of the Ru(II) complexes in water/dimethyl sulfoxide (49:1) using the myoglobin assay showed that they are stable under physiological conditions in the dark for at least 60 min and most of them even for up to 15 h. In contrast, photoinduced CO release was observed upon illumination at 365 nm, the low-energy shoulder of the main absorption maxiμm centered around 300 nm, establishing these compounds as a new class of PhotoCORMs. While the two 2,2'-bipyridine complexes release 1 equiv of CO per mole of complex, the terpyridine, 2-(2'-pyridyl)pyrimidine, and dipyrido[3,2-a:2',3'-c]phenazine complexes are less effective CO releasers. Attachment of the 2-(2'-pyridyl)pyrimidine complex to a PNA backbone as in [RuCl2(Cpp-L-PNA)CO2] did not significantly change the spectroscopic or CO-release properties compared to the parent complex. Thus, a novel class of Ru(II)-based PhotoCORMs has been established which can be coupled to carrier delivery vectors such as PNA to facilitate cellular uptake without loss of the inherent CORM properties of the parent compound.

Address: Lehrstuhl fur Anorganische Chemie I, Ruhr-Universitat Bochum, NC 3/74, Universitatsstrasse 150, D-44801 Bochum, Germany

3916096c: J.Biol.Inorg.Chem., 815-830(2013)
Link: [ PubMED ] [ DOI ]
Title: Hydrogen bonding to the cysteine ligand of superoxide reductase: acid-base control of the reaction intermediates.
Author: Tremey,E., Bonnot,F., Moreau,Y., Berthomieu,C., Desbois,A., Favaudon,V., Blondin,G., Houee-Levin,C., Niviere,V.
Abstract:
Superoxide reductase (SOR) is a non-heme iron metalloenzyme that detoxifies superoxide radical in microorganisms. Its active site consists of an unusual non-heme Fe2+ center in a [His4Cys1] square pyramidal pentacoordination, with the axial cysteine ligand proposed to be an essential feature in catalysis. Two NH peptide groups from isoleucine 118 and histidine 119 establish hydrogen bonds involving the sulfur ligand (Desulfoarculus baarsii SOR numbering). To investigate the catalytic role of these hydrogen bonds, the isoleucine 118 residue of the SOR from Desulfoarculus baarsii was mutated into alanine, aspartate, or serine residues. Resonance Raman spectroscopy showed that the mutations specifically induced an increase of the strength of the Fe3+-S(Cys) and S-Cβ(Cys) bonds as well as a change in conformation of the cysteinyl side chain, which was associated with the alteration of the NH hydrogen bonding involving the sulfur ligand. The effects of the isoleucine mutations on the reactivity of SOR with O2 (*-) were investigated by pulse radiolysis. These studies showed that the mutations induced a specific increase of the pK a of the first reaction intermediate, recently proposed to be an Fe2+-O2 (*-) species. These data were supported by density functional theory calculations conducted on three models of the Fe2+-O2 (*-) intermediate, with one, two, or no hydrogen bonds involving the sulfur ligand. Our results demonstrated that the hydrogen bonds between the NH (peptide) and the cysteine ligand tightly control the rate of protonation of the Fe2+-O2 (*-) reaction intermediate to form an Fe3+-OOH species.

Address: Laboratoire de Chimie et Biologie des Metaux, CEA, iRTSV, 17 avenue des Martyrs, 38054, Grenoble, France

3916147c: Inorg.Chem., 9166-9168(2013)
Link: [ PubMED ] [ DOI ]
Title: Controlled formation of emissive silver nanoclusters using rationally designed metal-binding proteins.
Author: Morozov,V.A., Ogawa,M.Y.
Abstract:
The metal-binding properties of rationally designed, synthetic proteins were used to prepare a series of emissive silver nanoclusters having predictable sizes and emission energies. Metal-binding α-helical coiled coils were designed to exist as peptide trimers, tetramers, and hexamers and found to uniquely bind 6, 8, and 12 Ag+ ions, respectively. Subsequent treatment with a chemical reducing agent produced a series of peptide-bound Ag(0) nanoclusters that display a strong visible fluorescence whose emission energies depend on the number of bound metal ions in excellent agreement with theory.

Address: Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio 43403, United States

3916155c: Chem.Eur.J., 12356-12375(2013)
Link: [ PubMED ] [ DOI ]
Title: Synthesis and self-assembly of NCN-pincer Pd-complex-bound norvalines.
Author: Ogata,K., Sasano,D., Yokoi,T., Isozaki,K., Yoshida,R., Takenaka,T., Seike,H., Ogawa,T., Kurata,H., Yasuda,N., Takaya,H., Nakamura,M.
Abstract:
The NCN-pincer Pd-complex-bound norvalines Boc-D/L-[PdCl(dpb)]Nva-OMe (1) were synthesized in multigram quantities. The molecular structure and absolute configuration of 1 were unequivocally determined by single-crystal X-ray structure analysis. The robustness of 1 under acidic/basic conditions provides a wide range of N-/C-terminus convertibility based on the related synthetic transformations. Installation of a variety of functional groups into the N-/C-terminus of 1 was readily carried out through N-Boc- or C-methyl ester deprotection and subsequent condensations with carboxylic acids, R(1)COOH, or amines, R(2)NH2 , to give the corresponding N-/C-functionalized norvalines R(1)-D/L-[PdCl(dpb)]Nva-R(2) 2-9. The dipeptide bearing two Pd units 10 was successfully synthesized through the condensation of C-free 1 with N-free 1. The robustness of these Pd-bound norvalines was adequately demonstrated by the preservation of the optical purity and Pd unit during the synthetic transformations. The lipophilic Pd-bound norvalines L-2, Boc-L-[PdCl(dpb)]Nva-NH-n-C11H23, and L-4, n-C4H9CO-L-[PdCl(dpb)]Nva-NH-n-C11H23, self-assembled in aromatic solvents to afford supramolecular gels. The assembled structures in a thermodynamically stable single crystal of L-2 and kinetically stable supramolecular aggregates of L-2 were precisely elucidated by cryo-TEM, WAX, SAXS, UV/Vis, IR analyses, and single-crystal X-ray crystallography. An antiparallel β-sheet-type aggregate consisting of an infinite one-dimensional hydrogen-bonding network of amide groups and π-stacking of PdCl(dpb) moieties was observed in the supramolecular gel fiber of L-2, even though discrete dimers are assembled through hydrogen bonding in the thermodynamically stable single crystal of L-2. The disparate DSC profiles of the single crystal and xerogel of L-2 indicate different thermodynamics of the molecular assembly process.

Address: International Research Center for Elements Science, Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011 (Japan), Fax: (+81) 774-38-3186

3916176c: Molecules, 12857-12876(2013)
Link: [ PubMED ] [ DOI ]
Title: Chemometric evaluation of urinary steroid hormone levels as potential biomarkers of neuroendocrine tumors.
Author: Plenis,A., Miekus,N., Oledzka,I., Baczek,T., Lewczuk,A., Wozniak,Z., Koszalka,P., Seroczynska,B., Skokowski,J.
Abstract:
Neuroendocrine tumors (NETs) are uncommon tumors which can secrete specific hormone products such as peptides, biogenic amines and hormones. So far, the diagnosis of NETs has been difficult because most NET markers are not specific for a given tumor and none of the NET markers can be used to fulfil the criteria of high specificity and high sensitivity for the screening procedure. However, by combining the measurements of different NET markers, they become highly sensitive and specific diagnostic tests. The aim of the work was to identify whether urinary steroid hormones can be identified as potential new biomarkers of NETs, which could be used as prognostic and clinical course monitoring factors. Thus, a rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection has been developed for the determination of cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone in human urine. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification were 0.5 and 1 ng mL-1 for each steroid hormone, respectively. Linearity was confirmed within a range of 1-300 ng mL-1 with a correlation coefficient greater than 0.9995 for all analytes. The described method was successfully applied for the quantification of six endogenous steroid levels in human urine. Studies were performed on 20 healthy volunteers and 19 patients with NETs. Next, for better understanding of tumor biology in NETs and for checking whether steroid hormones can be used as potential biomarkers of NETs, a chemometric analysis of urinary steroid hormone levels in both data sets was performed.

E-Mail: aplenis@gumed.edu.pl
Address: Department of Pharmaceutical Chemistry, Medical University of Gdansk, Hallera 107, Gdansk 80-416, Poland

3916188c: Open Biochem.J., 82-93(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Biophysical mechanisms of the neutralization of endotoxins by lipopolyamines.
Author: Sil,D., Heinbockel,L., Kaconis,Y., Rossle,M., Garidel,P., Gutsmann,T., David,S.A., Brandenburg,K.
Abstract:
Endotoxins (lipopolysaccharides, LPS) are one of the strongest immunostimulators in nature, responsible for beneficial effects at low, and pathophysiological effects at high concentrations, the latter frequently leading to sepsis and septic shock associated with high mortality in critical care settings. There are no drugs specifically targeting the pathophysiology of sepsis, and new therapeutic agents are therefore urgently needed. The lipopolyamines are a novel class of small molecules designed to sequester and neutralize LPS. To understand the mechanisms underlying the binding and neutralization of LPS toxicity, we have performed detailed biophysical analyses of the interactions of LPS with candidate lipopolyamines which differ in their potencies of LPS neutralization. We examined gel-to-liquid crystalline phase behavior of LPS and of its supramolecular aggregate structures in the absence and presence of lipopolyamines, the ability of such compounds to incorporate into different membrane systems, and the thermodynamics of the LPS:lipopolyamine binding. We have found that the mechanisms which govern the inactivation process of LPS obey similar rules as found for other active endotoxin neutralizers such as certain antimicrobial peptides.

Address: Department. of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA

3916194c: Chem.Commun., 10980-10982(2013)
Link: [ PubMED ] [ DOI ]
Title: Bispidine as a helix inducing scaffold: examples of helically folded linear peptides.
Author: Haridas,V., Sadanandan,S., Gopalakrishna,M.V., Bijesh,M.B., Verma,R.P., Chinthalapalli,S., Shandilya,A.
Abstract:
We designed and synthesized bispidine-anchored peptides and showed that these peptides as small as (containing four chiral α-amino acid residues) adopt a right handed helical conformation. Bispidine anchored linear peptide adopts a helical conformation in solution and in the solid state.

E-Mail: h_haridas@hotmail.com
Address: Department of Chemistry, Indian Institute of Technology Delhi, New Delhi-110016, India

3916233c: ACS Nano, 9489-9505(2013)
Link: [ PubMED ] [ DOI ]
Title: Competition between Forster Resonance Energy Transfer and Electron Transfer in Stoichiometrically Assembled Semiconductor Quantum Dot-Fullerene Conjugates.
Author: Stewart,M.H., Huston,A.L., Scott,A.M., Oh,E., Algar,W.R., Deschamps,J.R., Susumu,K., Jain,V., Prasuhn,D.E., Blanco-Canosa,J., Dawson,P.E., Medintz,I.L.
Abstract:
Understanding how semiconductor quantum dots (QDs) engage in photoinduced energy transfer with carbon allotropes is necessary for enhanced performance in solar cells and other optoelectronic devices along with the potential to create new types of (bio)sensors. Here, we systematically investigate energy transfer interactions between C60 fullerenes and four different QDs, composed of CdSe/ZnS (type I) and CdSe/CdS/ZnS (quasi type II), with emission maxima ranging from 530 to 630 nm. C60-pyrrolidine tris-acid was first coupled to the N-terminus of a hexahistidine-terminated peptide via carbodiimide chemistry to yield a C60-labeled peptide (pepC60). This peptide provided the critical means to achieve ratiometric self-assembly of the QD-(pepC60) nanoheterostructures by exploiting metal affinity coordination to the QD surface. Controlled QD-(pepC60)N bioconjugates were prepared by discretely increasing the ratio (N) of pepC60 assembled per QD in mixtures of dimethyl sulfoxide and buffer; this mixed organic/aqueous approach helped alleviate issues of C60 solubility. An extensive set of control experiments were initially performed to verify the specific and ratiometric nature of QD-(pepC60)N assembly. Photoinitiated energy transfer in these hybrid organic-inorganic systems was then interrogated using steady-state and time-resolved fluorescence along with ultrafast transient absorption spectroscopy. Coordination of pepC60 to the QD results in QD PL quenching that directly tracks with the number of peptides displayed around the QD. A detailed photophysical analysis suggests a competition between electron transfer and Forster resonance energy transfer from the QD to the C60 that is dependent upon a complex interplay of pepC60 ratio per QD, the presence of underlying spectral overlap, and contributions from QD size. These results highlight several important factors that must be considered when designing QD-donor/C60-acceptor systems for potential optoelectronic and biosensing applications.

Address: Optical Sciences Division, Code 5611, double daggerCenter for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory , Washington, DC 20375, United States

3916291c: Nanoscale, 9073-9080(2013)
Link: [ PubMED ] [ DOI ]
Title: A single-stage functionalization and exfoliation method for the production of graphene in water: stepwise construction of 2D-nanostructured composites with iron oxide nanoparticles.
Author: Ihiawakrim,D., Ersen,O., Melin,F., Hellwig,P., Janowska,I., Begin,D., Baaziz,W., Begin-Colin,S., Pham-Huu,C., Baati,R.
Abstract:
A practically simple top-down process for the exfoliation of graphene (GN) and few-layer graphene (FLG) from graphite is described. We have discovered that a biocompatible amphiphilic pyrene-based hexahistidine peptide is able to exfoliate, functionalize, and dissolve few layer graphene flakes in pure water under exceptionally mild, sustainable and virtually innocuous low intensity cavitation conditions. Large area functionalized graphene flakes with the hexahistidine oligopeptide (His(6)-TagGN = His(6)@GN) have been produced efficiently at room temperature and characterized by TEM, Raman, and UV spectroscopy. Conductivity experiments carried out on His(6)-TagGN samples revealed superior electric performances as compared to reduced graphene oxide (rGO) and non-functionalized graphene, demonstrating the non-invasive features of our non-covalent functionalization process. We postulated a rational exfoliation mechanism based on the intercalation of the peptide amphiphile under cavitational chemistry. We also demonstrated the ability of His6-TagGN nanoassemblies to self-assemble spontaneously with inorganic iron oxide nanoparticles generating magnetic two-dimensional (2D) His(6)-TagGN/Fe(3)O_4 nanocomposites under mild and non-hydrothermal conditions. The set of original experiments described here open novel perspectives in the facile production of water dispersible high quality GN and FLG sheets that will improve and facilitate the interfacing, processing and manipulation of graphene for promising applications in catalysis, nanocomposite construction, integrated nanoelectronic devices and bionanotechnology.

Address: Institut de Physique et Chimie des Materiaux de Strasbourg, Universite de Strasbourg, CNRS UMR 7504, 23 rue du Loess, BP 43, F-67034 Strasbourg Cedex 2, France

3916305c: Biopolymers, 519-526(2013)
Link: [ PubMED ] [ DOI ]
Title: Design, synthesis, and characterization of cyclic analogues of the iron regulatory peptide hormone hepcidin.
Author: Clark,R.J., Preza,G.C., Tan,C.C., Van Dijk,J.W., Fung,E., Nemeth,E., Ganz,T., Craik,D.J.
Abstract:
The peptide hormone hepcidin is a key regulator of iron homeostasis in vertebrates. Hepcidin acts by binding to ferroportin, the sole known iron exporter, causing it to be internalized and thus trapping iron within the cell. Dysregulation of hepcidin concentrations is associated with a range of iron-related diseases and hepcidin-based therapeutics could be developed as candidate treatments for these diseases. However peptide-based drugs, despite their many advantages, are often limited by their susceptibility to degradation within the body. Here we describe the design, synthesis and characterization of a series of backbone cyclized hepcidin analogues as an approach to produce stable hepcidin-based leads. The cyclic peptides were shown by NMR to be structurally analogous to native hepcidin. Comparison of the stability of hepcidin with one of the cyclic analogues in human serum revealed that 77% of the cyclic peptide but only 18% of linear hepcidin remained after 24 h. The cyclic peptides were tested for their ability to induce internalization of GFP-ferroportin in vitro but were all found to be inactive. This study demonstrates that backbone cyclization of disulfide-rich peptides is a suitable approach for increasing stability. However, careful consideration of a number of factors, including location of important residues and their bioactive conformation, is required to generate biologically active lead molecules.

Address: Institute for Molecular Bioscience, The University of Queensland, Institute for Molecular Bioscience, Brisbane QLD, 4072, Australia

3916308c: Dalton Trans., 16006-16013(2013)
Link: [ PubMED ] [ DOI ]
Title: Metallo-regulation of the bimolecular triplex formation of a peptide nucleic acid.
Author: Shimada,H., Sakurai,T., Kitamura,Y., Matsuura,H., Ihara,T.
Abstract:
Peptide nucleic acid (PNA) conjugates incorporating a bipyridine unit were prepared. The bipyridine was built into the loop moiety of PNAs that were designed to specifically form a hairpin and a PNA/DNA bimolecular triplex. While the thermal stability of the hairpin structure was only minimally affected by Cu2+ addition, the PNA/DNA bimolecular triplex structure was significantly destabilized by complexation with Cu2+. The melting temperature of the bimolecular triplex decreased by 17.4 degrees C in the presence of Cu2+. This corresponds to more than a 1000 fold decrease in the binding constant for bimolecular triplex formation. Upon complexation, the bipyridine unit underwent a drastic conformational change which accounts for the observed differences in the thermal stabilities of the triplex upon binding. The bipyridine-PNA conjugate may be useful as an allosteric DNA carrier that releases the DNA in response to a certain metal ion concentration.

E-Mail: toshi@chem.kumamoto-u.ac.jp
Address: Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan

D. Biochemistry and Analytical Chemistry

3915415d: BMC Microbiol., 194-194(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Identification of a novel infection-enhancing epitope on dengue prM using a dengue cross-reacting monoclonal antibody.
Author: Luo,Y.Y., Feng,J.J., Zhou,J.M., Yu,Z.Z., Fang,D.Y., Yan,H.J., Zeng,G.C., Jiang,L.F.
Abstract:
BACKGROUND: Dengue virus (DENV) infection is the most important arthropod- borne viral disease in human, but antiviral therapy and approved vaccines remain unavailable due to antibody-dependent enhancement (ADE) phenomenon. Many studies showed that pre-membrane (prM)-specific antibodies do not efficiently neutralize DENV infection but potently promote ADE infection. However, most of the binding epitopes of these antibodies remain unknown. RESULTS: In the present study, we characterized a DENV cross-reactive monoclonal antibody (mAb), 4D10, that neutralized poorly but potently enhanced infection of four standard DENV serotypes and immature DENV (imDENV) over a broad range of concentration. In addition, the epitope of 4D10 was successfully mapped to amino acid residues 14 to18 of DENV1-4 prM protein using a phage-displayed peptide library and comprehensive bioinformatics analysis. We found that the epitope was DENV serocomplex cross-reactive and showed to be highly immunogenic in Balb/c mice. Furthermore, antibody against epitope peptide PL10, like 4D10, showed broad cross-reactivity and weak neutralizing activtity with four standard DENV serotypes and imDENV but significantly promoted ADE infection. These results suggested 4D10 and anti-PL10 sera were infection-enhancing antibodies and PL10 was infection-enhancing epitope. CONCLUSIONS: We mapped the epitope of 4D10 to amino acid residues 14 to18 of DENV1-4 prM and found that this epitope was infection-enhancing. These findings may provide significant implications for future vaccine design and facilitate understanding the pathogenesis of DENV infection.

3915419d: Proteomics, 1871-1884(2013)
Link: [ PubMED ] [ DOI ]
Title: Proteomics in the fruit tree science arena: new insights into fruit defense, development, and ripening.
Author: Molassiotis,A., Tanou,G., Filippou,P., Fotopoulos,V.
Abstract:
Fruit tree crops are agricultural commodities of high economic importance, while fruits also represent one of the most vital components of the human diet. Therefore, a great effort has been made to understand the molecular mechanisms covering fundamental biological processes in fruit tree physiology and fruit biology. Thanks to the development of cutting-edge "omics" technologies such as proteomic analysis, scientists now have powerful tools to support traditional fruit tree research. Such proteomic analyses are establishing high-density 2DE reference maps and peptide mass fingerprint databases that can lead fruit science into a new postgenomic research era. Here, an overview of the application of proteomics in key aspects of fruit tree physiology as well as in fruit biology, including defense responses to abiotic and biotic stress factors, is presented. A panoramic view of ripening-related proteins is also discussed, as an example of proteomic application in fruit science.

Address: School of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece

3915423d: J.Virol., 11872-11883(2013)
Link: [ PubMED ] [ DOI ]
Title: Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase.
Author: Lamp,B., Riedel,C., Wentz,E., Tortorici,M.A., Rumenapf,T.
Abstract:
Classical swine fever virus (CSFV) is a positive-stranded RNA virus belonging to the genus Pestivirus within the Flaviviridae family. Pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (NS3) that also harbors helicase and NTPase activities essential for RNA replication. In CSFV-infected cells, NS3 appears as two forms, a fully processed NS3 of 80 kDa and the precursor molecule NS2-3 of 120 kDa. Here we report the identification and mapping of additional autocatalytic intramolecular cleavages. One cleavable peptide bond occurs between Leu1781 and Met1782, giving rise to a helicase subunit of 55 kDa and, depending on the substrate, a NS2-3 fragment of 78 kDa (NS2-3p) or a NS3 protease subunit of 26 kDa (NS3p). In trans-cleavage assays using NS4-5 as a substrate, NS3p acts as a fully functional protease that is able to process the polyprotein. NS3p comprises the minimal essential protease, as deletion of Leu1781 results in inactivation. A second intramolecular cleavage was mapped to the Leu1748/Lys1749 peptide bond that yields a proteolytically inactive NS3 fragment. Deletion of either of the cleavage site residues resulted in a loss of RNA infectivity, indicating the functional importance of amino acid identity at the respective positions. Our data suggest that internal cleavage within the NS3 moiety is a common process that further extends the functional repertoires of the multifunctional NS2-3 or NS3 and represents another level of the complex polyprotein processing of Flaviviridae.

Address: Institute of Virology, University of Veterinary Medicine, Vienna, Austria

3915424d: J.Biochem.(Tokyo), 419-427(2013)
Link: [ PubMED ] [ DOI ]
Title: A C69-family cysteine dipeptidase from Lactobacillus farciminis JCM1097 possesses strong Gly-Pro hydrolytic activity.
Author: Sakamoto,T., Otokawa,T., Kono,R., Shigeri,Y., Watanabe,K.
Abstract:
Dipeptide Gly-Pro, a hard-to-degrade and collagenous peptide, is thought to be hydrolysed by prolidases that can work on various X-Pro dipeptides. Here, we found an entirely different type of dipeptidase from Lactobacillus farciminis JCM1097 that cleaves Gly-Pro far more efficiently and with higher specificity than prolidases, and then investigated its properties by use of a recombinant enzyme. Although L. farciminis dipeptidase was expressed in the form of an inclusion body in Escherichia coli at 37 degrees C, it was smoothly over-expressed in a soluble form at a lower temperature. The maximal Gly-Pro hydrolytic activity was attained in E. coli at 30 degrees C. In contrast to prolidases that are metallopeptidases showing the modest or marginal activity toward Gly-Pro, this L. farciminis dipeptidase belongs to the cysteine peptidase family C69. Lactobacillus farciminis dipeptidase occurs in cytoplasm and utilizes the side chain of an amino-terminal cysteine residue to perform the nucleophilic attack on the target amide bond between Gly-Pro after processing eight amino acid residues at the N-terminus. Furthermore, L. farciminis dipeptidase is potent enough to synthesize Gly-Pro from Gly and Pro by a reverse reaction. These novel properties could be revealed by virtue of the success in preparing recombinant enzymes in higher yield and in a stable form.

Address: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University

3915425d: Mol.BioSyst., 2686-2695(2013)
Link: [ PubMED ] [ DOI ]
Title: Proteomic analysis of formalin-fixed paraffin-embedded colorectal cancer tissue using tandem mass tag protein labeling.
Author: Maes,E., Valkenborg,D., Mertens,I., Broeckx,V., Baggerman,G., Sagaert,X., Landuyt,B., Prenen,H., Schoofs,L.
Abstract:
In clinical research, repositories of biological samples form a rich source of clinical material for biomarker studies. Banked material, however, is often not stored in optimal conditions regarding the technology used for biomarker research. A case in point is formalin-fixed paraffin-embedded (FFPE) tissue that could be used to obtain large cohorts of samples over a short period of time, as these tissues are routinely prepared for pathological analysis. However, in the context of mass spectrometry based peptide-centric proteomics, protein extraction and identification can be hampered by formalin-induced crosslinking. Furthermore, the molecular formalin crosslinks might be entangled differently across various samples, making it more difficult to reproducibly extract the same proteins from different samples. In this study, we establish the crosslink variability using Tandem Mass Tag (TMT) protein labeling followed by digestion, separation, identification and quantification of proteins extracted from FFPE colorectal cancer and paired healthy tissues. Moreover, by applying de novo interpretation of tandem mass spectra and subsequent analysis by Peaks PTM, unspecified modifications could be elucidated, leading to increased protein and proteome coverage. This approach might be useful for future FFPE proteomics studies.

Address: Flemish Institute for Technological Research (VITO), Mol, Belgium

3915426d: Sci.Transl.Med., 0ra114-0ra114(2013)
Link: [ PubMED ] [ DOI ]
Title: Vaccine-induced anti-HA2 antibodies promote virus fusion and enhance influenza virus respiratory disease.
Author: Khurana,S., Loving,C.L., Manischewitz,J., King,L.R., Gauger,P.C., Henningson,J., Vincent,A.L., Golding,H.
Abstract:
Vaccine-induced disease enhancement has been described in connection with several viral vaccines in animal models and in humans. We investigated a swine model to evaluate mismatched influenza vaccine-associated enhanced respiratory disease (VAERD) after pH1N1 infection. Vaccinating pigs with whole inactivated H1N2 (human-like) virus vaccine (WIV-H1N2) resulted in enhanced pneumonia and disease after pH1N1 infection. WIV-H1N2 immune sera contained high titers of cross-reactive anti-pH1N1 hemagglutinin (HA) antibodies that bound exclusively to the HA2 domain but not to the HA1 globular head. No hemagglutination inhibition titers against pH1N1 (challenge virus) were measured. Epitope mapping using phage display library identified the immunodominant epitope recognized by WIV-H1N2 immune sera as amino acids 32 to 77 of pH1N1-HA2 domain, close to the fusion peptide. These cross-reactive anti-HA2 antibodies enhanced pH1N1 infection of Madin-Darby canine kidney cells by promoting virus membrane fusion activity. The enhanced fusion activity correlated with lung pathology in pigs. This study suggests a role for fusion-enhancing anti-HA2 antibodies in VAERD, in the absence of receptor-blocking virus-neutralizing antibodies. These findings should be considered during the evaluation of universal influenza vaccines designed to elicit HA2 stem-targeting antibodies.

Address: Division of Viral Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892, USA

3915434d: Mol.Biol.Cell, 3215-3226(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling.
Author: Eulitz,S., Sauer,F., Pelissier,M.C., Boisguerin,P., Molt,S., Schuld,J., Orfanos,Z., Kley,R.A., Volkmer,R., Wilmanns,M., Kirfel,G., Van der Ven,P.F., Furst,D.O.
Abstract:
The Xin actin-binding repeat-containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain-binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat-containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.

Address: Germany Department of Medicinal Immunology, Charite-University Medicine Berlin, D-13353 Berlin, Germany Department of Neurology, Neuromuscular Center Ruhrgebiet, University Hospital Bergmannsheil, Ruhr-University Bochum, D-44789 Bochum, Germany

3915435d: J.Proteome Res., 4577-4584(2013)
Link: [ PubMED ] [ DOI ]
Title: Serum Metabolic Signatures Induced By a Three-Day Intensified Exercise Period Persist After 14 h of Recovery in Runners.
Author: Nieman,D.C., Shanely,R.A., Gillitt,N.D., Pappan,K.L., Lila,M.A.
Abstract:
This study investigated changes in the human serum metabolome elicited by a 3-day period of intensified training. Runners (N = 15, mean +/- SD age, 35.2 +/- 8.7 years) ran for 2.5 h/day on treadmills at approximately 70% VO2max for 3 days in a row, with blood samples collected pre-exercise, and immediately and 14 h post-exercise. Samples were analyzed using gas and liquid chromatography/mass spectrometry (GC-MS, LC-MS), with compounds identified based on comparison to more than 2800 purified standards. Repeated measures ANOVA was used to identify metabolites that differed significantly across time, with multiple testing corrected by the false discovery rate (FDR) (q-value). Immediately following the 3-day exercise period, significant 2-fold or higher increases in 75 metabolites were measured, with all but 22 of these metabolites related to lipid/carnitine metabolism, 13 to amino acid/peptide metabolism, 4 to hemoglobin/porphyrin metabolism, and 3 to Krebs cycle intermediates (q-values < 0.001). After a 14 h overnight recovery period, 50 of the 75 metabolites remained elevated, with 8 decreased (primarily amino acid-related metabolites) (q-values < 0.05). Among the top 20 metabolites, the mean fold changes were 12.4 +/- 5.3 and 2.9 +/- 1.3 immediately and 14-h post-exercise, respectively. Significant decreases (40-70%, q < 0.01) in 22 metabolites (primarily related to lysolipid and bile acid metabolism) were measured post-exercise, with all but 4 of these still decreased after 14 h rest recovery (q < 0.025). Runners experienced a profound systemic shift in blood metabolites related to energy production especially from the lipid super pathway following 3 days of heavy exertion that was not fully restored to pre-exercise levels after 14 h recovery.

Address: Human Performance Laboratory, Appalachian State University , North Carolina Research Campus, Kannapolis, North Carolina, United States

3915441d: BioMed Res.Int., 283805-283805(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Immunoinformatic docking approach for the analysis of KIR3DL1/HLA-B interaction.
Author: Grifoni,A., Montesano,C., Patronov,A., Colizzi,V., Amicosante,M.
Abstract:
KIR3DL1 is among the most interesting receptors studied, within the killer immunoglobulin receptor (KIR) family. Human leukocyte antigen (HLA) class I Bw4 epitope inhibits strongly Natural Killer (NK) cell's activity through interaction with KIR3DL1 receptor, while Bw6 generally does not. This interaction has been indicated to play an important role in the immune control of different viral infectious diseases. However, the structural interaction between the KIR3DL1 receptor and different HLA-B alleles has been scarcely studied. To understand the complexity of KIR3DL1-HLA-B interaction, HLA-B alleles carrying Bw4/Bw6 epitope and KIR3DL1 *001 allele in presence of different peptides has been evaluated by using a structural immunoinformatic approach. Different energy minimization force fields (ff) have been tested and NOVA ff enables the successful prediction of ligand-receptor interaction. HLA-B alleles carrying Bw4 epitope present the highest capability of interaction with KIR3DL1 *001 compared to the HLA-B alleles presenting Bw6. The presence of the epitope Bw4 determines a conformational change which leads to a stronger interaction between nonpolymorphic arginine at position 79 of HLA-B and KIR3DL1 *001 136-142 loop. The data shed new light on the modalities of KIR3DL1 interaction with HLA-B alleles essential for the modulation of NK immune-mediated response.

Address: Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy

3915442d: BioMed Res.Int., 168438-168438(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Biomedical applications of fermenticin HV6b isolated from Lactobacillus fermentum HV6b MTCC10770.
Author: Kaur,B., Balgir,P.P., Mittu,B., Kumar,B., Garg,N.
Abstract:
Fermenticin HV6b is a class IIa antimicrobial peptide produced by Lactobacillus fermentum HV6b MTCC 10770 isolated from human vaginal ecosystem. It shows growth inhibition of a wide range of opportunistic pathogens of humans, for example, Bacteroides, Gardnerella vaginalis, Mobiluncus, Staphylococci, and Streptococci, associated with bacterial vaginosis in humans. It does possess an impressive sperm immobilization and spermicidal activity tested against human sperms which makes it an attractive proposition for formulating antibacterial vaginosis and contraceptive products. Apart from this, in vitro studies conducted against four different tissue models have indicated its potential to be used as a component of anticancerous drug therapy as it is reported to induce apoptosis in cancerous cells. This information could be integrated in future studies focusing on in vivo assessment of anticancerous activity of lactic acid bacterial toxins or bacteriocins.

E-Mail: baljinderbt@hotmail.com
Address: Department of Biotechnology, Punjabi University, Patiala, Punjab 147002, India

3915446d: Proc.Natl.Acad.Sci.U.S.A., 15037-15042(2013)
Link: [ PubMED ] [ DOI ]
Title: Inhibition of SMG-8, a subunit of SMG-1 kinase, ameliorates nonsense-mediated mRNA decay-exacerbated mutant phenotypes without cytotoxicity.
Author: Usuki,F., Yamashita,A., Shiraishi,T., Shiga,A., Onodera,O., Higuchi,I., Ohno,S.
Abstract:
Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that eliminates aberrant mRNAs containing premature termination codons (PTCs). NMD inhibits the production of aberrant proteins that still retain, at least in part, wild-type function as well as dominant-negative peptides. Therefore, the selective inhibition of NMD has the potential to ameliorate NMD-exacerbated mutant phenotypes. However, we do not have sufficient knowledge of how to effectively suppress NMD with miniμm cytotoxic effects. In this study, we aimed to identify NMD-related factors that can be targeted to efficiently inhibit NMD without causing significant cytotoxicity to restore the levels of truncated but partially functional proteins. We evaluated the knockdown of 15 NMD components in Ullrich congenital muscular dystrophy fibroblasts, which have a homozygous frameshift mutation causing a PTC in the collagen type VI α 2 gene. Of the 15 NMD factors tested, knockdown of SMG-8 produced the best effect for restoring defective mRNA and protein levels without affecting cell growth, cell-cycle progression, or endoplasmic reticulum stress. The efficacy of SMG-8 knockdown to improve the mutant phenotype was confirmed using another cell line, from a cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy patient who carries a PTC-containing mutation in HtrA serine peptidase 1. Our results suggest that SMG-8 is an appropriate target for inhibiting NMD to improve NMD-exacerbated mutant phenotypes. NMD inhibition by knockdown of SMG-8 may also be useful to induce synergy in combining the use of read-through drugs for patients with nonsense mutation-associated diseases.

Address: Department of Clinical Medicine, National Institute for Minamata Disease, Minamata, Kumamoto 867-0008, Japan

3915447d: J.Am.Soc.Mass Spectrom., 1755-1766(2013)
Link: [ PubMED ] [ DOI ]
Title: Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions.
Author: Miladi,M., Harper,B., Solouki,T.
Abstract:
Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH2 (where "A" denotes carbon thirteen (13C1) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH2), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maxiμm of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic "b-type" ions.

Address: Department of Chemistry and Biochemistry, Baylor University, Waco, TX, 76798, USA

3915454d: Oncol.Rep., 2063-2070(2013)
Link: [ PubMED ] [ DOI ]
Title: Ghrelin inhibits ovarian epithelial carcinoma cell proliferation in vitro.
Author: Xu,Y., Pang,X., Dong,M., Wen,F., Zhang,Y.
Abstract:
The only orexigenic peptide, ghrelin, which is primarily produced by the gastrointestinal tract, has been implicated in malignant cell proliferation and invasion. Ghrelin is a natural ligand of the growth hormone secretagogue receptor 1a (GHSR1a). However, the role of ghrelin in ovarian epithelial carcinoma remains unknown since the expression of GHSR1a in ovary is not confirmed. The aim of the present study was to assess expression of ghrelin and its receptor in human ovarian epithelial carcinoma and to examine the effect of ghrelin on carcinoma cell proliferation. Frozen sections of ovarian samples and the human ovarian epithelial carcinoma cell line, HO-8910, were used to characterize the expression of ghrelin/GHSR1a axis and the effect of ghrelin on proliferation. We found that ghrelin and GHSR1a are expressed in ovarian epithelial carcinoma in vivo and in vitro. Ghrelin inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, and this inhibition may be abolished by the ghrelin receptor antagonist D-Lys-3-GH-releasing peptide-6 and ghrelin neutralizing antibody. Ghrelin enhances HO-8910 cell apoptosis and autophagy. The activation of mammalian target of rapamycin (mTOR) signaling pathway blocks the effects of ghrelin-induced autophagy and apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation induced by ghrelin. In conclusion, the present study demonstrates that ghrelin inhibits the proliferation of human HO-8910 ovarian epithelial carcinoma cells by inducing apoptosis and autophagy via the mTOR signaling pathway. This study provides a novel regulatory signaling pathway of ghrelin-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy.

Address: Department of Gynecology, the First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China

3915455d: Mol.Imaging, 376-387(2013)
Link:
Title: Micro-positron emission tomography/contrast-enhanced computed tomography imaging of orthotopic pancreatic tumor-bearing mice using the α-v-β3 integrin tracer Cu64-labeled cyclam-RAFT-c(-RGDfK-)4.
Author: Aung,W., Jin,Z.H., Furukawa,T., Claron,M., Boturyn,D., Sogawa,C., Tsuji,A.B., Wakizaka,H., Fukumura,T., Fujibayashi,Y., Dumy,P., Saga,T.
Abstract:
The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, (6)4Cu-labeled cyclam-RAFT-c(-RGDfK-)4 peptide ((6)4Cu-RAFT-RGD), using this model. Varying degrees of αvβ(3) integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing (6)4Cu-RAFT-RGD and (1)(8)F-fluoro-2-deoxy-d-glucose ((1)(8)F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvβ(3) integrin specific. (6)4Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of (6)4Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, (6)4Cu-RAFT-RGD accumulation provided better tumor contrast to background than (1)(8)F-FDG. Our results suggest that (6)4Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvβ(3) integrin-expressing pancreatic tumors.

E-Mail: winn@nirs.go.jp
Address: Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan

3915461d: Hum.Gene Ther.Clin.Dev., 99-107(2013)
Link: [ PubMED ] [ DOI ]
Title: Gene Electrotransfer of Plasmid Antiangiogenic Metargidin Peptide (AMEP) in Disseminated Melanoma: Safety and Efficacy Results of a Phase I First-in-Man Study.
Author: Spanggaard,I., Snoj,M., Cavalcanti,A., Bouquet,C., Sersa,G., Robert,C., Cemazar,M., Dam,E., Vasseur,B., Attali,P., Mir,L.M., Gehl,J.
Abstract:
Antiangiogenic metargidin peptide (AMEP) is a novel anticancer agent exerting antiproliferative and antiangiogenic effects by binding to αvβ3 and α5β1 integrins. Electrotransfer designates the use of electric pulses (electroporation) to transfer plasmid DNA into tissues. This first-in-man phase I study investigated safety and tolerability of intratumoral plasmid AMEP electrotransfer into cutaneous metastatic melanoma. Secondary objectives were efficacy and pharmacokinetics. Five patients with disseminated melanoma without further treatment options were treated at two dose levels (1 and 2 mg DNA). In each patient, two cutaneous lesions were identified (one treated and one control). At day 1 and day 8, plasmid AMEP was injected intratumorally followed by electrotransfer. Patients were monitored weekly until day 29, and at day 64. Local efficacy was assessed at day 29 by direct measurement, and posttreatment biopsies for AMEP mRNA levels were evaluated by reverse transcriptase quantitative polymerase chain reaction. Plasmid copy number in blood and urine was determined by quantitative polymerase chain reaction. Minimal systemic toxicity was observed, including transient fever and transitory increase in C-reactive protein. No related serious adverse events occurred. Plasmid AMEP was detected in plasma but not in urine. AMEP mRNA was found in three of five treated lesions and none of the control lesions. At day 29, all five treated lesions were stable in diameter, whereas four of five control lesions increased more than 20%. No response occurred in distant lesions. This first-in-man study on electrotransfer of plasmid AMEP into cutaneous melanoma shows that the procedure and drug are safe and that local transfection was obtained.

Address: 1 Department of Oncology, Center for Experimental Drug and Gene Electrotransfer, Copenhagen University Hospital Herlev , Herlev 2730, Denmark

3915462d: FEMS Microbiol.Lett., 52-57(2013)
Link: [ PubMED ] [ DOI ]
Title: Human-β-defensins-1-3 and analogs do not require proton motive force for antibacterial activity against Escherichia coli.
Author: Krishnakumari,V., Packiyanathan,K.K., Nagaraj,R.
Abstract:
Human-β-defensins 1-3 (HBD-1-3) and their C-terminal analogs Phd-1-3 do not show antibacterial activity against Escherichia coli in the presence of mono- and divalent cations. Activity of peptides was examined against E. coli pretreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) and salt remedial Escherichia coli ftsEX, a deletion mutant of FtsEX complex [an ATP-binding cassette (ABC) transporter protein], in the presence of Na+ , Ca2+ , and Mg2+ . Activity was observed in the presence of Na+ and Ca2+ , although not in the presence of Mg2+ against E. coli, when proton motive force (PMF) was dissipated by CCCP. The peptides exhibited antibacterial activity against E. coli ftsEX even in the presence of Na+ and Ca2+ . Our results indicate that HBD-1-3 and Phd-1-3 do not require PMF for their antibacterial activity. The absence of activity against E. coli in the presence of Na+ and Ca2+ ions is due to not only weakened electrostatic interactions with anionic membrane components, but also involvement of electrochemical gradients. However, Mg2+ prevents electrostatic interaction of the peptides with the outer membrane resulting in loss of activity.

Address: CSIR - Centre for Cellular and Molecular Biology, Hyderabad, India

3915465d: J.Biomed.Nanotechnol., 1636-1643(2013)
Link: [ PubMED ] [ DOI ]
Title: Long-circulating, pH-sensitive liposomes versus long-circulating, non-pH-sensitive liposomes as a delivery system for tumor identification.
Author: De Barros,A.L.B., Mota,L.das G., Soares,D.C.F., De Souza,C.M., Cassali,G.D., Oliveira,M.C., Cardoso,V.N.
Abstract:
Bombesin (BBN) is a tetradecapeptide that binds specifically to gastrin-releasing peptide receptors in humans. Several forms of cancer, including lung, prostate, breast, and colon over-express receptors for bombesin-like peptides. Therefore, radiolabeled bombesin analogs might be useful for tumor identification. Nevertheless, it is well known that higher tumor uptake can yield images in higher quality. Hence, drug delivery systems, such as liposomes, can be used to achieve a higher concentration of radiotracer in tumor site, and also improve the radiotracer stability, since peptides can suffer easily degradation in vivo by natural plasma and tissue peptides. In this paper, we prepared long-circulating, pH-sensitive liposomes and long-circulation, non-pH sensitive liposomes. Both formulations were able to encapsulate the radiolabeled bombesin derivative (99mTc-BBN(7_14)), and also showing high in vitro stability. Biodistribution studies were performed in Ehrlich tumor bearing-mice to compare the ability of pH-sensitive and non-pH sensitive liposomes to deliver 99mTc-BBN(7_14) to tumor site. Results showed higher tumor uptake (2-fold) when pH-sensitive liposomes were used, suggesting that these vesicles can facilitate the access to the tumor by releasing the diagnostic agent into the ideal area. As a result, tumor-to-muscle ratio achieved with pH-sensitive liposomes was higher than that obtained with non-pH-sensitive formulation. In addition, scintigraphic images for pH-sensitive liposomes showed evident tumor uptake, corroborating with biodistribution data. Therefore, the results presented in this paper suggest that pH-sensitive liposomes are able to deliver more efficiently the radiolabeled bombesin analog. This finding poses a new possibility to improve images quality, since the tumor-to-muscle ratio was strongly enhanced.

Address: Faculdade de Farmacia, Universidade Federal de Minas Gerais, Av. AntOnio Carlos, 6627, 31279-901, Belo Horizonte, Minas Gerais, Brazil

3915466d: J.Biomed.Nanotechnol., 1509-1520(2013)
Link: [ PubMED ] [ DOI ]
Title: Magnetite nanoparticle with positively charged surface for immobilization of peptide nucleic acid and deoxyribonucleic acid.
Author: Theppaleak,T., Rutnakornpituk,B., Wichai,U., Vilaivan,T., Rutnakornpituk,M.
Abstract:
We herein report the surface modification of magnetite nanoparticle (MNP) with the (co)polymer of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and/or diethylamino ethyl methacrylate (DEAEMA) via atom transfer radical polymerization (ATRP) for use as anion exchanger solid support for detection of DNA sequence using peptide nucleic acid (PNA) probe. Molar ratio of the PEGMA:DEAEMA (co)polymer was systematically varied to tune the positive charges on the particle surface. Kinetic studies of the (co)polymerizations were investigated via 1HNMR to disclose the relative reactivity of the (co)polymers in the reaction. Zeta potential of the (co)polymer-grafted MNP was analyzed by photo correlation spectroscopy (PCS). Transmission electron microscopy (TEM) and PCS indicated an improvement in the particle dispersibility in water upon quaternization of the DEAEMA entities grafted on the particle surface. From the preliminary results, these (co)polymer-grafted MNPs can be used as a nanosolid support to differentiate between full match and single-base mismatch DNA sequences using an acpcPNA probe. These novel cationic MNPs might be efficiently applicable for use as a magnetically guidable tool for detection of DNA sequences.

Address: Faculty of Science, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 6500, Thailand

3915469d: Protein Eng.Des.Sel., 713-724(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: In vitro affinity screening of protein and peptide binders by megavalent bead surface display.
Author: Diamante,L., Gatti-Lafranconi,P., Schaerli,Y., Hollfelder,F.
Abstract:
The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead Kd measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

Address: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, CB2 1GA Cambridge, UK

3915472d: Proc.Natl.Acad.Sci.U.S.A., 14900-14905(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Protoribosome by quantum kernel energy method.
Author: Huang,L., Krupkin,M., Bashan,A., Yonath,A., Massa,L.
Abstract:
Experimental evidence suggests the existence of an RNA molecular prebiotic entity, called by us the "protoribosome," which may have evolved in the RNA world before evolution of the genetic code and proteins. This vestige of the RNA world, which possesses all of the capabilities required for peptide bond formation, seems to be still functioning in the heart of all of the contemporary ribosome. Within the modern ribosome this remnant includes the peptidyl transferase center. Its highly conserved nucleotide sequence is suggestive of its robustness under diverse environmental conditions, and hence on its prebiotic origin. Its twofold pseudosymmetry suggests that this entity could have been a dimer of self-folding RNA units that formed a pocket within which two activated amino acids might be accommodated, similar to the binding mode of modern tRNA molecules that carry amino acids or peptidyl moieties. Using quantum mechanics and crystal coordinates, this work studies the question of whether the putative protoribosome has properties necessary to function as an evolutionary precursor to the modern ribosome. The quantum model used in the calculations is density functional theory--B3LYP/3-21G*, implemented using the kernel energy method to make the computations practical and efficient. It occurs that the necessary conditions that would characterize a practicable protoribosome--namely (i) energetic structural stability and (ii) energetically stable attachment to substrates--are both well satisfied.

Address: Center for Computational Materials Science, Naval Research Laboratory, Washington, DC 20375-5341

3915474d: Proc.Natl.Acad.Sci.U.S.A., 14924-14929(2013)
Link: [ PubMED ] [ DOI ]
Title: Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.
Author: Sha,F., Gencer,E.B., Georgeon,S., Koide,A., Yasui,N., Koide,S., Hantschel,O.
Abstract:
The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

Address: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637 and Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland

3915478d: G3(Bethesda), 1753-1758(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Characterization of Two ENU-Induced Mutations Affecting Mouse Skeletal Morphology.
Author: Dauphinee,S.M., Eva,M.M., Yuki,K.E., Herman,M., Vidal,S.M., Malo,D.
Abstract:
Using the N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we have identified two skeletal morphology mutants, Skm1 and Skm2. Positional cloning and candidate gene sequencing localized the causative point mutations within the genes coding for natriuretic peptide receptor C (NPR-C) and filamin b (FLNB), respectively. Mice that carry a mutation in Npr3 exhibit a skeletal overgrowth phenotype, resulting in an elongated body and kyphosis. Skm2 mice, carrying a mutation in Flnb, present with scoliosis and lordosis. These mutant mice will serve as useful models for the study of vertebral malformations.

Address: Department of Human Genetics, McGill University, Montreal, Quebec H3G 0B1, Canada

3915479d: Analyst, 6385-6397(2013)
Link: [ PubMED ] [ DOI ]
Title: Forensic proteomics of poxvirus production.
Author: Wunschel,D., Tulman,E., Engelmann,H., Clowers,B.H., Geary,S., Robinson,A., Liao,X.
Abstract:
The field of microbial forensics has recently sought to develop methods to discern biological signatures to indicate production methods for biological agents. Viral agents have received less attention to date. Their obligate propagation in living cells makes purification from cellular material a challenge. This leads to potential carryover of protein-rich signatures of their production system. Here we have explored a proteomic analysis of vaccinia virus as a model poxvirus system in which to compare samples of virus propagated in different cell lines and subjected to different purification schemes. The proteomic data sets indicated viral, host cell and culture medium proteins. Several layers of data analysis were applied to build confidence in the peptide identification and capture information on the taxonomic utility of each. The analysis showed clear shifts in protein profiles with virus purification, with successive gradient purification steps showing different levels of viral protein enrichment. Peptides from cellular proteins, including those present in purified virus preparations, provided signatures which enabled discrimination of cell line substrates, including distinguishing between cells derived from different primate species. The ability to discern multiple aspects of viral production demonstrates the potential value of proteomic analysis as tool for microbial forensics.

E-Mail: David.Wunschel@pnnl.gov
Address: Chemical and Biological Signature Sciences Group, Pacific Northwest National Laboratory, Richland, Washington, USA

3915480d: Antimicrob.Agents Chemother., 5492-5499(2013)
Link: [ PubMED ] [ DOI ]
Title: A Novel C5a-Derived Immunobiotic Peptide Reduces Streptococcus agalactiae Colonization through Targeted Bacterial Killing.
Author: Cavaco,C.K., Patras,K.A., Zlamal,J.E., Thoman,M.L., Morgan,E.L., Sanderson,S.D., Doran,K.S.
Abstract:
Streptococcus agalactiae (group B Streptococcus [GBS]) is a Gram-positive bacterium that colonizes the cervicovaginal tract in approximately 25% of healthy women. Although colonization is asymptomatic, GBS can be vertically transmitted to newborns peripartum, causing severe disease such as pneumonia and meningitis. Current prophylaxis, consisting of late gestation screening and intrapartum antibiotics, has failed to completely prevent transmission, and GBS remains a leading cause of neonatal sepsis and meningitis in the United States. Lack of an effective vaccine and emerging antibiotic resistance necessitate exploring novel therapeutic strategies. We have employed a host-directed immunomodulatory therapy using a novel peptide, known as EP67, derived from the C-terminal region of human complement component C5a. Previously, we have demonstrated in vivo that EP67 engagement of the C5a receptor (CD88) effectively limits staphylococcal infection by promoting cytokine release and neutrophil infiltration. Here, using our established mouse model of GBS vaginal colonization, we observed that EP67 treatment results in rapid clearance of GBS from the murine vagina. However, this was not dependent on functional neutrophil recruitment or CD88 signaling, as EP67 treatment reduced the vaginal bacterial load in mice lacking CD88 or the major neutrophil receptor CXCr2. Interestingly, we found that EP67 inhibits GBS growth in vitro and in vivo and that antibacterial activity was specific to Streptococcus species. Our work establishes that EP67-mediated clearance of GBS is likely due to direct bacterial killing rather than to enhanced immune stimulation. We conclude that EP67 may have potential as a therapeutic to control GBS vaginal colonization.

Address: Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California, USA

3915481d: Antimicrob.Agents Chemother., 5559-5564(2013)
Link: [ PubMED ] [ DOI ]
Title: Bioengineered Lysozyme Reduces Bacterial Burden and Inflammation in a Murine Model of Mucoid Pseudomonas aeruginosa Lung Infection.
Author: Teneback,C.C., Scanlon,T.C., Wargo,M.J., Bement,J.L., Griswold,K.E., Leclair,L.W.
Abstract:
The spread of drug-resistant bacterial pathogens is a growing global concern and has prompted an effort to explore potential adjuvant and alternative therapies derived from nature's repertoire of bactericidal proteins and peptides. In humans, the airway surface liquid layer is a rich source of antibiotics, and lysozyme represents one of the most abundant and effective antimicrobial components of airway secretions. Human lysozyme is active against both Gram-positive and Gram-negative bacteria, acting through several mechanisms, including catalytic degradation of cell wall peptidoglycan and subsequent bacterial lysis. In the infected lung, however, lysozyme's dense cationic character can result in sequestration and inhibition by polyanions associated with airway inflammation. As a result, the efficacy of the native enzyme may be compromised in the infected and inflamed lung. To address this limitation, we previously constructed a charge-engineered variant of human lysozyme that was less prone to electrostatic-mediated inhibition in vitro. Here, we employ a murine model to show that this engineered enzyme is superior to wild-type human lysozyme as a treatment for mucoid Pseudomonas aeruginosa lung infections. The engineered enzyme effectively decreases the bacterial burden and reduces markers of inflammation and lung injury. Importantly, we found no evidence of acute toxicity or allergic hypersensitivity upon repeated administration of the engineered biotherapeutic. Thus, the charge-engineered lysozyme represents an interesting therapeutic candidate for P. aeruginosa lung infections.

Address: University of Vermont College of Medicine, Division of Pulmonary and Critical Care Medicine, Burlington, Vermont, USA

3915482d: Antimicrob.Agents Chemother., 5565-5571(2013)
Link: [ PubMED ] [ DOI ]
Title: High-Level Pacidamycin Resistance in Pseudomonas aeruginosa Is Mediated by an Opp Oligopeptide Permease Encoded by the opp-fabI Operon.
Author: Mistry,A., Warren,M.S., Cusick,J.K., Karkhoff-Schweizer,R.R., Lomovskaya,O., Schweizer,H.P.
Abstract:
Pacidamycins (or uridyl peptide antibiotics) possess selective in vivo activity against Pseudomonas aeruginosa. An important limitation for the therapeutic use of pacidamycins with P. aeruginosa is the high frequency (10(-6) to 10(-7)) at which resistant mutants emerge. To elucidate the mechanism(s) of this resistance, pacidamycin-resistant P. aeruginosa mutants were isolated. Two types of mutants were obtained. Type 1, or high-level resistance mutants with a pacidamycin MIC of 512 μg/ml, were more abundant, with a frequency of approximately 2 x 10(-6), and did not show cross-resistance with other antibiotics. Type 2, low-level resistance mutants, were isolated with a frequency of approximately 10(-8) and had a pacidamycin MIC of 64 μg/ml (the MIC for the wild-type strain was 4 to 16 μg/ml). These mutants were cross-resistant to levofloxacin, tetracycline, and erythromycin and were shown to overexpress either the MexAB-OprM or MexCD-OprJ multidrug resistance efflux pumps. High-level resistant mutants were isolated by transposon mutagenesis and one insertion was localized to oppB, one of two periplasmic binding protein components of an oligopeptide transport system which is encoded by the opp-fabI operon. The Opp system is required for uptake of pacidamycin across the inner membrane, since various opp, but not fabI, mutants were resistant to high levels of pacidamycin. Both of the two putative Opp periplasmic binding proteins, OppA and OppB, were required for pacidamycin uptake. Although both impaired uptake into and efflux from the cell can cause pacidamycin resistance in P. aeruginosa, our data suggest that impaired uptake is the primary reason for the high-frequency and high-level pacidamycin resistance.

Address: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA

3915483d: Antimicrob.Agents Chemother., 5572-5579(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of Bacteriocins on Methicillin-Resistant Staphylococcus aureus Biofilm.
Author: Okuda,K., Zendo,T., Sugimoto,S., Iwase,T., Tajima,A., Yamada,S., Sonomoto,K., Mizunoe,Y.
Abstract:
Control of biofilms formed by microbial pathogens is an important subject for medical researchers, since the development of biofilms on foreign-body surfaces often causes biofilm-associated infections in patients with indwelling medical devices. The present study examined the effects of different kinds of bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by certain bacteria, on biofilms formed by a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). The activities and modes of action of three bacteriocins with different structures (nisin A, lacticin Q, and nukacin ISK-1) were evaluated. Vancomycin, a glycopeptide antibiotic used in the treatment of MRSA infections, showed bactericidal activity against planktonic cells but not against biofilm cells. Among the tested bacteriocins, nisin A showed the highest bactericidal activity against both planktonic cells and biofilm cells. Lacticin Q also showed bactericidal activity against both planktonic cells and biofilm cells, but its activity against biofilm cells was significantly lower than that of nisin A. Nukacin ISK-1 showed bacteriostatic activity against planktonic cells and did not show bactericidal activity against biofilm cells. Mode-of-action studies indicated that pore formation leading to ATP efflux is important for the bactericidal activity against biofilm cells. Our results suggest that bacteriocins that form stable pores on biofilm cells are highly potent for the treatment of MRSA biofilm infections.

Address: Department of Bacteriology, The Jikei University School of Medicine, Tokyo, Japan

3915484d: Antimicrob.Agents Chemother., 5737-5739(2013)
Link: [ PubMED ] [ DOI ]
Title: New Structure of Phage-Related Islands Carrying fusB and a Virulence Gene in Fusidic Acid-Resistant Staphylococcus epidermidis.
Author: Chen,H.J., Chang,Y.C., Tsai,J.C., Hung,W.C., Lin,Y.T., You,S.J., Tseng,S.P., Teng,L.J.
Abstract:
Nucleotide sequencing of the fusB-flanking regions in two fusidic acid-resistant Staphylococcus epidermidis isolates with the type IV aj1-leader peptide (LP)-fusB structure (lacking aj1) revealed that their fusB gene was located on novel phage-related islands inserted downstream of smpB and are here referred to as SeRIfusB-3692 and SePIfusB-857. The novel SePIfusB-857 structure was followed by SeCI857, forming a composite pathogenicity island which contained a putative virulence gene, vapE. The linkage of fusB and vapE may contribute to bacterial adaption.

Address: Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan

3915487d: J.Vis.Exp., /50541-/50541(2013)
Link: [ PubMED ] [ DOI ]
Title: Protein purification-free method of binding affinity determination by microscale thermophoresis.
Author: Khavrutskii,L., Yeh,J., Timofeeva,O., Tarasov,S.G., Pritt,S., Stefanisko,K., Tarasova,N.
Abstract:
Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Address: Cancer and Inflammation Program, National Cancer Institute, USA

3915488d: Extremophiles, 921-930(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Experimental and computational analysis of the secretome of the hyperthermophilic archaeon Pyrococcus furiosus.
Author: Schmid,G., Mathiesen,G., Arntzen,M.O., Eijsink,V.G., Thomm,M.
Abstract:
Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC-MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.

Address: Hyperthermics Regensburg GmbH, Josef-Engert-Strasse 9, 93053, Regensburg, Germany

3915496d: J.Proteome Res., 4612-4616(2013)
Link: [ PubMED ] [ DOI ]
Title: Quantification of human kallikrein-2 in clinical samples by selected reaction monitoring.
Author: Vegvari,A., Sjodin,K., Rezeli,M., Marko-Varga,G.
Abstract:
Recently, the number of mass spectrometry-based quantification assays has been increased, partially due to the global efforts of chromosome-centric human proteome project (C-HPP). Our goal at the Chromosome 19 Consortium is to provide novel selected reaction monitoring (SRM) assays of proteins coded on chromosome 19. We have selected the two most useful signature peptides (NSQVWLGR and HNLFEPEDTGQR) of human kallikrein-2 (hK2 - NX_P20151) and developed an SRM assay. Details of the analytical parameters, including multiple transitions by peptides, are presented. The endogenous levels of hK2 were determined in clinical samples (n = 35). The limit of quantification was also estimated by spiking heavy isotope-labeled peptides into seminal plasma samples at various concentrations (LOQ approximately 29 ng/mL).

Address: Clinical Protein Science & Imaging, Biomedical Center, Department of Measurement Technology and Industrial Electrical Engineering, Lund University , BMC C13, 221 84 Lund, Sweden

3915500d: J.Proteome Res., 4402-4413(2013)
Link: [ PubMED ] [ DOI ]
Title: Targeted Quantitative Proteomics for the Analysis of 14 UGT1As and -2Bs in Human Liver Using NanoUPLC-MS/MS with Selected Reaction Monitoring.
Author: Fallon,J.K., Neubert,H., Hyland,R., Goosen,T.C., Smith,P.C.
Abstract:
Targeted quantitative proteomics using heavy isotope dilution techniques is increasingly being utilized to quantify proteins, including UGT enzymes, in biological matrices. Here we present a multiplexed method using nanoLC-MS/MS and multiple reaction monitoring (MRM) to quantify 14 UGT1As and UGT2Bs in liver matrices. Where feasible, we employ two or more proteotypic peptides per protein, with only four proteins quantified with only one proteotypic peptide. We apply the method to analysis of a library of 60 human liver microsome (HLM) and matching S9 samples. Ten of the UGT isoforms could be detected in liver, and the expression of each was consistent with mRNA expression reported in the literature. UGT2B17 was unusual in that approximately 30% of liver microsomes had no or little (<0.5 pmol/mg protein) content, consistent with a known common polymorphism. Liver S9 UGT concentrations were approximately 10-15% those of microsomes. The method was robust, precise, and reproducible and provides novel UGT expression data in human liver that will benefit rational approaches to evaluate metabolism in drug development.

Address: Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States

3915504d: PLoS One, e74016-e74016(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Elevation in sphingomyelin synthase activity is associated with increases in amyloid-β peptide generation.
Author: Hsiao,J.H., Fu,Y., Hill,A.F., Halliday,G.M., Kim,W.S.
Abstract:
A pathological hallmark of Alzheimer's disease (AD) is the presence of amyloid-β peptide (Aβ) plaques in the brain. Aβ is derived from a sequential proteolysis of the transmenbrane amyloid precursor protein (APP), a process which is dependent on the distribution of lipids present in the plasma membrane. Sphingomyelin is a major membrane lipid, however its role in APP processing is unclear. Here, we assessed the expression of sphingomyelin synthase (SGMS1; the gene responsible for sphingomyelin synthesis) in human brain and found that it was significantly elevated in the hippocampus of AD brains, but not in the cerebellum. Secondly, we assessed the impact of altering SGMS activity on Aβ generation. Inhibition of SGMS activity significantly reduced the level of Aβ in a dose- and time dependent manner. The decrease in Aβ level occurred without changes in APP expression or cell viability. These results when put together indicate that SGMS activity impacts on APP processing to produce Aβ and it could be a contributing factor in Aβ pathology associated with AD.

Address: Neuroscience Research Australia, Sydney, New South Wales, Australia

3915505d: PLoS One, e72584-e72584(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Analysis of human blood plasma proteome from ten healthy volunteers from Indian population.
Author: Gautam,P., Nair,S.C., Ramamoorthy,K., Swamy,C.V., Nagaraj,R.
Abstract:
Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used 'reference plasma sample', a pool of plasma from 10 healthy individuals from Indian population in the age group of 25-60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by >/= 2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.

E-Mail: gautamp@icmr.org.in
Address: CSIR- Centre for Cellular and Molecular Biology, Hyderabad, India

3915509d: PLoS One, e77595-e77595(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The Effect of N-acetylation and N-methylation of Lysine Residue of Tat Peptide on its Interaction with HIV-1 TAR RNA.
Author: Kumar,S., Maiti,S.
Abstract:
Post-translational modification (PTM) of RNA binding proteins (RBPs) play a very important role in determining their binding to cognate RNAs and therefore regulate the downstream effects. Lysine can undergo various PTMs and thereby contribute to the regulation of different cellular processes. It can be reversibly acetylated and methylated using a pool of respective enzymes, to act as a switch for controlling the binding efficiency of RBPs. Here we have delineated the thermodynamic and kinetic effects of N-acetylation and N-monomethylation of lysine on interaction between HIV-1 TAR RNA and its cognate binder Tat peptide ( a model system). Our results indicate that acetylation of lysine 50 (K50), leads to eight- fold reduction in binding affinity, originating exclusively from entropy changes whereas, lysine 51 (K51) acetylation resulted only in three fold decrease with large enthalpy-entropy compensation. The measurement of kinetic parameters indicated major change (4.5 fold) in dissociation rate in case of K50 acetylation however, K51 acetylation showed similar effect on both association and dissociation rates. In contrast, lysine methylation did not affect the binding affinity of Tat peptide to TAR RNA at K50, nonetheless three fold enhancement in binding affinity was observed at K51 position. In spite of large enthalpy-entropy compensation, lysine methylation seems to have more pronounced position specific effect on the kinetic parameters. In case of K50 methylation, simultaneous increase was observed in the rate of association and dissociation leaving binding affinity unaffected. The increased binding affinity for methylated Tat at K51 stems from faster association rate with slightly slower dissociation rate.

Address: Proteomics and Structural Biology Unit, CSIR-Institute of Genomics and Integrative Biology, CSIR, Delhi, India

3915510d: PLoS One, e77586-e77586(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Characterization of Niemann-Pick Type C2 Protein Expression in Multiple Cancers Using a Novel NPC2 Monoclonal Antibody.
Author: Liao,Y.-J., Lin,M.-W., Yen,C.-H., Lin,Y.-T., Wang,C.-K., Huang,S.-F., Chen,K.-H., Yang,C.-P., Chen,T.-L., Hou,M.-F., Arthur Chen,Y.-M.
Abstract:
Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor -, progesterone receptor - and human epidermal growth factor receptor +. On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.

Address: School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan

3915515d: PLoS One, e76733-e76733(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Four Amino Acids within a Tandem QxVx Repeat in a Predicted Extended α-Helix of the Smad-Binding Domain of Sip1 Are Necessary for Binding to Activated Smad Proteins.
Author: Conidi,A., Van den Berghe,V., Leslie,K., Stryjewska,A., Xue,H., Chen,Y.G., Seuntjens,E., Huylebroeck,D.
Abstract:
The zinc finger transcription factor Smad-interacting protein-1 (Sip1; Zeb2, Zfhx1b) plays an important role during vertebrate embryogenesis in various tissues and differentiating cell types, and during tumorigenesis. Previous biochemical analysis suggests that interactions with several partner proteins, including TGFβ family receptor-activated Smads, regulate the activities of Sip1 in the nucleus both as a DNA-binding transcriptional repressor and activator. Using a peptide aptamer approach we mapped in Sip1 its Smad-binding domain (SBD), initially defined as a segment of 51 amino acids, to a shorter stretch of 14 amino acids within this SBD. Modelling suggests that this short SBD stretch is part of an extended α-helix that may fit the binding to a hydrophobic corridor within the MH2 domain of activated Smads. Four amino acids (two polar Q residues and two non-polar V residues) that form the tandem repeat (QxVx)2 in this 14-residue stretch were found to be crucial for binding to both TGFβ/Nodal/Activin-Smads and BMP-Smads. A full-length Sip1 with collective mutation of these Q and V residues (to A) no longer binds to Smads, while it retains its binding activity to its cognate bipartite target DNA sequence. This missense mutant Sip1(AxAx)2 provides a new molecular tool to identify SBD (in)dependent target genes in Sip1-controlled TGFβ and/or BMP (de)regulated cellular, developmental and pathological processes.

Address: Laboratory of Molecular Biology (Celgen), Department of Development and Regeneration, Katholieke Universiteit Leuven, Leuven, Belgium

3915516d: PLoS One, e76350-e76350(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Regulation of the Expression of Chaperone gp96 in Macrophages and Dendritic Cells.
Author: Wolfram,L., Fischbeck,A., Frey-Wagner,I., Wojtal,K.A., Lang,S., Fried,M., Vavricka,S.R., Hausmann,M., Rogler,G.
Abstract:
The chaperone function of the ER-residing heat shock protein gp96 plays an important role in protein physiology and has additionally important immunological functions due to its peptide-binding capacity. Low amounts of gp96 stimulate immunity; high quantities induce tolerance by mechanisms not fully understood. A lack of gp96 protein in intestinal macrophages (IMACs) from Crohn`s disease (CD) patients correlates with loss of tolerance against the host gut flora, leading to chronic inflammation. Since gp96 shows dose-dependent direction of immunological reactions, we studied primary IMACs and developed cell models to understand the regulation of gp96 expression. Induction of gp96-expression was higher in in vitro differentiated dendritic cells (i.v.DCs) than in in vitro differentiated macrophages (i.v.MACs), whereas monocytes (MOs) expressed only low gp96 levels. The highest levels of expression were found in IMACs. Lipopolysaccharide (LPS), muramyl dipeptide (MDP), tumour necrosis factor (TNF), and Interleukin (IL)-4 induced gp96-expression, while IL12, IL-17, IL-23 and interferon (IFN)-γ were not effective indicating that Th1 and Th17 cells are probably not involved in the induction of gp96. Furthermore, gp96 was able to induce its own expression. The ER-stress inducer tunicamycin increased gp96-expression in a concentration- and time-dependent manner. Both ulcerative colitis (UC) and CD patients showed significantly elevated gp96 mRNA levels in intestinal biopsies which correlated positively with the degree of inflammation of the tissue. Since gp96 is highly expressed on the one hand upon stress induction as during inflammation and on the other hand possibly mediating tolerance, these results will help to understand the whether gp96 plays a role in the pathophysiology of inflammatory bowel disease (IBD).

Address: Division of Gastroenterology and Hepatology, University Hospital of Zurich, Zurich, Switzerland

3915517d: PLoS One, e76133-e76133(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Insulin-Like Growth Factor I (IGF-1) Ec/Mechano Growth Factor - A Splice Variant of IGF-1 within the Growth Plate.
Author: Schlegel,W., Raimann,A., Halbauer,D., Scharmer,D., Sagmeister,S., Wessner,B., Helmreich,M., Haeusler,G., Egerbacher,M.
Abstract:
Human insulin-like growth factor 1 Ec (IGF-1Ec), also called mechano growth factor (MGF), is a splice variant of insulin-like growth factor 1 (IGF-1), which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.

Address: Medical University of Vienna, Department of Paediatrics and Adolescent Medicine, Vienna, Austria

3915518d: PLoS One, e75528-e75528(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Biophysical investigation of the membrane-disrupting mechanism of the antimicrobial and amyloid-like peptide dermaseptin S9.
Author: Caillon,L., Killian,J.A., Lequin,O., Khemtemourian,L.
Abstract:
Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with 2H- and (31)P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.

Address: UPMC Univ Paris 06, UMR 7203 CNRS-UPMC-ENS, Laboratoire des Biomolecules, Paris, France

3915539d: Molecules, 13020-13026(2013)
Link: [ PubMED ] [ DOI ]
Title: Selection of a Mimotope Peptide of S-adenosyl-L-homocysteine and Its Application in Immunoassays.
Author: Wu,C., Tzertzinis,G.
Abstract:
A competitive immunoassay for S-adenosyl-l-homocysteine (SAH) has been used in the clinical test for homocysteine via an enzymatic conversion reaction. Since S-adenosyl-l-homocysteine is a relatively unstable compound, we have used peptide library phage display to select a new mimotope peptide that interacts with the anti-SAH antibody. By immobilizing the synthetic peptide on solid phase as a competitive surrogate for SAH, we demonstrate its utility in a competitive ELISA assay. The linear range of the assay for SAH was 0.4-6.4 μM, in good correlation to the conventional assay using an SAH-conjugated plate. Our results show that the mimotope peptide has potential to substitute for SAH in immunoassays.

E-Mail: tzertzinis@neb.com
Address: Health Research Institute, AIST, 1-8-31 Midorigaoka, Ikeda 563-8577, Osaka, Japan

3915549d: Asian Pac.J.Trop.Med., 934-941(2013)
Link: [ PubMED ] [ DOI ]
Title: Physical chemical and biological characterization of a new bacteriocin produced by Bacillus cereus NS02.
Author: Senbagam,D., Gurusamy,R., Senthilkumar,B.
Abstract:
OBJECTIVE: To screen the bacteriocinogenic isolate from buffalo milk and to characterize it on physical, chemical and biological aspects for the application in biopreservation. METHODS: Bacillus cereus (B. cereus) was isolated and assessed for its baceteriocinogenic activity. Bacteriocin was produced and purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography. Purified bacteriocin was used to check its antimicrobial activity against food borne bacteria. Effect and stability of bacteriocin was determined with the respect to temperature, pH, enzymes, organic solvents and chemicals. Bacteriocin was also subjected to SDS PAGE analysis to determine its molecular weight. In addition, functional groups exist in the bacteriocin was determined by FTIR analysis. RESULTS: B. cereus was identified by 16S rRNA sequence analysis. Bacteriocin showed increased activity against all the bacteria used and its activity unit was found to be 51, 200 AU/mL. It was stable to high temperature (100 degrees C) and wide range of pH (3-10), sensitive to proteolytic enzymes and resistant to nonproteolytic enzymes. It was low molecular weight (3.5 - 6 KDa) protein and FTIR study revealed the presence of amide group and NH stretching. CONCLUSIONS: Bacteriocin produced in this study possesses the highest antimicrobial activity against both gram positive and gram negative bacteria thereby it has immense application as biopreservative agent. FTIR proved its peptide nature.

E-Mail: nevithasenthil@gmail.com
Address: Department of Biotechnology, Vivekanandha College of Engineering for Women Tiruchengode - 637 205, Tamil Nadu, India. Electronic address:

3915552d: PLoS One, e77520-e77520(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Allatotropin: an ancestral myotropic neuropeptide involved in feeding.
Author: Alzugaray,M.E., Adami,M.L., Diambra,L.A., Hernandez-Martinez,S., Damborenea,C., Noriega,F.G., Ronderos,J.R.
Abstract:
BACKGROUND: Cell-cell interactions are a basic principle for the organization of tissues and organs allowing them to perform integrated functions and to organize themselves spatially and temporally. Peptidic molecules secreted by neurons and epithelial cells play fundamental roles in cell-cell interactions, acting as local neuromodulators, neurohormones, as well as endocrine and paracrine messengers. Allatotropin (AT) is a neuropeptide originally described as a regulator of Juvenile Hormone synthesis, which plays multiple neural, endocrine and myoactive roles in insects and other organisms. METHODS: A combination of immunohistochemistry using AT-antibodies and AT-Qdot nanocrystal conjugates was used to identify immunoreactive nerve cells containing the peptide and epithelial-muscular cells targeted by AT in Hydra plagiodesmica. Physiological assays using AT and AT- antibodies revealed that while AT stimulated the extrusion of the hypostome in a dose-response fashion in starved hydroids, the activity of hypostome in hydroids challenged with food was blocked by treatments with different doses of AT-antibodies. CONCLUSIONS: AT antibodies immunolabeled nerve cells in the stalk, pedal disc, tentacles and hypostome. AT-Qdot conjugates recognized epithelial-muscular cell in the same tissues, suggesting the existence of anatomical and functional relationships between these two cell populations. Physiological assays indicated that the AT-like peptide is facilitating food ingestion. SIGNIFICANCE: Immunochemical, physiological and bioinformatics evidence advocates that AT is an ancestral neuropeptide involved in myoregulatory activities associated with meal ingestion and digestion.

Address: Catedra Histologia y Embriologia Animal, Facultad de Ciencias Naturales y Museo

3915553d: PLoS One, e77516-e77516(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Inhibition of Human MCF-7 Breast Cancer Cells and HT-29 Colon Cancer Cells by Rice-Produced Recombinant Human Insulin-Like Growth Binding Protein-3 (rhIGFBP-3).
Author: Cheung,S.C., Long,X., Liu,L., Liu,Q., Lan,L., Tong,P.C., Sun,S.S.
Abstract:
BACKGROUND: Insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I) to form a complex (IGF-I/IGFBP-3) that can treat growth hormone insensitivity syndrome (GHIS) and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3) in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL) were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 +/- 1.72% vs 45.00 +/- 0.86%, p < 0.05; 50.84 +/- 1.97% vs 45.00 +/- 0.86%, p < 0.01 respectively) and HT-29 colon cancer cells (65.14 +/-3.84% vs 18.01 +/- 13.81%, p < 0.05 and 54.7 +/- 9.44% vs 18.01 +/- 13.81%, p < 0.05 respectively) when compared with wild type rice. CONCLUSION/SIGNIFICANCE: These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future.

Address: State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China

3915557d: Int.J.Nanomed., 3855-3866(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: A novel application of maleimide for advanced drug delivery: in vitro and in vivo evaluation of maleimide-modified pH-sensitive liposomes.
Author: Li,T., Takeoka,S.
Abstract:
Maleimide is a stable and easy-to-handle moiety that rapidly and covalently conjugates thiol groups of cysteine residues in proteins or peptides. Herein, we use maleimide to modify the surface of liposomes in order to obtain an advanced drug delivery system. Employing a small amount (0.3 mol%) of maleimide-polyethylene glycol (PEG) to modify the surface of the liposomes M-GGLG-liposomes, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG)/cholesterol/poly(ethylene glycol) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG5000-DSPE)/maleimide-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03, drug delivery efficiency was remarkably improved both in vitro and in vivo compared to unmodified liposomes (GGLG-liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). Moreover, this modification did not elicit any detectable increase in cytotoxicity. The maleimide-modification did not alter the physical characteristics of the liposomes such as size, zeta potential, pH sensitivity, dispersibility and drug encapsulation efficiency. However, M-GGLG-liposomes were more rapidly (>/=2-fold) internalized into HeLa, HCC1954, and MDA-MB-468 cells compared to GGLG-liposomes. In vivo, M-GGLG-liposomes encapsulating doxorubicin (M-GGLG-DOX-liposomes) also showed a more potent antitumor effect than GGLG-DOX-liposomes and the widely used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-DOX-liposomes after two subcutaneous injections around breast cancer tissue in mice. The biodistribution of liposomes in this model was observed using an in vivo imaging system, which showed that M-GGLG-liposomes were present for significantly longer at the injection site compared to GGLG-liposomes. The outstanding biological functions of the maleimide-modified liposomes as a novel drug delivery system make them ideally suited to a wide range of applications.

Address: Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns), Shinjuku-ku, Tokyo, Japan

3915559d: J.Huazhong Univ.Sci.Technol.Med.Sci., 735-742(2013)
Link: [ PubMED ] [ DOI ]
Title: HPV16 E5 peptide vaccine in treatment of cervical cancer in vitro and in vivo.
Author: Liao,S.J., Deng,D.R., Zeng,D., Zhang,L., Hu,X.J., Zhang,W.N., Li,L., Jiang,X.F., Wang,C.Y., Zhou,J.F., Wang,S.X., Zhang,H.W., Ma,D.
Abstract:
Human papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide. Despite the encouraging development of the preventive vaccine for HPV, a vaccine for both prevention and therapy or pre-cancerous lesions remains in high priority. Thus far, most of the HPV therapeutic vaccines are focused on HPV E6 and E7 oncogene. However these vaccines could not completely eradicate the lesions. Recently, HPV E5, which is considered as an oncogene, is getting more and more attention. In this study, we predicted the epitopes of HPV16 E5 by bioinformatics as candidate peptide, then, evaluated the efficacy and chose an effective one to do the further test. To evaluate the effect of vaccine, rTC-1 (TC-1 cells infected by rAAV-HPV16E5) served as cell tumor model and rTC-1 loading mice as an ectopic tumor model. We prepared vaccine by muscle injection. The vaccine effects were determined by evaluating the function of tumor-specific T cells by cell proliferation assay and ELISPOT, calculating the tumor volume in mice and estimating the survival time of mice. Our in vitro and in vivo studies revealed that injection of E5 peptide+CpG resulted in strong cell-mediated immunity (CMI) and protected mice from tumor growth, meanwhile, prolonged the survival time after tumor cell loading. This study provides new insights into HPV16 E5 as a possible target on the therapeutic strategies about cervical cancer.

E-Mail: sjliao@tjh.tjmu.edu.cn
Address: Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China,

3915560d: Oncotarget, 1819-1828(2013)
Link: [ PubMED ]
Title: Identification of a novel peptide that blocks basic fibroblast growth factor-mediated cell proliferation.
Author: Wu,X., Huang,H., Wang,C., Lin,S., Huang,Y., Wang,Y., Liang,G., Yan,Q., Xiao,J., Wu,J., Yang,Y., Li,X.
Abstract:
Basic fibroblast growth factor (bFGF) has been implicated in tumor growth via interactions with its receptors (FGFRs) on the cell surface and therefore, bFGF/FGFRs are considered essential targets for cancer therapy. Herein, a consensus heptapeptide (LSPPRYP) was identified for the first time from a phage display heptapeptide library after three sequential rounds of biopanning against FGFR-expressing cells with competitive displacement of phage by bFGF, followed by subtraction of non-specific binding by FGFR-deficient cells. Phage bearing LSPPRYP showed high levels of binding to Balb/c 3T3 cells expressing high-affinity bFGF-binding FGFR (bFGFR), but not to the cells that do not express bFGFR (Cos-7), or express a very low affinity bFGFR (HaCat). The selected-phage-derived peptide synthesized by solid phase method using a rapid and practical Fmoc strategy was found to specifically compete with bFGF for binding to its receptors, inhibit bFGF-stimulated cell proliferation by inducing cell cycle arrest, and block bFGF-induced activation of Erk1 and Erk2 kinase in B16-F10 melanoma cells. Importantly, treatment of melanoma-bearing mice with the synthetic peptide significantly suppressed tumor growth. The results demonstrate a strong anticancer activity of the isolated bFGFR-binding peptide (and its future derivatives), which may have great potential for cancer therapy.

Address: School of Pharmaceutical Science, Key Laboratory of Biotechnology and Pharmaceutical Engineering of Zhejiang Province, Wenzhou Medical College, Wenzhou, PR China

3915563d: Spectrochim.Acta A, 707-712(2013)
Link: [ PubMED ] [ DOI ]
Title: Interactions of fish gelatin and chitosan in uncrosslinked and crosslinked with EDC films: FT-IR study.
Author: Staroszczyk,H., Sztuka,K., Wolska,J., Wojtasz-Pajak,A., Kolodziejska,I.
Abstract:
Films based on fish gelatin, chitosan and blend of fish gelatin and chitosan before and after cross-linking with EDC have been characterized by FT-IR spectroscopy. The FT-IR spectrum of fish gelatin film showed the characteristic amide I, amide II and amide III bands, and the FT-IR spectrum of chitosan film confirmed that the polymer was only a partially deacetylated product, and included CH3CO and NH2 groups, the latter both in their free -NH2 and protonated -NH3+ form. Analysis of FT-IR spectra of two-component, fish gelatin-chitosan film revealed the formation not only of hydrogen bonds within and between chains of polymers, but also of electrostatic interactions between -COO- of gelatin and -NH3+ of chitosan. Modification with EDC provided cross-linking of composites of the film. New iso-peptide bonds formed between activated carboxylic acid groups of glutamic or aspartic acid residue of gelatin and amine groups of gelatin or/and chitosan.

E-Mail: hanna.staroszczyk@pg.gda.pl
Address: Department of Food Chemistry, Technology and Biotechnology, Gdansk University of Technology, G. Narutowicza 11/12, 80-233 Gdansk, Poland. Electronic address:

3915564d: J.Biol.Eng., 24-24(2013)
Link: [ PubMED ] [ DOI ]
Title: Secretion of polyhydroxybutyrate in Escherichia coli using a synthetic biological engineering approach.
Author: Rahman,A., Linton,E., Hatch,A.D., Sims,R.C., Miller,C.D.
Abstract:
BACKGROUND: Polyhydroxyalkanoates (PHAs) are a group of biodegradable plastics that are produced by a wide variety of microorganisms, mainly as a storage intermediate for energy and carbon. Polyhydroxybutyrate (PHB) is a short-chain-length PHA with interesting chemical and physical properties. Large scale production of PHB is not wide-spread mainly due to the downstream processing of bacterial cultures to extract the PHB. Secretion of PHB from Escherichia coli could reduce downstream processing costs. PHB are non-proteinaceous polymers, hence cannot be directly targeted for secretion. Phasin, PhaP1, is a lower molecular weight protein that binds to PHB, reducing PHB granule size. In this study PHB is indirectly secreted with PhaP1 from E. coli via type I secretion using HlyA signal peptides. RESULTS: This study demonstrated the successful secretion of Phasin and Phasin bound PHB outside of the cell and into the culture medium. The secretion of PHB was initiated between 24 and 48 h after induction. After 48 h of culturing, 36% of the total PHB produced in the secreting strain was collected in the secreted fraction and 64% remained in the internal fraction. To further support the findings of this study, the PHB secretion phenomenon was observed using SEM. CONCLUSIONS: From this study, the ability to use type I secretion to: 1) secrete phasin and 2) successfully secrete PHB has been shown.

3915565d: J.Neuroinflamm., 127-127(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Antimicrobial peptide β-defensin-1 expression is upregulated in Alzheimer's brain.
Author: Williams,W.M., Torres,S., Siedlak,S.L., Castellani,R.J., Perry,G., Smith,M.A., Zhu,X.
Abstract:
BACKGROUND: The human β-defensins (hBDs) are a highly conserved family of cationic antimicrobial and immunomodulatory peptides expressed primarily by epithelial cells in response to invasion by bacteria, fungi and some viruses. To date, the most studied members of this family of peptides are hBD-1, -2, and -3. Expression of hBD-1 and -2 has been demonstrated previously in cultured microglia and astrocytes of both mouse and human brain. Unlike inducible hBD-2 and -3, hBD-1 is constitutively expressed and is not generally upregulated by proinflammatory factors. In this study, we investigated whether hBDs, as active components of the innate immune response, are affected by pathological events in the Alzheimer's disease (AD) brain. We assessed the expression of hBD-1, -2, and -3 in tissue obtained at autopsy from AD and age-matched control brains. METHODS: Fixed and frozen choroid plexus and the CA1 region of the hippocampus were obtained at autopsy from individuals diagnosed with AD, or from age-matched control brains without diagnosed neurodegenerative disease. Histopathologically diagnosed AD brain tissue was obtained for our study. Immunocytochemical analysis was performed using affinity purified polyclonal antibodies directed against hBD-1, -2, or -3. TaqMan gene expression assays were used to quantify the mRNA of hBD-1, -2, and -3 in the choroid plexus and hippocampus. Immunocytochemical detection of iron deposits was achieved using a modified Perl's stain for redox-active iron. In vitro experiments were performed on human primary oral epithelial cells to model the human choroid plexus epithelial response to ferric chloride. Cells were then exposed to ferric chloride added to selected wells at 0, 1, or 10 mM concentrations for 24 h at 37[degree sign]C. Total mRNA was isolated to quantify hBD-1 mRNA expression by RTqPCR. RESULTS: hBD-1 peptide is apparent in astrocytes of the AD hippocampus and hippocampal neurons, notably within granulovacuolar degeneration structures (GVD). A higher level of hBD-1 was also seen in the choroid plexus of AD brain in comparison to age-matched control tissue. Increased expression of hBD-1 mRNA was observed only in the choroid plexus of the AD brain when compared to expression level in age-matched control brain. Redox-active iron was also elevated in the AD choroid plexus and in vitro addition of Fe+3Cl3 to cultured epithelial cells induced hBD-1 mRNA expression. CONCLUSIONS: Our findings suggest interplay between hBD-1 and neuroimmunological responses in AD, marked by microglial and astrocytic activation, and increased expression of the peptide within the choroid plexus and accumulation within GVD. As a constitutively expressed component of the innate immune system, we propose that hBD-1 may be of considerable importance early in the disease process. We also demonstrate that increased iron deposition in AD may contribute to the elevated expression of hBD-1 within the choroid plexus. These findings represent a potentially important etiological aspect of Alzheimer's disease neuropathology not previously reported.

3915567d: Bioanalysis, 2567-2580(2013)
Link: [ PubMED ] [ DOI ]
Title: Recent developments in microfluidic chip-based separation devices coupled to MS for bioanalysis.
Author: Lin,S.L., Lin,T.Y., Fuh,M.R.
Abstract:
In recent years, the development of microfluidic chip separation devices coupled to MS has dramatically increased for high-throughput bioanalysis. In this review, advances in different types of microfluidic chip separation devices, such as electrophoresis- and LC-based microchips, as well as 2D design of microfluidic chip-based separation devices will be discussed. In addition, the utilization of chip-based separation devices coupled to MS for analyzing peptides/proteins, glycans, drug metabolites and biomarkers for various bioanalytical applications will be evaluated.

Address: Department of Chemistry, Soochow University, No. 70, Linhsi Road, Shihlin, 11102 Taipei, Taiwan

3915568d: Bioanalysis, 2509-2520(2013)
Link: [ PubMED ] [ DOI ]
Title: Validation of hepcidin quantification in plasma using LC-HRMS and discovery of a new hepcidin isoform.
Author: Rochat,B., Peduzzi,D., McMullen,J., Favre,A., Kottelat,E., Favrat,B., Tissot,J.D., Angelillo-Scherrer,A., Bromirski,M., Waldvogel,S.
Abstract:
Background: Hepcidin, a 25 amino acid peptide, plays an important role in iron homeostasis. Some hepcidin truncated peptides have antibiotic effects. Results: A new analytical method for hepcidin determination in human plasma using LC-HRMS operating in full-scan acquisition mode has been validated. The extraction consists of protein precipitation and a drying reconstitution step; a 2.1 x 50 mm (idxL) C18 analytical column was used. Detection specificity, stability, accuracy, precision and recoveries were determined. The LOQ/LOD were 0.25/0.1 nM, respectively. More than 600 injections of plasma extracts were performed, allowing evaluation of the assay robustness. Hepcidin-20, hepcidin-22 and a new isoform, hepcidin-24, were detected in patients. Conclusion: The data underscore the usefulness of LC-HRMS for in-depth investigations related to hepcidin levels and pathways.

Address: Quantitative Mass Spectrometry Facility University of Lausanne -CHUV, Route du Bugnon, BH18-218, 1011 Lausanne, Switzerland

3915573d: Bull.Exp.Biol.Med., 370-372(2013)
Link: [ PubMED ] [ DOI ]
Title: Antimutagenic activity of wheat polypeptides in human cells exposed to cadmium chloride.
Author: Zasukhina,G.D., Vasilyeva,I.M., Kadnikov,I.A., Voronin,M.V., Odintsova,T.I., Korostileva,T.V., Pukhalskii,V.A.
Abstract:
Antimutagenic effects of polypeptides isolated from Triticum kiharae wheat plantule extracts have been studied on human cells exposed to cadmium chloride. The most effective polypeptide Tk-AMP-BP β -purothionin exhibited higher antimutagenic activity than wheat water extract and another peptide isolated from the same wheat species, Tk-AMP-γ 2 defensin; it also produced a pronounced antioxidant effect. This polypeptide can be used as a preventive agent for reducing the mutagenic potential of some environmental pollutants and for correction of human diseases associated with the defense system defects.

E-Mail: Zasukhina@vigg.ru
Address: N. I. Vavilov Institute of General Genetics, the Russian Academy of Sciences, Moscow, Russia

3915575d: PLoS One, e73386-e73386(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Alpha-Helical Destabilization of the Bcl-2-BH4-Domain Peptide Abolishes Its Ability to Inhibit the IP3 Receptor.
Author: Monaco,G., Decrock,E., Nuyts,K., Wagner,L.E.II, Luyten,T., Strelkov,S.V., Missiaen,L., De Borggraeve,W.M., Leybaert,L., Yule,D.I., De Smedt,H., Parys,J.B., Bultynck,G.
Abstract:
The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the primary Ca2+-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca2+ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine "hinges" replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.

Address: Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Leuven, Belgium

3915577d: Exp.Ther.Med., 1049-1053(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Recombinant expression and downstream processing of the disulfide-rich tumor-targeting peptide chlorotoxin.
Author: Wang,X.M., Luo,X., Guo,Z.Y.
Abstract:
Chlorotoxin (CTX) is a scorpion-derived disulfide-rich peptide that targets malignant tumors by binding the cell surface matrix metalloproteinase-2 and annexin A2. Various CTXs labeled with functional moieties have shown great potential for tumor diagnosis and treatment. In the present study, we established an efficient approach for preparing mature CTX that may be used for experimental and therapeutic purposes. The designed CTX precursors carried either a 6xHis-tag or a 6xHis-tag and a glutathione transferase (GST)-tag and were recombinantly expressed in Escherichia coli. Following S-sulfonation, the precursors were purified using immobilized metal-ion affinity chromatography. Subsequent to the removal of the tag by enterokinase cleavage and in vitro oxidative refolding, mature CTX was obtained with a considerable yield. The yield of mature CTX whose precursors carried a 6xHis-tag and a GST-tag (2 mg per liter of culture) was approximately 10-fold that of the mature CTX whose precursors carried a 6xHis-tag (150-200 μg per liter of culture). The folded CTX inhibited the migration of glioma cells in a concentration-dependent manner, suggesting it was biologically active.

Address: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, P.R. China

3915578d: Exp.Ther.Med., 1000-1004(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Synergistic effect of clinically used antibiotics and peptide antibiotics against Gram-positive and Gram-negative bacteria.
Author: Zhou,Y., Peng,Y.
Abstract:
Ribosomally synthesized (natural) peptides demonstrate antimicrobial potency and may represent a novel therapeutic approach for the treatment of infections. The aim of the present study was to investigate the interaction between polycationic peptides and clinically used antimicrobial agents in the treatment of clinical isolates of Gram-positive and Gram-negative aerobic bacteria in vitro, using the microbroth dilution method. The combination studies demonstrated synergies between ranalexin and polymyxin E, doxycycline and clarithromycin. Similarly, magainin II was demonstrated to be synergistic with ceftriaxone, amoxicillin clavulanate, ceftazidime, meropenem, piperacillin and β-lactam antibiotics. Buforin II, cecropin P1 and indolicidin were not observed to be synergistic with the clinically used antibiotics, but demonstrated additive effects with them. Notably, no antagonistic effects were identified in all the combinations examined.

Address: Department of Respiratory Medicine, Xinxiang Central Hospital of Henan Province, Xinxiang, Henan 453000, P.R. China

3915584d: Exp.Ther.Med., 611-615(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Reduction of leptin levels by four cardiac hormones: Implications for hypertension in obesity.
Author: Lane,M.L., Vesely,D.L.
Abstract:
Circulating levels of leptin are increased in obesity and have been proposed to contribute to the development of hypertension in obese individuals. Four cardiac hormones, specifically, vessel dilator, long-acting natriuretic peptide (LANP), kaliuretic peptide and atrial natriuretic peptide (ANP), have blood pressure-lowering properties and correlate with the presence of hypertension in obesity. The objective of this study was to determine whether one or more of these cardiac hormones was able to decrease the levels of leptin in the hypothalamus, an area of the brain that has been demonstrated to synthesize more than 40% of leptin in the circulation. The effects of these four cardiac hormones on leptin were examined using dose-response curves in the rat hypothalamus, which synthesizes leptin. Vessel dilator, LANP, kaliuretic peptide and ANP maximally decreased the levels of leptin in hypothalamic cells by 79, 76, 80 and 62%, respectively (P<0.0001 for each). The cardiac hormones decreased leptin levels over a concentration range of 100 pM to 10 μM, with the most significant reductions in leptin levels occurring when the concentrations of the hormones were at micromolar levels. The results of the study suggest that the four cardiac hormones lead to significant reductions in hypothalamic leptin levels, which may be an important mechanism for alleviating leptin-induced hypertension in obesity.

Address: Departments of Medicine, Molecular Pharmacology and Physiology, and James A. Haley VA Medical Center, University of South Florida Morsani School of Medicine, Tampa, FL 33612, USA

3915592d: PLoS One, e72242-e72242(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: A tumor-penetrating peptide modification enhances the antitumor activity of thymosin α 1.
Author: Lao,X., Liu,M., Chen,J., Zheng,H.
Abstract:
A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC) fragments, would enhance the activity of thymosin α 1 (Tα1). The modified Tα1 (Tα1-iRGD) was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activity as Tα1 in promoting proliferation of mouse splenocytes. Meanwhile, cell adhesion analysis revealed that Tα1-iRGD exhibited more specific and greater binding with tumor cells compared with Tα1. Furthermore, the iRGD fragment evidently enhanced the basal ability of Tα1 to inhibit proliferation of cancer cells in vitro, particularly of mouse melanoma cell line B16F10 and human lung cancer cell line H460. Our findings indicated that the addition of an iRGD fragment increased the anti-proliferative activity of Tα1 against cancer cells by improving the ability of Tα1 to penetrate the tumor cells. This study highlighted the important roles of an iRGD sequence in the therapeutic strategy of Tα1-iRGD. Thus, Tα1-iRGD could be a novel drug candidate for cancer treatment.

Address: Department of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiang Su Province, PR China

3915593d: PLoS One, e72177-e72177(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The amyloid precursor protein (APP) does not have a ferroxidase site in its E2 domain.
Author: Honarmand Ebrahimi,K., Dienemann,C., Hoefgen,S., Than,M.E., Hagedoorn,P.-L., Hagen,W.R.
Abstract:
The ubiquitous 24-meric iron-storage protein ferritin and multicopper oxidases such as ceruloplasmin or hephaestin catalyze oxidation of Fe(II) to Fe(III), using molecular oxygen as oxidant. The ferroxidase activity of these proteins is essential for cellular iron homeostasis. It has been reported that the amyloid precursor protein (APP) also has ferroxidase activity. The activity is assigned to a ferroxidase site in the E2 domain of APP. A synthetic 22-residue peptide that carries the putative ferroxidase site of E2 domain (FD1 peptide) has been claimed to encompass the same activity. We previously tested the ferroxidase activity of the synthetic FD1 peptide but we did not observe any activity above the background oxidation of Fe(II) by molecular oxygen. Here we used isothermal titration calorimetry to study Zn(II) and Fe(II) binding to the natural E2 domain of APP, and we employed the transferrin assay and oxygen consumption measurements to test the ferroxidase activity of the E2 domain. We found that this domain neither in the presence nor in the absence of the E1 domain binds Fe(II) and it is not able to catalyze the oxidation of Fe(II). Binding of Cu(II) to the E2 domain did not induce ferroxidase activity contrary to the presence of redox active Cu(II) centers in ceruloplasmin or hephaestin. Thus, we conclude that E2 or E1 domains of APP do not have ferroxidase activity and that the potential involvement of APP as a ferroxidase in the pathology of Alzheimer's disease must be re-evaluated.

Address: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

3915595d: PLoS One, e71933-e71933(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Impact of Hfq on global gene expression and intracellular survival in Brucella melitensis.
Author: Cui,M., Wang,T., Xu,J., Ke,Y., Du,X., Yuan,X., Wang,Z., Gong,C., Zhuang,Y., Lei,S., Su,X., Wang,X., Huang,L., Zhong,Z., Peng,G., Yuan,J., Chen,Z., Wang,Y.
Abstract:
Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in B. melitensis. A B. melitensis hfq deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the hfq mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the hfq mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of Brucella to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated hfq mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of Brucella gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of B. melitensis.

Address: Department of Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China

3915597d: PLoS One, e71646-e71646(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.
Author: Takakuma,K., Ogo,N., Uehara,Y., Takahashi,S., Miyoshi,N., Asai,A.
Abstract:
Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Address: Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan

3915599d: PLoS One, e71509-e71509(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Genome-wide association study reveals genetic architecture of eating behavior in pigs and its implications for humans obesity by comparative mapping.
Author: Do,D.N., Strathe,A.B., Ostersen,T., Jensen,J., Mark,T., Kadarmideen,H.N.
Abstract:
This study was aimed at identifying genomic regions controlling feeding behavior in Danish Duroc boars and its potential implications for eating behavior in humans. Data regarding individual daily feed intake (DFI), total daily time spent in feeder (TPD), number of daily visits to feeder (NVD), average duration of each visit (TPV), mean feed intake per visit (FPV) and mean feed intake rate (FR) were available for 1130 boars. All boars were genotyped using the Illumina Porcine SNP60 BeadChip. The association analyses were performed using the GenABEL package in the R program. Sixteen SNPs were found to have moderate genome-wide significance (p<5E-05) and 76 SNPs had suggestive (p<5E-04) association with feeding behavior traits. MSI2 gene on chromosome (SSC) 14 was very strongly associated with NVD. Thirty-six SNPs were located in genome regions where QTLs have previously been reported for behavior and/or feed intake traits in pigs. The regions: 64-65 Mb on SSC 1, 124-130 Mb on SSC 8, 63-68 Mb on SSC 11, 32-39 Mb and 59-60 Mb on SSC 12 harbored several signifcant SNPs. Synapse genes (GABRR2, PPP1R9B, SYT1, GABRR1, CADPS2, DLGAP2 and GOPC), dephosphorylation genes (PPM1E, DAPP1, PTPN18, PTPRZ1, PTPN4, MTMR4 and RNGTT) and positive regulation of peptide secretion genes (GHRH, NNAT and TCF7L2) were highly significantly associated with feeding behavior traits. This is the first GWAS to identify genetic variants and biological mechanisms for eating behavior in pigs and these results are important for genetic improvement of pig feed efficiency. We have also conducted pig-human comparative gene mapping to reveal key genomic regions and/or genes on the human genome that may influence eating behavior in human beings and consequently affect the development of obesity and metabolic syndrome. This is the first translational genomics study of its kind to report potential candidate genes for eating behavior in humans.

Address: Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

3915600d: PLoS One, e71383-e71383(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Single domain antibody multimers confer protection against rabies infection.
Author: Boruah,B.M., Liu,D., Ye,D., Gu,T.J., Jiang,C.L., Qu,M., Wright,E., Wang,W., He,W., Liu,C., Gao,B.
Abstract:
Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naive llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90-95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection.

Address: CAS Key Laboratory for Pathogenic Microbiology and Immunology (CASPMI), Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

3915602d: PLoS One, e70918-e70918(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Collagen peptides in urine: a new promising biomarker for the detection of colorectal liver metastases.
Author: Broker,M.E., Lalmahomed,Z.S., Roest,H.P., Van Huizen,N.A., Dekker,L.J., Calame,W., Verhoef,C., Ijzermans,J.N., Luider,T.M.
Abstract:
INTRODUCTION: For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls. MATERIALS AND METHODS: Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM's) were developed on a UPLC-MS/MS system. RESULTS: Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%. CONCLUSION: Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner.

Address: Department of Surgery, Division of Transplantation and Hepatobiliary Surgery, Erasmus University Medical Center, Rotterdam, The Netherlands

3915603d: PLoS One, e69228-e69228(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Disruption of hydrogen bonds between major histocompatibility complex class II and the peptide N-terminus is not sufficient to form a human leukocyte antigen-DM receptive state of major histocompatibility complex class II.
Author: Schulze,M.S., Anders,A.K., Sethi,D.K., Call,M.J.
Abstract:
Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306-318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.

Address: Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America

3915616d: Protein J., 484-492(2013)
Link: [ PubMED ] [ DOI ]
Title: Thymosin β4 and tissue transglutaminase. molecular characterization of cyclic thymosin β4.
Author: App,C., Knop,J., Huff,T., Sticht,H., Hannappel,E.
Abstract:
Thymosin β4 is the prototype of β-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an ε-amino group of thymosin β4 serves also as an aminyl substrate and an intramolecular bond is formed concomitantly NH3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of thymosin β4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming isopeptide bond. In spite of 3 glutaminyl and 9 lysyl residues of thymosin β4 only one isopeptide bond between Lys16 and Gln36 was formed (cyclic thymosin β4). These two amino acid residues are conserved in all β-thymosins. Cyclic thymosin β4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the thymosin β4 x G-actin complex.

E-Mail: thymosin@biochem.uni-erlangen.de
Address: Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-University, Fahrstr.17, 91054, Erlangen, Germany

3915617d: Nat.Methods, 989-991(2013)
Link: [ PubMED ] [ DOI ]
Title: DMSO enhances electrospray response, boosting sensitivity of proteomic experiments.
Author: Hahne,H., Pachl,F., Ruprecht,B., Maier,S.K., Klaeger,S., Helm,D., Medard,G., Wilm,M., Lemeer,S., Kuster,B.
Abstract:
We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware or software and at no additional cost.

Address: Chair of Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universitat Munchen, Freising, Germany

3915621d: Biotechnol.Lett., 1831-1838(2013)
Link: [ PubMED ] [ DOI ]
Title: Proteome profile of bovine ruminal epithelial tissue based on GeLC-MS/MS.
Author: Yang,Y., Wang,J., Yuan,T., Bu,D., Yang,J., Sun,P.
Abstract:
The proteome of rumen epithelial tissue was analysed by SDS-PAGE coupled with LC-MS/MS. 813 non-redundant proteins were identified of which 7.4 % featured membrane-spanning domains and 15.4 % harboured a signal peptide. According to the gene ontology annotation, the most abundant proteins exhibited binding activities related to their molecular functions, were proteins of cellular components or belonged to various metabolic processes. A predominant group of canonical pathways in the rumen epithelial tissue was identified using the IPA software. The GeLC-MS/MS approach was used to characterise the entire protein expression repertoire in rumen tissue, providing a more detailed understanding of the important biological processes in the rumen.

Address: State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

3915622d: Plant Physiol.Biochem.(Paris), 247-253(2013)
Link: [ PubMED ] [ DOI ]
Title: Globular body production, their anatomy, DNase gel analysis and NDP kinase activity in root tips of Poncirus trifoliata L.
Author: Tzatzani,T.T., Dimassi-Theriou,K., Yupsanis,T., Bosabalidis,A., Therios,I., Sarropoulou,V.
Abstract:
Green globular bodies were developed from Poncirus trifoliata L. root tip explants as a response to addition in the substrate of different growth regulators. From the globular bodies, shoots initiated and grew. Median section of the globular bodies reveals that they are composed of parenchyma cells and originate from the pericycle. The activity of DNases during shoot formation from globular bodies was influenced by the type and concentration of plant growth regulators that were added in the nutrient substrate. Peptide bands formation was also influenced by the increase of BA concentration. Consequently, BA, NAA and IAA combination influenced 5'-triphosphonucleosides (NTPs) appearance and activity in the presence of metal. Peptide bands resulted from the electrophoretic analysis of endogenous protein phosphorylation, proved to be catalytic subunits of NDP kinases, as they all phosphorylate diphosphonucleosides. The enzymes DNases and NDP kinases could be used as a scientific tool for the study of shoot formation from P. trifoliata L. green globular bodies.

Address: Department of Horticulture, Laboratory of Pomology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

3915626d: Appl.Environ.Microbiol., 6637-6646(2013)
Link: [ PubMED ] [ DOI ]
Title: Stationary Phase and Nutrient Levels Trigger Transcription of a Genomic Locus Containing a Novel Peptide (TM1316) in the Hyperthermophilic Bacterium Thermotoga maritima.
Author: Frock,A.D., Montero,C.I., Blumer-Schuette,S.E., Kelly,R.M.
Abstract:
The genome of the hyperthermophilic bacterium Thermotoga maritima encodes numerous putative peptides/proteins of 100 amino acids or less. While most of these open reading frames (ORFs) are transcribed during growth, their corresponding physiological roles are largely unknown. The onset of stationary phase in T. maritima was accompanied by significant morphological changes and upregulation of several ORFs located in the TM1298-TM1336 genome locus. This region contains putative HicAB toxin-antitoxin pairs, hypothetical proteins, radical S-adenosylmethionine (SAM) enzymes, and ABC transporters. Of particular note was the TM1315-TM1319 operon, which includes a putative 31-amino-acid peptide (TM1316) that was the most highly transcribed gene in the transcriptome during stationary phase. Antibodies directed against a synthetic version of TM1316 were used to track its production, which correlated closely with transcriptomic data. Immunofluorescence microscopy revealed that TM1316 was localized to the cell envelope and prominent in cell aggregates formed during stationary phase. The only functionally characterized locus with an organization similar to that of TM1315-TM1319 is in Bacillus subtilis, which contains subtilosin A, a cyclic peptide with Cys-to-α-carbon linkages that functions as an antilisterial bacteriocin. While the organization of TM1316 resembled that of the Bacillus peptide (e.g., in its number of amino acids and spacing of Cys residues), preparations containing high levels of TM1316 affected the growth of neither Thermotoga species nor Pyrococcus furiosus, a hyperthermophilic archaeon isolated from the same locale as T. maritima. Several other putative Cys-rich peptides could be identified in the TM1298-TM1336 locus, and while their roles are also unclear, they merit examination as potential antimicrobial agents in hyperthermophilic biotopes.

Address: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA

3915627d: Appl.Environ.Microbiol., 6670-6676(2013)
Link: [ PubMED ] [ DOI ]
Title: Bimodular Peptide Synthetase SidE Produces Fumarylalanine in the Human Pathogen Aspergillus fumigatus.
Author: Steinchen,W., Lackner,G., Yasmin,S., Schrettl,M., Dahse,H.M., Haas,H., Hoffmeister,D.
Abstract:
The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[32P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Using full-length holo-SidE, fumarylalanine (FA) formation was observed in vitro. Furthermore, FA was identified in A. fumigatus culture supernatants under inducing conditions, unless sidE was genetically inactivated. As FA is structurally related to established pharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulence of A. fumigatus.

Address: Department of Pharmaceutical Biology at the Hans-Knoll-Institute, Friedrich-Schiller-Universitat, Jena, Germany

3915628d: Bioresour.Technol., 543-548(2013)
Link: [ PubMED ] [ DOI ]
Title: High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements.
Author: Zhang,J., Kang,Z., Ling,Z., Cao,W., Liu,L., Wang,M., Du,G., Chen,J.
Abstract:
The present work aims to construct a robust recombinant Bacillus subtilis to achieve secretory production of alkaline polygalacturonate lyase (PGL). First, 6 signal peptides (amyX, bpr, vpr, yvgO, wapA and nprE) were screened with a semi-rational approach and comparatively investigated their effects on the production of PGL. The signal peptide bpr directed efficient PGL secretory expression and increased PGL titer to 313.7 U mL(-1). By optimizing and applying strong promoter P43 and Shine-Dalgarno sequence, higher titer of 446.3 U mL(-1) PGL was achieved. Finally, the capacity of the recombinant B. subtilis WB43CB was evaluated with a fed-batch strategy in 3 L fermentor. The PGL titer reached 632.6 U mL(-1) with a productivity of 17.6 U mL(-1) h(-1), which was the highest secretory production of PGL by the B. subtilis system. The recombinant B. subtilis strain WB43CB constructed in the present work has great potential in production of alkaline PGL.

Address: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

3915633d: J.Inorg.Biochem., 99-106(2013)
Link: [ PubMED ] [ DOI ]
Title: Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering: active sequence optimization.
Author: Protas,A.M., Ariani,H.H., Bonna,A., Polkowska-Nowakowska,A., Poznanski,J., Bal,W.
Abstract:
In previous studies we showed that Ni(II) ions can hydrolytically cleave a peptide bond preceding Ser/Thr in peptides of a general sequence RN-(Ser/Thr)-Xaa-His-Zaa-RC, where RN and RC are any peptide sequences. A peptide library screening, assisted by accurate measurements of reaction kinetics for selected peptides, demonstrated the preference for bulky and aromatic residues at variable positions Xaa and Zaa [A. Krezel, E. Kopera, A.M. Protas, A. Wyslouch-Cieszynska, J. Poznanski, W. Bal, J. Am. Chem. Soc., 132 (2010) 3355-3366]. In this work we used a similar strategy to find out whether the next residue downstream to Zaa may influence the reaction rate. Using an Ac-Gly-Ala-Ser-Arg-His-Zaa-Baa-Arg-Leu-NH2 library, with Zaa and Baa positions containing all common amino acids except of Cys, we found a very strong preference for aromatic residues in both variable positions. This finding significantly limits the range of useful Xaa, Zaa and Baa substitutions, thus facilitating the search for optimal sequences for protein engineering applications [E. Kopera, A. Belczyk-Ciesielska, W. Bal, PLoS One 7 (2012) e36350].

Address: Lodz Regional Park of Science and Technology Ltd., 114/116 Dubois Street, 93-465 Lodz, Poland

3915641d: Chem.Biol., 1179-1186(2013)
Link: [ PubMED ] [ DOI ]
Title: Chemical Activators of Protein Phosphatase-1 Induce Calcium Release inside Intact Cells.
Author: Reither,G., Chatterjee,J., Beullens,M., Bollen,M., Schultz,C., Kohn,M.
Abstract:
Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase that is involved in numerous cellular processes. PP1-disrupting peptides (PDPs) are selective chemical tools used to study PP1. They generate catalytically active PP1 inside cells but do not bind to the closely related PP2A. Here, we show that PDPs also do not act directly on PP2B, thus demonstrating the selectivity of PDPs toward PP1. We present PDPs with different properties, enabling reversible versus permanent activation of PP1. We also show that Ca2+ spiking is an acute effect caused by PDP-induced activation of PP1. The Ca2+ is released from internal stores. Our data show that PDPs can be used as selective chemical genetics tools to study acute and long-term effects of PP1 activation in intact cells, and PDPs will therefore be valuable tools to study PP1 biology.

Address: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany

3915652d: Structure, 1769-1777(2013)
Link: [ PubMED ] [ DOI ]
Title: Transient Domain Interactions Enhance the Affinity of the Mitotic Regulator Pin1 toward Phosphorylated Peptide Ligands.
Author: Matena,A., Sinnen,C., Van den Boom,J., Wilms,C., Dybowski,J.N., Maltaner,R., Mueller,J.W., Link,N.M., Hoffmann,D., Bayer,P.
Abstract:
The mitotic regulator Pin1 plays an important role in protein quality control and age-related medical conditions such as Alzheimer disease and Parkinson disease. Although its cellular role has been thoroughly investigated during the past decade, the molecular mechanisms underlying its function remain elusive. We provide evidence for interactions between the two domains of Pin1. Several residues displayed unequivocal peak splits in nuclear magnetic resonance spectra, indicative of two different conformational states in equilibrium. Pareto analysis of paramagnetic relaxation enhancement data demonstrates that the two domains approach each other upon addition of a nonpeptidic ligand. Titration experiments with phosphorylated peptides monitored by fluorescence anisotropy and chemical shift perturbation indicate that domain interactions increase Pin1's affinity toward peptide ligands. We propose this interplay of the domains and ligands to be a general mechanism for a large class of two-domain proteins.

Address: Research Group Structural and Medicinal Biochemistry, ZMB, University of Duisburg-Essen, 45117 Essen, Germany

3915663d: BioMed Res.Int., 280432-280432(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Secretion of biologically active heterologous oxalate decarboxylase (OxdC) in Lactobacillus plantarum WCFS1 using homologous signal peptides.
Author: Sasikumar,P., Gomathi,S., Anbazhagan,K., Selvam,G.S.
Abstract:
Current treatment options for patients with hyperoxaluria and calcium oxalate stone diseases are limited and do not always lead to sufficient reduction in urinary oxalate excretion. Oxalate degrading bacteria have been suggested for degrading intestinal oxalate for the prevention of calcium oxalate stone. Here, we reported a recombinant Lactobacillus plantarum WCFS1 (L. plantarum) secreting heterologous oxalate decarboxylase (OxdC) that may provide possible therapeutic approach by degrading intestinal oxalate. The results showed secretion and functional expression of OxdC protein in L. plantarum driven by signal peptides Lp_0373 and Lp_3050. Supernatant of the recombinant strain containing pLp_0373sOxdC and pLp_3050sOxdC showed OxdC activity of 0.05 U/mg and 0.02 U/mg protein, while the purified OxdC from the supernatant showed specific activity of 18.3 U/mg and 17.5 U/mg protein, respectively. The concentration of OxdC protein in the supernatant was 8-12 μg/mL. The recombinant strain showed up to 50% oxalate reduction in medium containing 10 mM oxalate. In conclusion, the recombinant L. plantarum harboring pLp_0373sOxdC and pLp_3050sOxdC can express and secrete functional OxdC and degrade oxalate up to 50% and 30%, respectively.

Address: Department of Biochemistry, Centre for Advanced Studies in Organismal and Functional Genomics, School of Biological Sciences, Madurai Kamaraj University, Madurai, India

3915664d: Oxidative Med.Cell.Longevity, 932472-932472(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Thiol redox sensitivity of two key enzymes of heme biosynthesis and pentose phosphate pathways: uroporphyrinogen decarboxylase and transketolase.
Author: McDonagh,B., Pedrajas,J.R., Padilla,C.A., Barcena,J.A.
Abstract:
Uroporphyrinogen decarboxylase (Hem12p) and transketolase (Tkl1p) are key mediators of two critical processes within the cell, heme biosynthesis, and the nonoxidative part of the pentose phosphate pathway (PPP). The redox properties of both Hem12p and Tkl1p from Saccharomyces cerevisiae were investigated using proteomic techniques (SRM and label-free quantification) and biochemical assays in cell extracts and in vitro with recombinant proteins. The in vivo analysis revealed an increase in oxidized Cys-peptides in the absence of Grx2p, and also after treatment with H2O2 in the case of Tkl1p, without corresponding changes in total protein, demonstrating a true redox response. Out of three detectable Cys residues in Hem12p, only the conserved residue Cys52 could be modified by glutathione and efficiently deglutathionylated by Grx2p, suggesting a possible redox control mechanism for heme biosynthesis. On the other hand, Tkl1p activity was sensitive to thiol redox modification and although Cys622 could be glutathionylated to a limited extent, it was not a natural substrate of Grx2p. The human orthologues of both enzymes have been involved in certain cancers and possess Cys residues equivalent to those identified as redox sensitive in yeast. The possible implication for redox regulation in the context of tumour progression is put forward.

Address: Department of Biochemistry and Molecular Biology, University of Cordoba and Cordoba Maimonides Institute for Biomedical Research (IMIBIC), 14071 Cordoba, Spain

3915665d: Oxidative Med.Cell.Longevity, 414393-414393(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: 50 Hz electromagnetic field produced changes in FTIR spectroscopy associated with mitochondrial transmembrane potential reduction in neuronal-like SH-SY5Y cells.
Author: Calabro,E., Condello,S., Curro,M., Ferlazzo,N., Vecchio,M., Caccamo,D., Magazu,S., Ientile,R.
Abstract:
SH-SY5Y neuroblastoma cells were used as an experimental model to study the effects of 50 Hz electromagnetic field, in the range from 50 micro T to 1.4 mT. Fourier transform infrared spectroscopy analysis evidenced a reduction in intensity of the amide A band and a slight increase of vibration bands at 2921 cm-1 and 2853 cm-1 corresponding to methylene groups. A further increase of the magnetic field intensity of exposure up to 0.8 mT and 1.4 mT produced a clear increase in intensity of CH2 vibration bands. Moreover, it has been observed some alterations in the amide I region, such as a shifted peak of the amide I band to a smaller wavenumber, probably due to protein conformational changes. These results suggested that exposure to extremely low electromagnetic fields influenced lipid components of cellular membrane and the N-H in-plane bending and C-N stretching vibrations of peptide linkages, modifying the secondary structures of α -helix and β -sheet contents and producing unfolding process in cell membrane proteins. The observed changes after exposure to 50 Hz electromagnetic field higher than 0.8 mT were associated with a significant reduction of cell viability and reduced mitochondrial transmembrane potential.

Address: Department of Physics, University of Messina, 98165 Messina, Italy

3915667d: Int.J.Pept., 590329-590329(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Antigenic Peptides Capable of Inducing Specific Antibodies for Detection of the Major Alterations Found in Type 2B Von Willebrand Disease.
Author: Paro,M.de O., Ferreira,C.S., Vieira,F.S., De Santana,M.A., Castro-Borges,W., Namen-Lopes,M.S.S., Leclercq,S.Y., Velloso-Rodrigues,C., Guerra de Andrade,M.H.
Abstract:
Von Willebrand disease (VWD) is an inherited hemorrhagic disorder promoted by either quantitative or qualitative defects of the von Willebrand factor (VWF). The disease represents the most common human coagulopathy afflicting 1.3% of the population. Qualitative defects are subdivided into four subtypes and classified according to the molecular dysfunction of the VWF. The differential diagnosis of the VWD is a difficult task, relying on a panel of tests aimed to assess the plasma levels and function of the VWF. Here, we propose biochemical approaches for the identification of structural variants of the VWF. A bioinformatic analysis was conducted to design seven peptides among which three were representatives of specific amino acid sequences belonging to normal VWF and four encompassed sequences found in the most common VWD subtype 2B. These peptides were used to immunize mice, after which, peptide-specific immunoglobulins were purified. This resulted in four Ig preparations capable of detecting alterations in the subtype 2B VWD plus additional three antibody fractions targeting the normal VWF. The panel of antibodies could serve many applications among them (1) assessment of VWF: antigen interaction, (2) VWF multimer analysis, and (3) production of monoclonal antibodies against VWF for therapeutic purposes as in thrombotic thrombocytopenic purpura.

Address: Departamento de Ciencias Biologicas/DECBI, Instituto de Ciencias Exatas e Biologicas/ICEB, Universidade Federal de Ouro Preto, Nucleo de Pesquisas em Ciencias Biologicas/NUPEB, Campus Universitario Morro do Cruzeiro, 35400-000 Ouro Preto, MG, Brazil

3915671d: Proteomics, 3040-3045(2013)
Link: [ PubMED ] [ DOI ]
Title: Proteomic identification of Listeria monocytogenes surface-associated proteins.
Author: Zhang,C.X., Creskey,M.C., Cyr,T.D., Brooks,B., Huang,H., Pagotto,F., Lin,M.
Abstract:
This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano-LC and online-MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy-four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface-exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.

Address: Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, ON, Canada

3915672d: Proteomics, 2976-2979(2013)
Link: [ PubMED ] [ DOI ]
Title: Development of a novel assay for proprotein converting enzyme activity on a multiplex bead-based array system.
Author: Albrecht,A., Rahmoune,H., Leedjarv,K., Knorpp,T., Joos,T., Stocki,P., Guest,P.C., Bahn,S.
Abstract:
We describe the development of a novel, robust assay system for determining the changes in activity of proprotein converting enzymes. An assay for prolyl oligopeptidase (POP) activity was constructed using a peptide-streptavidin substrate coupled to magnetic microspheres and cleavage was detected by loss of streptavidin on the MAGPIX reader. Test analysis of postmortem pituitary extracts from schizophrenia patients showed an increase in POP activity compared to controls. The results were validated using both fluorometric and Western blot analyses for POP activity and immunoreactivity, respectively. The assays can be multiplexed for measuring the activity of multiple proprotein cleaving enzymes simultaneously in laboratory and clinical settings and should add valuable new information for conditions such as neuropsychiatric diseases, diabetes, endocrine dysfunction, and cancer, where effects on proteolysis of biologically active peptides play a key role.

Address: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK

3915674d: Proteomics, 2989-2997(2013)
Link: [ PubMED ] [ DOI ]
Title: Probing the acetylation code of histone H4.
Author: Lang,D., Schumann,M., Gelato,K., Fischle,W., Schwarzer,D., Krause,E.
Abstract:
Histone modifications play crucial roles in genome regulation with lysine acetylation being implicated in transcriptional control. Here we report a proteome-wide investigation of the acetylation-dependent protein-protein interactions of the N-terminal tail of histone H4. Quantitative peptide-based affinity MS experiments using the SILAC approach determined the interactomes of H4 tails monoacetylated at the four known acetylation sites K5, K8, K12, and K16, bis-acetylated at K5/K12, triple-acetylated at K8/12/16 and fully tetra-acetylated. A set of 29 proteins was found enriched on the fully acetylated H4 tail while specific binders of the mono and bis-acetylated tails were barely detectable. These observations are in good agreement with earlier reports indicating that the H4 acetylation state establishes its regulatory effects in a cumulative manner rather than via site-specific recruitment of regulatory proteins.

Address: Laboratory of Mass Spectrometry, Leibniz-Institut fur Molekulare Pharmakologie, Berlin, Germany

3915678d: Nanoscale, 8973-8976(2013)
Link: [ PubMED ] [ DOI ]
Title: Peptide functionalized gold nanoparticles for colorimetric detection of matrilysin (MMP-7) activity.
Author: Chen,P., Selegard,R., Aili,D., Liedberg,B.
Abstract:
A peptide with two cleavage sites for MMP-7 has been synthesized and immobilized on gold nanoparticles (AuNPs) through a cysteine residue. Digestion of the peptide by MMP-7 decreases its size and net charge, which leads to the aggregation of the AuNPs. The color shift caused by aggregation enables a direct and quantitative measurement of the concentration and activity of MMP-7 with an estimated limit of detection of approximately 5 nM (0.1 μg mL(-1)).

E-Mail: bliedberg@ntu.edu.sg
Address: Centre for Biomimetic Sensor Science, Nanyang Technological University, 50 Nanyang Drive, Singapore 637553, Singapore

3915686d: Biomaterials, 8979-8990(2013)
Link: [ PubMED ] [ DOI ]
Title: A DNA microarray-based analysis of immune-stimulatory and transcriptional responses of dendritic cells to KALA-modified nanoparticles.
Author: Akita,H., Ishii,S., Miura,N., Shaheen,S.M., Hayashi,Y., Nakamura,T., Kaji,N., Baba,Y., Harashima,H.
Abstract:
Technologies for the transfection of antigen-encoding genes into the dendritic cells, and subsequent immune-activation are both prerequisites for a successful DNA vaccine. We herein report on the density-dependent enhancement of transgene expression by the simple modification by stearyl-conjugated KALA, an α-helical peptide (STR-KALA), onto a lipid envelope-type nanoparticle (the R8-MEND, an octaarginine-modified multifunctional envelope-type nano device). The enhanced transgene expression in the KALA-modified R8-MEND (R8/KALA-MEND) cannot be explained by cellular uptake and nuclear delivery efficacy. Thus, the post-nuclear delivery process (i.e. transcription), but not intracellular trafficking processes attributed the enhanced transfection efficacy. Microarray analyses revealed that transfection with the R8/KALA-MEND resulted in a greater perturbation in host genes expression in comparison with the R8-MEND and that this effect was time-dependent. Further pathway analyses in the category of transcription-related genes and a gene ontology analysis indicated that the R8/KALA-MEND stimulated the expression of transcription factors that are closely related to immune-activation (i.e. NF-kB and STAT). Inhibition of the transfection efficacy by blockage of the STAT pathways revealed that the enhanced transcription activity is the result of immune-stimulation. Collectively, the R8/KALA-MEND mounts a "switch-on" function that triggers signal transduction forward to the immune-stimulation analogous to an adjuvant, and consequently elicits active transcription.

E-Mail: akita@pharm.hokudai.ac.jp
Address: Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan

3915690d: Anal.Chem., 8513-8517(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Simultaneous light-directed synthesis of mirror-image microarrays in a photochemical reaction cell with flare suppression.
Author: Sack,M., Kretschy,N., Rohm,B., Somoza,V., Somoza,M.M.
Abstract:
The use of photolabile protecting groups is a versatile and well-established means of synthesizing high complexity microarrays of biopolymers, such as nucleic acids and peptides, for high-throughput analysis. The synthesis takes place in a photochemical reaction cell which positions the microarray substrate at the focus of the optical system delivering the light and which can be connected to a fluidics system which delivers appropriate reagents to the surface in synchrony with the light exposure. Here we describe a novel photochemical reaction cell which allows for the simultaneous synthesis of microarrays on two substrates. The reaction cell positions both substrates within the limited depth-of-focus of the optical system while maintaining the necessary reagent flow conditions. The resulting microarrays are mirror images of each other but otherwise essentially identical. The new reaction cell doubles the throughput of microarray synthesis without increasing the consumption of reagents. In addition, a secondary flow chamber behind the reaction cell can be filled with an absorbent and index-matching fluid to eliminate reflections from light exiting the reaction cell assembly, greatly reducing unintended light exposure that reduces the sequence fidelity of the microarray probes.

Address: Institute of Inorganic Chemistry, University of Vienna , Wahringer Strasse 42, A-1090 Vienna, Austria

3915698d: Anal.Chem., 8498-8502(2013)
Link: [ PubMED ] [ DOI ]
Title: Electrospray ionization from nanopipette emitters with tip diameters of less than 100 nm.
Author: Yuill,E.M., Sa,N., Ray,S.J., Hieftje,G.M., Baker,L.A.
Abstract:
Work presented here demonstrates application of nanopipettes pulled to orifice diameters of less than 100 nm as electrospray ionization emitters for mass spectrometry. Mass spectrometric analysis of a series of peptides and proteins electrosprayed from pulled-quartz capillary nanopipette emitters with internal diameters ranging from 37 to 70 nm is detailed. Overall, the use of nanopipette emitters causes a shift toward the production of ions of higher charge states and leads to a reduction in width of charge-state distribution as compared to typical nanospray conditions. Further, nanopipettes show improved S/N and the same signal precision as typical nanospray, despite the much smaller dimensions. As characterized by SEM images acquired before and after spray, nanopipettes are shown to be robust under conditions employed. Analytical calculations and numerical simulations are used to calculate the electric field at the emitter tip, which can be significant for the small diameter tips used.

Address: Department of Chemistry, Indiana University , 800 E. Kirkwood Avenue, Bloomington, Indiana 47405, United States

3915703d: PLoS One, e73563-e73563(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: A double WAP domain-containing protein Es-DWD1 from Eriocheir sinensis exhibits antimicrobial and proteinase inhibitory activities.
Author: Li,S., Jin,X.K., Guo,X.N., Yu,A.Q., Wu,M.H., Tan,S.J., Zhu,Y.T., Li,W.W., Wang,Q.
Abstract:
Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides, which are critical in the host immune response against microbial invasion. The common feature of these proteins is a single WAP domain maintained by at least one four-disulfide core (4-DSC) structure rich in cysteine residues. In this study, a double WAP domain (DWD)-containing protein, Es-DWD1, was first cloned from the Chinese mitten crab (Eriocheirsinensis). The full-length Es-DWD1cDNA was 1193 bp, including a 411 bp open reading frame (ORF) encoding 136 amino acids with a signal peptide of 22 amino acids in the N-terminus. A comparison with other reported invertebrate and vertebrate sequences revealed the presence of WAP domains characteristic of WAP superfamilies. As determined by quantitative real-time RT-PCR, Es-DWD1 transcripts were ubiquitously expressed in all tissues, but it was up-regulated in hemocytes post-challenge with pathogen-associated molecular patterns (PAMPs). The mature recombinant Es-DWD1 (rEs-DWD1) protein exhibited different binding activities to bacteria and fungus. Moreover, rEs-DWD1 could exert agglutination activities against Bacillus subtilis and Pichiapastoris and demonstrated inhibitory activities against the growth of Staphylococcus aureus, Aeromonas hydrophila and P. pastoris. Furthermore, rEs-DWD1 showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1 onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1 is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans.

Address: School of Life Science, East China Normal University, Shanghai, China

3915704d: PLoS One, e72613-e72613(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Competence in Streptococcus pneumoniae is a response to an increasing mutational burden.
Author: Gagne,A.L., Stevens,K.E., Cassone,M., Pujari,A., Abiola,O.E., Chang,D.J., Sebert,M.E.
Abstract:
Competence for genetic transformation in Streptococcus pneumoniae has previously been described as a quorum-sensing trait regulated by a secreted peptide pheromone. Recently we demonstrated that competence is also activated by reduction in the accuracy of protein biosynthesis. We have now investigated whether errors upstream of translation in the form of random genomic mutations can provide a similar stimulus. Here we show that generation of a mutator phenotype in S. pneumoniae through deletions of mutX, hexA or hexB enhanced the expression of competence. Similarly, chemical mutagenesis with the nucleotide analog dPTP promoted development of competence. To investigate the relationship between mutational load and the activation of competence, replicate lineages of the mutX strain were serially passaged under conditions of relaxed selection allowing random accumulation of secondary mutations. Competence increased with propagation in these lineages but not in control lineages having wild-type mutX. Resequencing of these derived strains revealed between 1 and 9 single nucleotide polymorphisms (SNPs) per lineage, which were broadly distributed across the genome and did not involve known regulators of competence. Notably, the frequency of competence development among the sequenced strains correlated significantly with the number of nonsynonymous mutations that had been acquired. Together, these observations provide support for the hypothesis that competence in S. pneumoniae is regulated in response to the accumulated burden of coding mutations in the bacterial genome. In contrast to previously described DNA damage response systems that are activated by physical lesions in the chromosome, this pneumococcal pathway may represent a unique stress response system that monitors the coding integrity of the genome.

Address: Division of Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

3915705d: PLoS One, e72592-e72592(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Analysis of expression, cellular localization, and function of three inhibitors of apoptosis (IAPs) from Litopenaeus vannamei during WSSV infection and in regulation of antimicrobial peptide genes (AMPs).
Author: Wang,P.H., Wan,D.H., Gu,Z.H., Qiu,W., Chen,Y.G., Weng,S.P., Yu,X.Q., He,J.G.
Abstract:
Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei. LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrioalginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V. alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibriopenaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.

E-Mail: wph326@gmail.com
Address: MOE Key Laboratory of Aquatic Product Safety/State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, People's Republic of China

3915708d: PLoS One, e70933-e70933(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Preventive effect of dipeptidyl peptidase-4 inhibitor on atherosclerosis is mainly attributable to incretin's actions in nondiabetic and diabetic apolipoprotein E-null mice.
Author: Terasaki,M., Nagashima,M., Nohtomi,K., Kohashi,K., Tomoyasu,M., Sinmura,K., Nogi,Y., Katayama,Y., Sato,K., Itoh,F., Watanabe,T., Hirano,T.
Abstract:
AIM: Several recent reports have revealed that dipeptidyl peptidase (DPP)-4 inhibitors have suppressive effects on atherosclerosis in apolipoprotein E-null (Apoe (-/-)) mice. It remains to be seen, however, whether this effect stems from increased levels of the two active incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). METHODS: Nontreated Apoe (-/-) mice, streptozotocin-induced diabetic Apoe (-/-) mice, and db/db diabetic mice were administered the DPP-4 inhibitor vildagliptin in drinking water and co-infused with either saline, the GLP-1 receptor blocker, exendin(9-39), the GIP receptor blocker, (Pro(3))GIP, or both via osmotic minipumps for 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages were determined. RESULTS: Vildagliptin increased plasma GLP-1 and GIP levels without affecting food intake, body weight, blood pressure, or plasma lipid profile in any of the animals tested, though it reduced HbA1c in the diabetic mice. Diabetic Apoe (-/-) mice exhibited further-progressed atherosclerotic lesions and foam cell formation compared with nondiabetic counterparts. Nondiabetic and diabetic Apoe (-/-) mice showed a comparable response to vildagliptin, namely, remarkable suppression of atherosclerotic lesions with macrophage accumulation and foam cell formation in peritoneal macrophages. Exendin(9-39) or (Pro(3))GIP partially attenuated the vildagliptin-induced suppression of atherosclerosis. The two blockers in combination abolished the anti-atherosclerotic effect of vildagliptin in nondiabetic mice but only partly attenuated it in diabetic mice. Vildagliptin suppressed macrophage foam cell formation in nondiabetic and diabetic mice, and this suppressive effect was abolished by infusions with exendin(9-39)+(Pro(3))GIP. Incubation of DPP-4 or vildagliptin in vitro had no effect on macrophage foam cell formation. CONCLUSIONS: Vildagliptin confers a substantial anti-atherosclerotic effect in both nondiabetic and diabetic mice, mainly via the action of the two incretins. However, the partial attenuation of atherosclerotic lesions by the dual incretin receptor antagonists in diabetic mice implies that vildagliptin confers a partial anti-atherogenic effect beyond that from the incretins.

Address: Department of Medicine, Division of Diabetes, Metabolism, and Endocrinology, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan

3915714d: PLoS One, e68772-e68772(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.
Author: Lee,J.H., Park,D.Y., Lee,K.J., Kim,Y.K., So,Y.K., Ryu,J.S., Oh,S.H., Han,Y.S., Ko,K., Choo,Y.K., Park,S.J., Brodzik,R., Lee,K.K., Oh,D.B., Hwang,K.A., Koprowski,H., Lee,Y.S., Ko,K.
Abstract:
Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(P)s), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P) SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAb(P) SO57 with KDEL (mAb(P)K) were significantly higher than those of mAb(P) SO57 without KDEL (mAb(P)) regardless of the transcription level. The Fc domains of both purified mAb(P) and mAb(P)K and hybridoma-derived mAb (mAb(H)) had similar levels of binding activity to the FcγRI receptor (CD64). The mAb(P)K had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAb(P) had mainly Golgi type glycans (96.8%) similar to those seen with mAb(H). Confocal analysis showed that the mAb(P)K was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P) with KDEL in the ER. Both mAb(P) and mAb(P)K disappeared with similar trends to mAb(H) in BALB/c mice. In addition, mAb(P)K was as effective as mAb(H) at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P) by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

Address: Department of Medicine, Medical Research Institute, College of Medicine, Chung-Ang University, Seoul, Korea

3915723d: J.Virol., 11591-11603(2013)
Link: [ PubMED ] [ DOI ]
Title: Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of "Self-Tagged" Particles.
Author: Gullberg,M., Polacek,C., Botner,A., Belsham,G.J.
Abstract:
The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

Address: National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark

3915724d: Chirality, 726-734(2013)
Link: [ PubMED ] [ DOI ]
Title: Various polar tripeptides as asymmetric organocatalyst in direct aldol reactions in aqueous media.
Author: Bayat,S., Tejo,B.A., Salleh,A.B., Abdmalek,E., Normi,Y.M., Rahman,M.B.
Abstract:
A series of tripeptide organocatalysts containing a secondary amine group and two amino acids with polar side chain units were developed and evaluated in the direct asymmetric intermolecular aldol reaction of 4-nitrobenzaldehyde and cyclohexanone. The effectiveness of short polar peptides as asymmetric catalysts in aldol reactions to attain high yields of enantio- and diastereoselective isomers were investigated. In a comparison, glutamic acid and histidine produced higher % ee and yields when they were applied as the second amino acid in short trimeric peptides. These short polar peptides were found to be efficient organocatalysts for the asymmetric aldol addition reaction in aqueous media.

Address: Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

3915726d: Macromol.Biosci., 1404-1412(2013)
Link: [ PubMED ] [ DOI ]
Title: Improved MIN6 β-Cell Function on Self-Assembled Peptide Amphiphile Nanomatrix Inscribed with Extracellular Matrix-Derived Cell Adhesive Ligands.
Author: Lim,D.J., Antipenko,S.V., Vines,J.B., Andukuri,A., Hwang,P.T., Hadley,N.T., Rahman,S.M., Corbett,J.A., Jun,H.W.
Abstract:
Understanding the role of the pancreatic extracellular matrix (ECM) in supporting islet survival and function drives the pursuit to create biomaterials that imitate and restore the pancreatic ECM microenvironment. To create an ECM mimic holding bioinductive cues for β-cells, self-assembled peptide amphiphiles (PAs) inscribed with four selected ECM-derived cell adhesive ligands are synthesized. After 7 days, compared to control groups cultured on biologically inert substrates, MIN6 β-cells cultured on PAs functionalized with YIGSR and RGDS cell adhesive ligands exhibit elevated insulin secretion in responses to glucose and also form β-cell clusters. These findings suggest that the self-assembled PA nanomatrix may be utilized to improve pancreatic islet transplantation for treating type 1 diabetes.

Address: Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama 35233, USA

3915730d: Mar.Drugs, 3025-3045(2013)
Link: [ PubMED ] [ DOI ]
Title: Structural characterization of new microcystins containing tryptophan and oxidized tryptophan residues.
Author: Puddick,J., Prinsep,M.R., Wood,S.A., Miles,C.O., Rise,F., Cary,S.C., Hamilton,D.P., Wilkins,A.L.
Abstract:
Microcystins are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Eight of the (more than) 90 microcystin variants presently characterized, contain the amino acid tryptophan. The well-researched oxidation products of tryptophan; kynurenine, oxindolylalanine, and N-formylkynurenine, have been previously identified in intact polypeptides but microcystin congeners containing oxidized tryptophan moieties have not been reported. Liquid chromatography-tandem mass spectrometric analysis of an extract of Microcystis CAWBG11 led to the tentative identification of two new tryptophan-containing microcystins (MCWAba and MC-WL), as well as eight other microcystin analogs containing kynurenine, oxindolylalanine and Nformylkynurenine (Nfk). Investigation of one of these congeners (MCNfkA) by nuclear magnetic resonance spectroscopy was used to verify the presence of Nfk in the microcystin. Liquid chromatography-mass spectrometry analysis of a tryptophan oxidation experiment demonstrated that tryptophan-containing microcystins could be converted into oxidized tryptophan analogs and that low levels of oxidized tryptophan congeners were present intracellularly in CAWBG11. MC-NfkR and MC-LNfk were detected in standards of MC-WR and MC-LW, indicating that care during storage of tryptophan-containing microcystins is required.

E-Mail: jonathan.puddick@cawthron.org.nz
Address: Cawthron Institute, Private Bag 2, Nelson 7042, New Zealand

3915732d: J.Biol.Chem., 28567-28580(2013)
Link: [ PubMED ] [ DOI ]
Title: Recruitment of the Mint3 Adaptor Is Necessary for Export of the Amyloid Precursor Protein (APP) from the Golgi Complex.
Author: Caster,A.H., Kahn,R.A.
Abstract:
The amyloid precursor protein (APP) is a ubiquitously expressed single-pass transmembrane protein that undergoes proteolytic processing by secretases to generate the pathogenic amyloid-β peptide, the major component in Alzheimer plaques. The traffic of APP through the cell determines its exposure to secretases and consequently the cleavages that generate the pathogenic or nonpathogenic peptide fragments. Despite the likely importance of APP traffic to Alzheimer disease, we still lack clear models for the routing and regulation of APP in cells. Like the traffic of most transmembrane proteins, the binding of adaptors to its cytoplasmic tail, which is 47 residues long and contains at least four distinct sorting motifs, regulates that of APP. We tested each of these for effects on the traffic of APP from the Golgi by mutating key residues within them and examining adaptor recruitment at the Golgi and traffic to post-Golgi site(s). We demonstrate strict specificity for recruitment of the Mint3 adaptor by APP at the Golgi, a critical role for Tyr-682 (within the YENPTY motif) in Mint3 recruitment and export of APP from the Golgi, and we identify LAMP1+ structures as the proximal destination of APP after leaving the Golgi. Together, these data provide a detailed view of the first sorting step in its route to the cell surface and processing by secretases and further highlight the critical role played by Mint3.

Address: From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322

3915733d: Oncotarget, 1427-1437(2013)
Link: [ PubMED ] [ OpenAccess]
Title: Mdig de-represses H19 large intergenic non-coding RNA (lincRNA) by down-regulating H3K9me3 and heterochromatin.
Author: Chen,B., Yu,M., Chang,Q., Lu,Y., Thakur,C., Ma,D., Yi,Z., Chen,F.
Abstract:
Mineral dust-induced gene (mdig) had been linked to the development of human lung cancers associated with environmental exposure to mineral dust, tobacco smoke or other carcinogens. In the present studies, we demonstrated that the overexpression of mdig in A549 adenocarcinomic human alveolar type II epithelial cells decreases the heterochromatin conformation of the cells and de-represses the transcription of genes in the tandemly repeated DNA regions. Although mdig can only cause a marginal decrease of the total histone H3 lysine 9 trimethylation (H3K9me3), a significant reduction of H3K9me3 in the promoter region of H19, the paternally imprinted but maternally expressed gene transcribing a large intergenic non-coding RNA (lincRNA), was observed in the cells with mdig overexpression. Silencing mdig by either shRNA or siRNA not only increased the level of H3K9me3 in the promoter region of H19 but also attenuated the transcription of H19 long non-coding RNA. Demethylation assays using immunoprecipitated mdig and histone H3 peptide substrate suggested that mdig is able to remove the methyl groups from H3K9me3. Clinically, we found that higher levels of mdig and H19 expression correlate with poorer survival of the lung cancer patients. Taken together, our results imply that mdig is involved in the regulation of H3K9me3 to influence the heterochromatin structure of the genome and the expression of genes important for cell growth or transformation.

Address: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy, Wayne State University, 259 Mack Avenue, Detroit, MI, USA

3915736d: Nature(London), 52-57(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Bacteria activate sensory neurons that modulate pain and inflammation.
Author: Chiu,I.M., Heesters,B.A., Ghasemlou,N., Von Hehn,C.A., Zhao,F., Tran,J., Wainger,B., Strominger,A., Muralidharan,S., Horswill,A.R., Bubeck Wardenburg,J., Hwang,S.W., Carroll,M.C., Woolf,C.J.
Abstract:
Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.

Address: Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

3915737d: Cell Struct.Funct., 205-221(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Decrease of ATP by Mitochondrial m-calpain Inhibitory Peptide in the Rat Retinas.
Author: Ozaki,T., Nakazawa,M., Yamashita,T., Tomita,H., Ebina,Y., Ishiguro,S.
Abstract:
Activations of mitochondrial calpains cause apoptosis-inducing factor-dependent apoptosis of retinal photoreceptor cells in the Royal College of Surgeons (RCS) rat, an animal model of retinitis pigmentosa. In the present study, we attempted to develop specific inhibitors of mitochondrial calpains that would prevent the retinal degeneration. We examined the inhibitory potency of 20-mer peptides of the m-calpain for mitochondrial calpains activity, determined the inhibitory regions, and conjugated the cell-penetrating peptides (CPP). The cytotoxicity and delivery of the peptide was evaluated using mouse photoreceptor-derived 661W cells. After intravitreal injection of the peptide in RCS rats, we examined the peptide delivery to the retina, photoreceptor cell death numbers, responses of the electroretinogram (ERG), concentrations of intracellular ATP, and changes of retinal morphology. Results showed that one of the peptides inhibited the activity of the mitochondrial m-calpain. The HIV-1 tat-conjugated m-calpain peptide, HIV-Nm, could preserve the inhibitory potency of the mitochondrial m-calpain, and penetrate into the 661W cells. While intravitreal injection of HIV-Nm made it possible to deliver to the retina, it did not prevent photoreceptor cell death. Furthermore, it caused the ERG attenuation and the decrease in the intracellular ATP only a day after the injection. Although HIV-Nm did not cause histological change of the retina after 1 or 2 days of the administration, the morphological abnormality of the retina was observed after 3-14 days. Our results demonstrated that HIV-Nm failed to prevent the photoreceptor cell death, but rather caused the attenuation of ERG response and the decrease of ATP.

Address: Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University

3915740d: J.Biotechnol., 40-45(2013)
Link: [ PubMED ] [ DOI ]
Title: Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli.
Author: Ye,W., Wang,H., Ma,Y., Luo,X., Zhang,W., Wang,J., Wang,X.
Abstract:
Glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL) is widely used in peptide recovery and synthesis because of its unique substrate specificity. However, the mechanism underlying its specificity is still not thoroughly understood. In this study, the roles of the prosegment and key amino acids involved in the proteolytic activity of GSE-BL were investigated. Loss of the GSE-BL prosegment severely restricted enzymatic activity toward Z-Phe-Leu-Glu-pNA. A homologous model of GSE-BL revealed that it contains the catalytic triad "His47, Asp96 and Ser 167", which was further confirmed by site-directed mutagenesis. In vitro mutagenesis further indicated that Val2, Arg89 and His190 are essential for enzymatic activity toward Z-Phe-Leu-Glu-pNA. Moreover, the catalytic efficiency of Phe57Ala GSE-BL toward Z-Phe-Leu-Glu-pNA was 50% higher than that of the native mature GSE-BL. This is the first study to fully elucidate the key amino acids for proteolytic activity of GSE-BL. Mature GSE-BL could be obtained through self-cleavage alone when Lys at -1 position was replaced by Glu, providing a new strategy for the preparation of mature GSE-BL. This study yielded some valuable insights into the substrate specificity of glutamate-specific endopeptidase, establishing a foundation for broadening the applications of GSE-BL.

Address: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China

3915742d: ACS Appl.Mater.Interfaces, 8267-8272(2013)
Link: [ PubMED ] [ DOI ]
Title: High throughput detection of human neutrophil peptides from serum, saliva, and tear by anthrax lethal factor-modified nanoparticles.
Author: Zhao,Y., Zhang,X., Xue,X., Li,Z., Chen,F., Li,S., Kumar,A., Zou,G., Liang,X.J.
Abstract:
Human α defensins human neutrophil peptide 1-3 (HNP 1-3) are potential prognostic cancer biomarkers. Metalloprotein anthrax lethal factor (ALF) binds to HNP 1-3 in a Zn2+-dependent manner. We conjugated ALF to the surface of magnetic nanoparticles (MNP) to magnetically isolate the HNPs, and used Zn2+ to control the capture and release HNPs.

Address: CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology , 11 Beiyitiao, Zhongguancun, Beijing 100190, China

3915743d: FEMS Microbiol.Lett., 19-25(2013)
Link: [ PubMED ] [ DOI ]
Title: Chitooligosaccharides are converted to N-acetylglucosamine by N-acetyl-β-hexosaminidase from Stenotrophomonas maltophilia.
Author: Katta,S., Ankati,S., Podile,A.R.
Abstract:
The Stenotrophomonas maltophilia k279a (Stm) Hex gene encodes a polypeptide of 785 amino acid residues, with an N-terminal signal peptide. StmHex was cloned without signal peptide and expressed as an 83.6 kDa soluble protein in Escherichia coli BL21 (DE3). Purified StmHex was optimally active at pH 5.0 and 40 degrees C. The Vmax , Km and kcat /Km for StmHex towards chitin hexamer were 10.55 nkat (mg protein)(-1) , 271 μM and 0.246 s(-1) mM(-1) , while the kinetic values with chitobiose were 30.65 nkat (mg protein)(-1) , 2365 μM and 0.082 s(-1) mM(-1) , respectively. Hydrolytic activity on chitooligosaccharides indicated that StmHex was an exo-acting enzyme and yielded N-acetyl-d-glucosamine (GlcNAc) as the final product. StmHex hydrolysed chitooligosaccharides (up to hexamer) into GlcNAc within 60 min, suggesting that this enzyme has potential for use in large-scale production of GlcNAc from chitooligosaccharides.

Address: Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India

3915745d: J.Agric.Food Chem., 8807-8813(2013)
Link: [ PubMED ] [ DOI ]
Title: Ingestion of low dose pyroglutamyl leucine improves dextran sulfate sodium-induced colitis and intestinal microbiota in mice.
Author: Wada,S., Sato,K., Ohta,R., Wada,E., Bou,Y., Fujiwara,M., Kiyono,T., Park,E.Y., Aoi,W., Takagi,T., Naito,Y., Yoshikawa,T.
Abstract:
Inflammatory bowel diseases (IBD) are based on chronic inflammation in the gastrointestinal tract. We previously found anti-inflammatory peptide pyroGlu-Leu in the enzymatic hydrolysate of wheat gluten. The objective of present study is to elucidate improvement of colitis by oral administration of pyroGlu-Leu in an animal model. Acute colitis was induced by dextran sulfate sodium (DSS), and various concentrations of pyroGlu-Leu were administrated by oral gavage for 7 days. A dose of 0.1 mg/kg body weight/day showed the most significant improvement. The pyroGlu-Leu concentration was significantly increased 24 h after oral administration both in the small intestine and the colon compared with the baseline. It was 20-fold higher in the small intestine than the colon. Administration of pyroGlu-Leu normalized population of Bacteroidetes and Firmicutes in the colon. These results indicate that pyroGlu-Leu has a potential therapeutic effect against IBD at a practical dose.

Address: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University , Shimogamo, Kyoto, 606 8522, Japan

3915746d: ACS Appl.Mater.Interfaces, 8430-8439(2013)
Link: [ PubMED ] [ DOI ]
Title: S-nitroso-N-acetylpenicillamine (SNAP) derivatization of peptide primary amines to create inducible nitric oxide donor biomaterials.
Author: VanWagner,M., Rhadigan,J., Lancina,M., Lebovsky,A., Romanowicz,G., Holmes,H., Brunette,M.A., Snyder,K.L., Bostwick,M., Lee,B.P., Frost,M.C., Rajachar,R.M.
Abstract:
An S-nitroso-N-acetylpenicillamine (SNAP) derivatization approach was used to modify existing free primary amines found in fibrin (a natural protein-based biomaterial) to generate a controlled nitric oxide (NO) releasing scaffold material. The duration of the derivatization reaction affects the NO release kinetics, the induction of controlled NO-release, hydrophobicity, swelling behavior, elastic moduli, rheometric character, and degradation behavior. These properties were quantified to determine changes in fibrin hydrogels following covalent attachment of SNAP. NO-releasing materials exhibited minimal cytotoxicity when cultured with fibroblasts or osteoblasts. Cells maintained viability and proliferative character on derivatized materials as demonstrated by Live/Dead cell staining and counting. In addition, SNAP-derivatized hydrogels exhibited an antimicrobial character indicative of NO-releasing materials. SNAP derivatization of natural polymeric biomaterials containing free primary amines offers a means to generate inducible NO-releasing biomaterials for use as an antimicrobial and regenerative support for tissue engineering.

Address: Department of Biomedical Engineering, College of Engineering, Michigan Technological University , 1400 Townsend Drive, Houghton, Michigan 49931, United States

3915748d: Biochemistry, 6313-6323(2013)
Link: [ PubMED ] [ DOI ]
Title: Bifacial peptide nucleic acid directs cooperative folding and assembly of binary, ternary, and quaternary DNA complexes.
Author: Piao,X., Xia,X., Bong,D.
Abstract:
We report herein the structuring of single-stranded thymine-rich DNA sequences into peptide-DNA hairpin triplex structures via designed melamine-thymine nucleobase recognition. Melamine-displaying α-peptides were synthesized with the general form (EM*)n, where M* denotes a lysine residue side chain derivatized with melamine, a bifacial hydrogen bond complement for thymine. We have found that (EM*)n peptides, which we term bifacial peptide nucleic acid (bPNA), function as a noncovalent template for thymine-rich DNA tracts. Unstructured DNA of the general form dTnCmTn are bound to (EM*)n peptides and fold into cooperatively melting 1:1 bPNA-DNA hairpin complexes with dissociation constants in the submicromolar to low nanomolar range for n = 4-10. As the length of the interface (n) is decreased, the melting temperature of the bPNA-DNA complex drops significantly, though Kd increases are less substantial, suggestive of strong enthalpy-entropy compensation. This is borne out by differential scanning calorimetry analysis, which indicates enthalpically driven bPNA-DNA base-stacking that becomes markedly less exothermic as the recognition surface n decreases in size. The recognition interface tolerates a high number of "mismatches" and indicates half-site, or monofacial, recognition between melamine and thymine may occur if only 1 complementary nucleobase is available. Association correlates directly with fractional thymine content, with optimal binding when the number of T-T sites match the number of melamine units. Interestingly, when a DNA host has more T-T sites than melamine sites on bPNA, two or three bPNAs can bind to a single DNA, resulting in ternary and quaternary complexes that have higher thermal stability than the binary (1:1) bPNA-DNA complex, suggestive of cooperative multisite binding. In contrast, when two bPNAs of different lengths bind to the same DNA host, a ternary complex is formed with two melting transitions, corresponding to independent melting of each bPNA component from the complex. These data demonstrate that melamine-displaying bPNA recognize thymine-rich DNA in predictable and multifaceted ways that allow binding affinity, structure stability, and stoichiometry to be tuned through simple bPNA length modification and matching with DNA length. Synthetic bPNA structuring elements may be useful tools for biotechnology.

Address: Department of Chemistry and Biochemistry, The Ohio State University , 100 West 18th Avenue, Columbus, Ohio 43210, United States

3915750d: J.Drug Target., 926-939(2013)
Link: [ PubMED ] [ DOI ]
Title: Strategies on the nuclear-targeted delivery of genes.
Author: Yao,J., Fan,Y., Li,Y., Huang,L.
Abstract:
To improve the nuclear-targeted delivery of non-viral vectors, extensive effort has been carried out on the development of smart vectors which could overcome multiple barriers. The nuclear envelope presents a major barrier to transgene delivery. Viruses are capable of crossing the nuclear envelope to efficiently deliver their genome into the nucleus through the specialized protein components. However, non-viral vectors are preferred over viral ones because of the safety concerns associated with the latter. Non-viral delivery systems have been designed to include various types of components to enable nuclear translocation at the periphery of the nucleus. This review summarizes the progress of research regarding nuclear transport mechanisms. "Smart" non-viral vectors that have been modified by peptides and other small molecules are able to facilitate the nuclear translocation and enhance the efficacy of gene expression. The resulting technology may also enhance delivery of other macromolecules to the nucleus.

Address: Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill , Chapel Hill, NC , USA and

3915760d: Mol.Cell.Biochem., 29-38(2013)
Link: [ PubMED ] [ DOI ]
Title: Structure-activity relationships of αs-casein peptides with multifunctional biological activities.
Author: Sistla,S.
Abstract:
Multifunctional bioactive peptides have a wider role in modulating physiological functions and possess multiple biological activities. Peptides from bovine milk with sequences QKALNEINQF [p10] and TKKTKLTEEEKNRL [p14] from α-S2 casein f (79-88) and α-S2 casein f (148-161) were identified to be having multifunctional biological activities and were synthesized. These synthesized peptides show various biological activities like angiotensin-converting enzyme inhibition, prolyl endopeptidase inhibition, antioxidant, and antimicrobial activities. The mode of antimicrobial mechanism was studied and p10 shows depolarization of cell membrane, whereas p14 was found to display DNA-binding activity. Structural studies envisaged backbone flexibility, for differences in their mode of action. Peptide structure function studies were correlated to understand their multifunctional biological activity.

E-Mail: sistlasrinivas@gmail.com
Address: Department of Protein Chemistry and Technology, Central Food Technological Research Institute (CSIR), Mysore, 570020, Karnataka, India,

3915769d: Protein Sci., 1552-1562(2013)
Link: [ PubMED ] [ DOI ]
Title: Interaction of HMG proteins and H1 with hybrid PNA-DNA junctions.
Author: Totsingan,F., Bell,A.J.Jr.
Abstract:
The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein-binding surface of an immobilized four-way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ-PNA1 and 4WJ-PNA3 ). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two-state equilibrium between: (i) an open-x state favored by absence of Mg2+ , low salt, and protein binding, and (ii) a compact stacked-x state favored by Mg2+ . 4WJ-PNA3 , like J1, shifts readily from an open to stacked conformation in the presence of Mg(+2) , while 4WJ-PNA1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ-PNA3 with nearly identical affinity (KD s) and 4WJ-PNA1 with two-fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.

Address: Department of Chemistry, New York University, New York, New York

3915770d: J.Am.Soc.Mass Spectrom., 1690-1699(2013)
Link: [ PubMED ] [ DOI ]
Title: Energy Dependence of HCD on Peptide Fragmentation: Stepped Collisional Energy Finds the Sweet Spot.
Author: Diedrich,J.K., Pinto,A.F., Yates,J.R.III
Abstract:
An understanding of the process of peptide fragmentation and what parameters are best to obtain the most useful information is important. This is especially true for large-scale proteomics where data collection and data analysis are most often automated, and manual interpretation of spectra is rare because of the vast amounts of data generated. We show herein that collisional cell peptide fragmentation, in this case higher collisional dissociation (HCD) in the Q Exactive, is significantly affected by the normalized energy applied. Both peptide sequence and energy applied determine what ion fragments are observed. However, by applying a stepped normalized collisional energy scheme and combining ions from low, medium, and high collision energies, we are able to increase the diversity of fragmentation ions generated. Application of stepped collision energy to HEK293T lysate demonstrated a minimal effect on peptide and protein identification in a large-scale proteomics dataset, but improved phospho site localization through increased sequence coverage. Stepped HCD is also beneficial for tandem mass tagged (TMT) experiments, increasing intensity of TMT reporters used for quantitation without adversely effecting peptide identification.

Address: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, 92037, USA

3915774d: J.Biol.Chem., 29530-29538(2013)
Link: [ PubMED ] [ DOI ]
Title: Eukaryotic release factor 3 is required for multiple turnovers of peptide release catalysis by eukaryotic release factor 1.
Author: Eyler,D.E., Wehner,K.A., Green,R.
Abstract:
Eukaryotic peptide release factor 3 (eRF3) is a conserved, essential gene in eukaryotes implicated in translation termination. We have systematically measured the contribution of eRF3 to the rates of peptide release with both saturating and limiting levels of eukaryotic release factor 1 (eRF1). Although eRF3 modestly stimulates the absolute rate of peptide release ( approximately 5-fold), it strongly increases the rate of peptide release when eRF1 is limiting (>20-fold). This effect was generalizable across all stop codons and in a variety of contexts. Further investigation revealed that eRF1 remains associated with ribosomal complexes after peptide release and subunit dissociation and that eRF3 promotes the dissociation of eRF1 from these post-termination complexes. These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release. A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.

Address: From the Howard Hughes Medical Institute and the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

3915775d: Microbiology, 2200-2211(2013)
Link: [ PubMED ] [ DOI ]
Title: Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus.
Author: Vosloo,J.A., Stander,M.A., Leussa,A.N., Spathelf,B.M., Rautenbach,M.
Abstract:
A group of non-ribosomally produced antimicrobial peptides, the tyrocidines from the tyrothricin complex, have potential as antimicrobial agents in both medicine and industry. Previous work by our group illustrated that the more polar tyrocidines rich in Trp residues in their structure were more active toward Gram-positive bacteria, while the more non-polar tyrocidines rich in Phe residues had greater activity toward Plasmodium falciparum, one of the major causative pathogens of malaria in humans. Our group also found that the tyrocidines have pronounced antifungal activity, dictated by the primary sequence of the tyrocidine. By simply manipulating the Phe or Trp concentration in the culture medium of the tyrothricin producer, Bacillus aneurinolyticus ATCC 10068, we were able to modulate the production of subsets of tyrocidines, thereby tailoring the tyrothricin complex to target specific pathogens. We optimized the tailored tyrothricin production using a novel, small-scale, high-throughput deep 96-well plate culturing method followed by analyses of the peptide mixtures using ultra-performance liquid chromatography linked to mass spectrometry. We were able to gradually shift the production profile of the tyrocidines and analogues, as well as the gramicidins between two extremes in terms of peptide subsets and peptide hydrophobicity. This study demonstrated that tyrothricin peptide subsets with targeted activity can be efficiently produced by simple manipulation of the aromatic amino acid profile of the culture medium.

Address: BIOPEP Peptide Group, Department of Biochemistry, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa

3915781d: Dev.Comp.Immunol., 703-714(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification and functional studies of Akirin, a potential positive nuclear factor of NF-κB signaling pathways in the Pacific white shrimp, Litopenaeus vannamei.
Author: Hou,F., Wang,X., Qian,Z., Liu,Q., Liu,Y., He,S., Mi,X., Bai,C., Sun,C., Liu,X.
Abstract:
As conserved nuclear factors, Akirins play critical roles in regulating antimicrobial peptides (AMPs) transcription downstream of NF-κB dependent signaling pathways in insects and mammals. However, no any functional studies was reported in penaeid shrimp. The identification and functional analysis of Akirin in the Pacific white shrimp, Litopenaeus vannamei were made in this research. The 833 nucleotides cDNA of Litopenaeus vannamei Akirin (LvAkirin) was obtained with an open reading frame of 639bp, which encodes a putative protein of 212 amino acids. The molecular weight of LvAkirin is about 23.7kDa with theoretical pI of 9.05. Two predicted nuclear localization signals (NLSs) were found and amino acid sequence alignments showed that Akirins are highly conserved between insects and mammals. The constitutive expression of LvAkirin mRNA was confirmed in all the examined tissues and high level appeared in testis followed by hemocytes and gill. LvAkirin mRNA was strongly induced in response to Vibrio parahaemolyticus infection. Silencing LvAkirin by dsRNA significantly reduced the expression of NF-κB dependent anti-lipopolysaccharide factor, crustin and penaeidin3a as well as transcription factors, Dorsal and Relish post Vibrio anguillarum (V. anguillarum) and Micrococcus lysodeikticus (M. lysodeikticus) challenge. Antibacterial activities of shrimp plasma was analyzed and high cumulative mortality was found in LvAkirin-silenced shrimps post bacteria challenge. Hence, we proposed LvAkirin might function as a positive nuclear factor of NF-κB dependent signaling pathways in shrimp innate immunity.

Address: College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, China

3915783d: Biosens.Bioelectron., 208-212(2014)
Link: [ PubMED ] [ DOI ]
Title: Competitive electrochemical immunoassay for detection of β-amyloid (1-42) and total β-amyloid peptides using p-aminophenol redox cycling.
Author: Liu,L., He,Q., Zhao,F., Xia,N., Liu,H., Li,S., Liu,R., Zhang,H.
Abstract:
β-Amyloid (1-42) peptide (Aβ(1-42)) is believed to be important for diagnosis and prognosis of Alzheimer's disease (AD) serving as a reliable molecular biomarker. However, the levels of Aβ(1-42) may differ by gender and age; thus, assay of Aβ(1-42) only might be unable to discriminate between AD and health or other types of dementia. In this work, we reported a sensitive and selective electrochemical method for detection of both Aβ(1-42) and total Aβ using p-aminophenol (p-AP) redox cycling on antibody-modified gold electrodes. Specifically, the conjugates performed between streptavidin-conjugated alkaline phosphatase (SA-ALP) and biotinylated Aβ peptides were captured by the antibody-modified electrodes, which induced the production of electrochemically active p-AP from the p-aminophenyl phosphate (p-APP) substrate. In the presence of tris(2-carboxyethyl)phosphine (TCEP), p-AP could be cycled after its electro-oxidization on the electrode, enabling the increase of the anodic current. Because native Aβ competed with the conjugates to bind the anchored antibody, the signal decreased with the increase of native Aβ concentration. A detection limit of 5pM was achieved. To demonstrate the viability of the method for analysis of Aβ(1-42) and total Aβ in real sample, artificial cerebrospinal fluid (aCSF) containing Aβ(1-40), Aβ(1-42) and Aβ(1-16) was tested. We believe that the simultaneous detection of Aβ(1-42) and total Aβ would be valuable for the early diagnosis of AD.

Address: College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan 455000, People's Republic of China

3915786d: Biochemistry, 6515-6524(2013)
Link: [ PubMED ] [ DOI ]
Title: Surface reengineering of RPA70N enables cocrystallization with an inhibitor of the replication protein A interaction motif of ATR interacting protein.
Author: Feldkamp,M.D., Frank,A.O., Kennedy,J.P., Patrone,J.D., Vangamudi,B., Waterson,A.G., Fesik,S.W., Chazin,W.J.
Abstract:
Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and 15N-1H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors.

Address: Department of Biochemistry, double daggerDepartment of Chemistry, section signDepartment of Pharmacology, and parallelCenter for Structural Biology, Vanderbilt University , Nashville, Tennessee 37232, United States

3915788d: Biochem.J., 99-108(2013)
Link: [ PubMED ] [ DOI ]
Title: Ribonucleases as a host-defence family: evidence of evolutionarily conserved antimicrobial activity at the N-terminus.
Author: Torrent,M., Pulido,D., Valle,J., Nogues,M.V., Andreu,D., Boix,E.
Abstract:
Vertebrate secreted RNases (ribonucleases) are small proteins that play important roles in RNA metabolism, angiogenesis or host defence. In the present study we describe the antimicrobial properties of the N-terminal domain of the hcRNases (human canonical RNases) and show that their antimicrobial activity is well conserved among their lineage. Furthermore, all domains display a similar antimicrobial mechanism, characterized by bacteria agglutination followed by membrane permeabilization. The results of the present study show that, for all antimicrobial hcRNases, (i) activity is retained at the N-terminus and (ii) the antimicrobial mechanism is conserved. Moreover, using computational analysis we show that antimicrobial propensity may be conserved at the N-terminus for all vertebrate RNases, thereby suggesting that a defence mechanism could be a primary function in vertebrate RNases and that the N-terminus was selected to ensure this property. In a broader context, from the overall comparison of the peptides' physicochemical and biological properties, general correlation rules could be drawn to assist in the structure-based development of antimicrobial agents.

Address: *Department of Biochemistry and Molecular Biology, Universitat Autonoma de Barcelona, Cerdanyola del Valles, 08193 Barcelona, Spain

3915790d: Mol.Microbiol., 400-414(2013)
Link: [ PubMED ] [ DOI ]
Title: Signal peptide etiquette during assembly of a complex respiratory enzyme.
Author: James,M.J., Coulthurst,S.J., Palmer,T., Sargent,F.
Abstract:
Salmonella enterica serovar Typhimurium is a Gram-negative pathogen capable of respiration with a number of terminal electron acceptors. Tetrathionate reductase is important for the infection process and is encoded by the ttrBCA operon where TtrA and TtrB are metallocofactor-containing proteins targeted to the periplasmic side of the membrane by two different Tat targeting peptides. In this work, the inter-relationship between these two signal peptides has been explored. Molecular genetics and biochemical approaches reveal that the processing of the TtrB Tat signal peptide is dependent on the successful assembly of its partner protein, TtrA. Inactivation of either the TtrA or the TtrB Tat targeting peptides individually was observed to have limited overall effects on assembly of the enzyme or on cellular tetrathionate reductase activity. However, inactivation of both signal peptides simultaneously was found to completely abolish physiological tetrathionate reductase activity. These data suggest both signals are normally active during assembly of the enzyme, and imply a code of conduct exists between the signal peptides where one can compensate for inactivity in the other. Since it appears likely that tetrathionate reductase presents itself for export as a multi-signal complex, these observations also have implications for the mechanism of the bacterial Tat translocase.

Address: College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK

3915792d: SpringerPlus, 352-352(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Energy intake, nutritional status and weight reduction in patients one year after laparoscopic sleeve gastrectomy.
Author: Gjessing,H.R., Nielsen,H.J., Mellgren,G., Gudbrandsen,O.A.
Abstract:
BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is increasingly popular due to its efficiency in reducing excess weight, however little is known about the nutritional status in patients after surgery. PURPOSE: To investigate how LSG affects energy intake, nutritional status and body weight one year after surgery. METHODS: A total of 150 patients (116 women) were enrolled in the study. Data on body weight, waist circumference and blood samples were registered preoperatively and after surgery. Food intake was reported 3 and 12 months postoperatively. RESULTS: The preoperative median BMI was 44.3 (inter quartile range 41.4-47.1), and was significantly reduced to 35.4 (32.6-38.6) after 3 months and further reduced to 30.5 (27.4-33.8) 12 months after surgery (p < 0.05). The median post surgery daily energy intake was significantly increased from 2971 (1982-3687) kJ after three months to 3840 (3046-4625) kJ twelve months postoperatively. One year after surgery, serum levels of folate, cobalamin, PTH and HDL cholesterol were significantly increased, whereas calcium, albumin, haemoglobin, creatinine, uric acid, CRP, glucose, insulin, insulin c-peptide, HOMA-IR, HbA1c and triacylglycerol were significantly decreased. Serum levels of vitamins E and D were unchanged after one year. The prevalence of patients with medically regulated type 2 diabetes was significantly reduced one year post surgery however no changes were seen in the prevalence of patients taking lipid lowering drugs or thyroxin. CONCLUSION: Based on the data obtained 12 months after surgery, LSG appears to be an effective treatment of morbid obesity without worsening the nutritional status despite the very low energy intake.

Address: Department of Clinical Science, University of Bergen, Bergen, Norway

3915796d: J.Biol.Chem., 29143-29150(2013)
Link: [ PubMED ] [ DOI ]
Title: Structural and Functional Properties of the Membranotropic HIV-1 Glycoprotein gp41 Loop Region Are Modulated by Its Intrinsic Hydrophobic Core.
Author: Qiu,J., Ashkenazi,A., Liu,S., Shai,Y.
Abstract:
The gp41 disulfide loop region switches from a soluble state to a membrane-bound state during the human immunodeficiency virus type 1 (HIV-1) envelope-mediated membrane fusion process. The loop possesses a hydrophobic core at the center of the region with an unusual basic residue (Lys-601). Furthermore, two loop core mutations, K601A and L602A, are found to inhibit HIV-1 infectivity while keeping wild type-like levels of the envelope, implying that they exert an inhibitory effect on gp41 during the membrane fusion event. Here, we investigated the mode of action of these mutations on the loop region. We show that the K601A mutation, but not the L602A mutation, abolished the binding of a loop-specific monoclonal antibody to a loop domain peptide. Additionally, the K601A, but not the L602A, impaired disulfide bond formation in the peptides. This was correlated with changes in the circular dichroism spectrum imposed by the K601A mutation. In the membrane, however, the L602A, but not the K601A, reduced the lipid mixing ability of the loop peptides, which was correlated with decreased α-helical content of the L602A mutant. The results suggest that the Lys-601 residue provides a moderate hydrophobicity level within the gp41 loop core that contributes to the proper structure and function of the loop inside and outside the membrane. Because basic residues are found between the loop Cys residues of several lentiviral fusion proteins, the findings may contribute to understanding the fusion mechanism of other viruses as well.

Address: From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100 Israel and

3915818d: Anal.Chem., 8507-8512(2013)
Link: [ PubMED ] [ DOI ]
Title: Biphasic microreactor for efficient membrane protein pretreatment with a combination of formic acid assisted solubilization, on-column pH adjustment, reduction, alkylation, and tryptic digestion.
Author: Zhao,Q., Liang,Y., Yuan,H., Sui,Z., Wu,Q., Liang,Z., Zhang,L., Zhang,Y.
Abstract:
Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an "SCX-SAX" biphasic capillary column. To evaluate the performance of the developed microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome c was pretreated. Compared with the results obtained by the traditional in-solution process, the peptide recovery (93% vs 83%) and analysis throughput (3.5 vs 14 h) were obviously improved. The microreactor was further applied for the pretreatment of 14 μg of membrane proteins extracted from rat cerebellums, and 416 integral membrane proteins (IMPs) (43% of total protein groups) and 103 transmembrane peptides were identified by two-dimensional nanoliquid chromatography-electrospray ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate analysis. With the starting sample preparation amount decreased to as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified confidently, while those obtained by the traditional in-solution method were 10 and 1, respectively. All these results demonstrated that such an "SCX-SAX" based biphasic microreactor could offer a promising tool for the pretreatment of trace membrane proteins with high efficiency and throughput.

Address: National Chromatographic R. & A. Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science , Dalian 116023, China

3915827d: Int.J.Pept., 849303-849303(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: High-throughput peptide epitope mapping using carbon nanotube field-effect transistors.
Author: Stefansson,S., Knight,M., Kwon,H.H., Stefansson,L.A., Ahn,S.N.
Abstract:
Label-free and real-time detection technologies can dramatically reduce the time and cost of pharmaceutical testing and development. However, to reach their full promise, these technologies need to be adaptable to high-throughput automation. To demonstrate the potential of single-walled carbon nanotube field-effect transistors (SWCNT-FETs) for high-throughput peptide-based assays, we have designed circuits arranged in an 8 x 12 (96-well) format that are accessible to standard multichannel pipettors. We performed epitope mapping of two HIV-1 gp160 antibodies using an overlapping gp160 15-mer peptide library coated onto nonfunctionalized SWCNTs. The 15-mer peptides did not require a linker to adhere to the non-functionalized SWCNTs, and binding data was obtained in real time for all 96 circuits. Despite some sequence differences in the HIV strains used to generate these antibodies and the overlapping peptide library, respectively, our results using these antibodies are in good agreement with known data, indicating that peptides immobilized onto SWCNT are accessible and that linear epitope mapping can be performed in minutes using SWCNT-FET.

Address: Fuzbien Technology Institute, 9700 Great Seneca Highway, Rockville, MD 20850, USA

3915830d: ChemBioChem, 1640-1647(2013)
Link: [ PubMED ] [ DOI ]
Title: Substrate selection influences molecular recognition in a screen for lymphoid tyrosine phosphatase inhibitors.
Author: Kulkarni,R.A., Vellore,N.A., Bliss,M.R., Stanford,S.M., Falk,M.D., Bottini,N., Baron,R., Barrios,A.M.
Abstract:
Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000-member library of drugs and drug-like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac-ARLIEDNE-pCAP-TAREG-NH_2, peptide 1) and a small-molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin-3,5-digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC_5(0) of 50 nM and significant activity in T-cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.

Address: Department of Medicinal Chemistry, University of Utah, 30 South 2000 East, Salt Lake City, UT 84112 (USA)

3915831d: Proteomics, 2956-2966(2013)
Link: [ PubMED ] [ DOI ]
Title: High pH reversed-phase chromatography as a superior fractionation scheme compared to off-gel isoelectric focusing for complex proteome analysis.
Author: Stein,D.R., Hu,X., McCorrister,S.J., Westmacott,G.R., Plummer,F.A., Ball,T.B., Carpenter,M.S.
Abstract:
MS/MS is the technology of choice for analyzing complex protein mixtures. However, due to the intrinsic complexity and dynamic range present in higher eukaryotic proteomes, prefractionation is an important step to maximize the number of proteins identified. Off-gel IEF (OG-IEF) and high pH RP (Hp-RP) column chromatography have both been successfully utilized as a first-dimension peptide separation technique in shotgun proteomic experiments. Here, a direct comparison of the two methodologies was performed on ex vivo peripheral blood mononuclear cell lysate. In 12-fraction replicate analysis, Hp-RP resulted in more peptides and proteins identified than OG-IEF fractionation. Distributions of peptide pIs and hydropathy did not reveal any appreciable bias in either technique. Resolution, defined here as the ability to limit a specific peptide to one particular fraction, was significantly better for Hp-RP. This leads to a more uniform distribution of total and unique peptides for Hp-RP across all fractions collected. These results suggest that fractionation by Hp-RP over OG-IEF is the better choice for typical complex proteome analysis.

Address: Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada

3915832d: Insect Sci., 575-584(2013)
Link: [ PubMED ] [ DOI ]
Title: Characterization of a β-1,3-glucan recognition protein from the beet armyworm, Spodoptera exigua (Insecta: Lepidoptera: Noctuidae).
Author: Bang,K., Park,S., Cho,S.
Abstract:
The β-1,3-glucan recognition protein gene from Spodoptera exigua (SeβGRP) was cloned and characterized. The cDNA of this gene is 1 644 nucleotides in length and the predicted polypeptide is 491 amino acids (aa) in length, with a calculated molecular mass of 54.8 kDa. The first 22 aa encode a predicted secretion signal peptide. A BLAST search, multiple sequence alignment, and phylogenetic analysis of the aa sequence of SeβGRP revealed that this protein is most similar to the β-1,3-glucan recognition protein (βGRP) family of pattern recognition proteins. Using reverse-transcription polymerase chain reaction, we detected the presence of SeβGRP transcripts in the egg, larval, pupal, and adult stages of S. exigua. In addition, the SeβGRP transcript was expressed in all the tissues examined including the brain, hemocytes, fat body, intestine, and cuticle. There were no changes in SeβGRP mRNA levels in larvae infected with ultraviolet (UV)-killed Escherichia coli DH5α compared with the control larvae inoculated with the water; however, SeβGRP mRNA levels were markedly elevated 4-8 h after infection and slightly induced 12-24 h after infection in larvae injected with UV-killed Fusarium oxysporum. This may be because β-1,3-glucan is the main component of the cell wall of F. oxysporum, but not E. coli DH5α.

Address: Department of Applied Biology, College of Agriculture and Life Science, Agriculture and Life Sciences Research Institute, Kangwon National University, Chuncheon

3915834d: Insect Sci., 485-496(2013)
Link: [ PubMED ] [ DOI ]
Title: Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and Cry1Ab-resistant strains of sugarcane borer, Diatraea saccharalis.
Author: Yang,Y., Zhu,Y.C., Ottea,J., Husseneder,C., Leonard,B.R., Abel,C., Luttrell,R., Huang,F.
Abstract:
Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin-like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.

Address: Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA, USA

3915838d: Biopolymers, 897-907(2013)
Link: [ PubMED ] [ DOI ]
Title: Differential recognition of syk-binding sites by each of the two phosphotyrosine-binding pockets of the Vav SH2 domain.
Author: Chen,C.H., Piraner,D., Gorenstein,N.M., Geahlen,R.L., Beth Post,C.
Abstract:
The association of spleen tyrosine kinase (Syk), a central tyrosine kinase in B cell signaling, with Vav SH2 domain is controlled by phosphorylation of two closely spaced tyrosines in Syk linker B: Y342 and Y346. Previous studies established both singly phosphorylated and doubly phosphorylated forms play a role in signaling. The structure of the doubly phosphorylated form identified a new recognition of phosphotyrosine whereby two phosphotyrosines bind simultaneously to the Vav SH2 domain, one in the canonical pTyr pocket and one in the specificity pocket on the opposite side of the central β-sheet. It is unknown if the specificity pocket can bind phosphotyrosine independent of phosphotyrosine binding the pTyr pocket. To address this gap in knowledge, we determined the structure of the complex between Vav1 SH2 and a peptide (SykLB-YpY) modeling the singly phosphorylated-Y346 form of Syk with unphosphorylated Y342. The nuclear magnetic resonance (NMR) data conclusively establish that recognition of phosphotyrosine is swapped between the two pockets; phosphorylated pY346 binds the specificity pocket of Vav1 SH2, and unphosphorylated Y342 occupies what is normally the pTyr binding pocket. Nearly identical changes in chemical shifts occurred upon binding all three forms of singly and doubly phosphorylated peptides; however, somewhat smaller shift perturbations for SykLB-YpY from residues in regions of high internal mobility suggest that internal motions are coupled to binding affinity. The differential recognition that includes this swapped binding of phosphotyrosine to the specificity pocket of Vav SH2 increases the repertoire of possible phosphotyrosine binding by SH2 domains in regulating protein-protein interactions in cellular signaling.

Address: Department of Medicinal Chemistry and Molecular Pharmacology, and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, 47907

3915843d: Nat.Biotechnol., 916-921(2013)
Link: [ PubMED ] [ DOI ]
Title: Nature-inspired design of motif-specific antibody scaffolds.
Author: Koerber,J.T., Thomsen,N.D., Hannigan,B.T., Degrado,W.F., Wells,J.A.
Abstract:
Aberrant changes in post-translational modifications (PTMs) such as phosphate groups underlie a majority of human diseases. However, detection and quantification of PTMs for diagnostic or biomarker applications often require PTM-specific monoclonal antibodies (mAbs), which are challenging to generate using traditional antibody-selection methods. Here we outline a general strategy for producing synthetic, PTM-specific mAbs by engineering a motif-specific 'hot spot' into an antibody scaffold. Inspired by a natural phosphate-binding motif, we designed and selected mAb scaffolds with hot spots specific for phosphoserine, phosphothreonine or phosphotyrosine. Crystal structures of the phospho-specific mAbs revealed two distinct modes of phosphoresidue recognition. Our data suggest that each hot spot functions independently of the surrounding scaffold, as phage display antibody libraries using these scaffolds yielded >50 phospho- and target-specific mAbs against 70% of target peptides. Our motif-specific scaffold strategy may provide a general solution for rapid, robust development of anti-PTM mAbs for signaling, diagnostic and therapeutic applications.

Address: Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA

3915846d: J.Bacteriol., 4735-4741(2013)
Link: [ PubMED ] [ DOI ]
Title: The D3 Bacteriophage α-Polymerase Inhibitor (Iap) Peptide Disrupts O-Antigen Biosynthesis through Mimicry of the Chain Length Regulator Wzz in Pseudomonas aeruginosa.
Author: Taylor,V.L., Udaskin,M.L., Islam,S.T., Lam,J.S.
Abstract:
Lysogenic bacteriophage D3 causes seroconversion of Pseudomonas aeruginosa PAO1 from serotype O5 to O16 by inverting the linkage between O-specific antigen (OSA) repeat units from α to β. The OSA units are polymerized by Wzy to modal lengths regulated by Wzz1 and Wzz2. A key component of the D3 seroconversion machinery is the inhibitor of α-polymerase (Iap) peptide, which is able to solely suppress α-linked long-chain OSA production in P. aeruginosa PAO1. To establish the target specificity of Iap for Wzyα, changes in OSA phenotypes were examined via Western immunoblotting for wzz1 and wzz2 single-knockout strains, as well as a wzz1 wzz2 double knockout, following the expression of iap from a tuneable vector. Increased induction of Iap expression completely abrogated OSA production in the wzz1 wzz2 double mutant, while background levels of OSA production were still observed in either of the single mutants. Therefore, Iap inhibition of OSA biosynthesis was most effective in the absence of both Wzz proteins. Sequence alignment analyses revealed a high degree of similarity between Iap and the first transmembrane segment (TMS) of either Wzz1 or Wzz2. Various topology prediction analyses of the Iap sequence consistently predicted the presence of a single TMS, suggesting a propensity for Iap to insert itself into the inner membrane (IM). The compromised ability of Iap to abrogate Wzyα function in the presence of Wzz1 or Wzz2 provides compelling evidence that inhibition occurs after Wzyα inserts itself into the IM and is achieved through mimicry of the first TMS from the Wzz proteins of P. aeruginosa PAO1.

Address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada

3915847d: Artif.DNA PNA XNA, 49-57(2013)
Link: [ PubMED ] [ DOI ]
Title: Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5.
Author: Bahal,R., McNeer,N.A., Ly,D.H., Saltzman,W.M., Glazer,P.M.
Abstract:
The development of a new class of peptide nucleic acids (PNAs), i.e., γ PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based γ peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of chemokine receptor 5 (CC R5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism.

3915848d: Cell.Mol.Immunol., 423-436(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The shrimp IKK-NF-κB signaling pathway regulates antimicrobial peptide expression and may be subverted by white spot syndrome virus to facilitate viral gene expression.
Author: Wang,P.H., Gu,Z.H., Wan,D.H., Liu,B.D., Huang,X.D., Weng,S.P., Yu,X.Q., He,J.G.
Abstract:
The IκB kinases IKKα and IKKβ and the IKK-related kinases TANK-binding kinase 1 (TBK1) and IKKε are the master regulators of the NF-κB signaling pathway. Although this pathway has been extensively studied in mammals, less attention has been paid in crustaceans, which have significant economic value. Here, we report the cloning and functional studies of two IKK homologs, LvIKKβ and LvIKKε, from Pacific white shrimp, Litopenaeus vannamei. LvIKKβ and LvIKKε mRNAs are widely expressed in different tissues and are responsive to white spot syndrome virus (WSSV) infection. When overexpressed in Drosophila S2 cells, LvIKKβ but not LvIKKε activates the promoters of NF-κB pathway-controlled antimicrobial peptide genes (AMPs), such as the Penaeidins (PENs). In HEK 293T cells, both LvIKKβ and LvIKKε activate an NF-κB reporter. The silencing of LvIKKβ or LvIKKε using double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) decreases the expression of L. vannamei AMPs, including PENs, lysozyme and crustins. Intriguingly, LvIKKβ- or LvIKKε-silenced L. vannamei are resistant to WSSV infection. We hypothesized that successful infection with WSSV requires the activation of the IKK-NF-κB signaling pathway to modulate viral gene expression. We constructed luciferase reporters for 147 WSSV genes. By screening, we found that the WSV051, WSV059, WSV069, WSV083, WSV090, WSV107, WSV244, WSV303, WSV371 and WSV445 promoters can be activated by LvIKKβ or LvIKKε in Drosophila S2 cells. Taken together, our results reveal that LvIKKβ and LvIKKε may participate in the regulation of shrimp AMPs and that WSSV may subvert the L. vannamei IKK-NF-κB signaling pathway to facilitate viral gene expression.

Address: MOE Key Laboratory of Aquatic Product Safety/State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China

3915850d: Biochim.Biophys.Acta, 2757-2762(2013)
Link: [ PubMED ] [ DOI ]
Title: The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains.
Author: Ros,U., Edwards,M.A., Epand,R.F., Lanio,M.E., Schreier,S., Yip,C.M., Alvarez,C., Epand,R.M.
Abstract:
Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.

Address: Center for Protein Studies, Biology Faculty, University of Havana, Havana CP 10400, Cuba

3915854d: J.Mol.Biol., 4496-4507(2013)
Link: [ PubMED ] [ DOI ]
Title: Plasticity in the Contribution of T Cell Receptor Variable Region Residues to Binding of Peptide-HLA-A2 Complexes.
Author: Smith,S.N., Sommermeyer,D., Piepenbrink,K.H., Blevins,S.J., Bernhard,H., Uckert,W., Baker,B.M., Kranz,D.M.
Abstract:
One hypothesis accounting for major histocompatibility complex (MHC) restriction by T cell receptors (TCRs) holds that there are several evolutionary conserved residues in TCR variable regions that contact MHC. While this "germline codon" hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα region and recognize the same MHC allele (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key germline codon residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. With the use of single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type germline codon residues, but several alternative residues could also accommodate binding and, hence, MHC restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e., MHC restriction), there is significant plasticity in requirements for particular side chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs.

Address: Department of Biochemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA

3915856d: Exp.Eye Res., 96-108(2013)
Link: [ PubMED ] [ DOI ]
Title: Novel compstatin family peptides inhibit complement activation by drusen-like deposits in human retinal pigmented epithelial cell cultures.
Author: Gorham,R.D.Jr., Forest,D.L., Tamamis,P., Lopez de Victoria,A., Kraszni,M., Kieslich,C.A., Banna,C.D., Bellows-Peterson,M.L., Larive,C.K., Floudas,C.A., Archontis,G., Johnson,L.V., Morikis,D.
Abstract:
We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment.

Address: Department of Bioengineering, University of California, Riverside, CA 92521, USA

3915859d: Cell Host Microbe, 195-206(2013)
Link: [ PubMED ] [ DOI ]
Title: Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin.
Author: Rubinstein,M.R., Wang,X., Liu,W., Hao,Y., Cai,G., Han,Y.W.
Abstract:
Fusobacterium nucleatum (Fn) has been associated with colorectal cancer (CRC), but causality and underlying mechanisms remain to be established. We demonstrate that Fn adheres to, invades, and induces oncogenic and inflammatory responses to stimulate growth of CRC cells through its unique FadA adhesin. FadA binds to E-cadherin, activates β-catenin signaling, and differentially regulates the inflammatory and oncogenic responses. The FadA-binding site on E-cadherin is mapped to an 11-amino-acid region. A synthetic peptide derived from this region of E-cadherin abolishes FadA-induced CRC cell growth and oncogenic and inflammatory responses. The fadA gene levels in the colon tissue from patients with adenomas and adenocarcinomas are >10-100 times higher compared to normal individuals. The increased FadA expression in CRC correlates with increased expression of oncogenic and inflammatory genes. This study unveils a mechanism by which Fn can drive CRC and identifies FadA as a potential diagnostic and therapeutic target for CRC.

Address: Department of Periodontics, Case Western Reserve University, Cleveland, OH 44106, USA

3915863d: Biomaterials, 8776-8785(2013)
Link: [ PubMED ] [ DOI ]
Title: The use of self-adjuvanting nanofiber vaccines to elicit high-affinity B cell responses to peptide antigens without inflammation.
Author: Chen,J., Pompano,R.R., Santiago,F.W., Maillat,L., Sciammas,R., Sun,T., Han,H., Topham,D.J., Chong,A.S., Collier,J.H.
Abstract:
Balancing immunogenicity with inflammation is a central tenet of vaccine design, especially for subunit vaccines that utilize traditional pro-inflammatory adjuvants. Here we report that by using a nanoparticulate peptide-based vaccine, immunogenicity and local inflammation could be decoupled. Self-assembled β-sheet-rich peptide nanofibers, previously shown to elicit potent antibody responses in mice, were found to be non-cytotoxic in vitro and, remarkably, elicited no measurable inflammation in vivo-with none of the swelling at the injection site, accumulation of inflammatory cells or cytokines, or production of allergic IgE that were elicited by an alum-adjuvanted vaccine. Nanofibers were internalized by dendritic cells and macrophages at the injection site, and only dendritic cells that acquired the material increased their expression of the activation markers CD80 and CD86. Immunization with epitope-bearing nanofibers elicited antigen-specific differentiation of T cells into T follicular helper cells and B cells into germinal center cells, as well as high-titer, high-affinity IgG that cross-reacted with the native protein antigen and was neutralizing in an in vitro influenza hemagglutination inhibition assay. These responses were superior to those induced by alum and comparable to those induced by complete Freund's adjuvant. Thus, nanoparticulate assemblies may provide a new route to non-inflammatory immunotherapies and vaccines.

Address: Committee on Immunology, Department of Surgery, The University of Chicago, Chicago, IL 60637, USA

3915878d: FEBS Open Bio, 310-320(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin.
Author: Faraj,S.E., Venturutti,L., Roman,E.A., Marino-Buslje,C.B., Mignone,A., Tosatto,S.C., Delfino,J.M., Santos,J.
Abstract:
The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42-80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56-210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81-210) is monomeric. Thus, cumulative experimental evidence points to the N-terminal moiety as an essential element for the assembly of a high molecular weight oligomer. The secondary structure propensity of peptide 56-81, the moiety putatively responsible for promoting protein-protein interactions, was also studied. Depending on the environment (TFE or SDS), this peptide adopts α-helical or β-strand structure. In this context, we explored the conformation and stability of hFXN56-210. The biophysical characterization by fluorescence, CD and SEC-FPLC shows that subunits are well folded, sharing similar stability to hFXN90-210. However, controlled proteolysis indicates that the N-terminal stretch is labile in the context of the multimer, whereas the FXN domain (residues 81-210) remains strongly resistant. In addition, guanidine hydrochloride at low concentration disrupts intermolecular interactions, shifting the ensemble toward the monomeric form. The conformational plasticity of the N-terminal tail might impart on hFXN the ability to act as a recognition signal as well as an oligomerization trigger. Understanding the fine-tuning of these activities and their resulting balance will bear direct relevance for ultimately comprehending hFXN function.

Address: Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB), School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956,C1113AAD Buenos Aires, Argentina

3915879d: PLoS One, e73303-e73303(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Interactions between Aβ and mutated Tau lead to polymorphism and induce aggregation of Aβ-mutated tau oligomeric complexes.
Author: Raz,Y., Miller,Y.
Abstract:
One of the main hallmarks of the fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) is the accumulation of neurofibrillary tangles in the brain as an outcome of the aggregation of mutated tau protein. This process occurs due to a number of genetic mutations in the MAPT gene. One of these mutations is the K280 mutation in the tau R2 repeat domain, which promotes the aggregation vis-a-vis that for the wild-type tau. Experimental studies have shown that in Alzheimer's disease Aβ peptide forms aggregates both with itself and with wild-type tau. By analogy, in FTDP-17, it is likely that there are interactions between Aβ and mutated tau, but the molecular mechanisms underlying such interactions remain to be elucidated. Thus, to investigate the interactions between Aβ and mutated tau, we constructed fourteen K280 mutated tau-Aβ17-42 oligomeric complexes. In seven of the mutated tau-Aβ17-42 oligoemric complexes the mutated tau oligomers exhibited hydrophobic interactions in their core domain, and in the other seven mutated tau-Aβ17-42 oligoemric complexes the mutated tau oligomers exhibited salt-bridge interactions in their core domain. We considered two types of interactions between mutated tau oligomers and Aβ oligomers: interactions of one monomer of the Aβ oligomer with one monomer of the mutated tau oligomer to form a single-layer conformation, and interactions of the entire Aβ oligomer with the entire mutated tau oligomer to form a double-layer conformation. We also considered parallel arrangements of Aβ trimers alternating with mutated tau trimers in a single-layer conformation. Our results demonstrate that in the interactions of Aβ and mutated tau oligomers, polymorphic mutated tau-Aβ17-42 oligomeric complexes were observed, with a slight preference for the double-layer conformation. Aβ trimers alternating with mutated tau trimers constituted a structurally stable confined β-structure, albeit one that was energetically less stable than all the other constructed models.

Address: Department of Chemistry, BenGurion University of the Negev, Beer-Sheva, Israel

3915880d: PLoS One, e73124-e73124(2013)
Link: [ PubMED ] [ DOI ]
Title: Equivalent T cell epitope promiscuity in ecologically diverse human pathogens.
Author: Wiens,K.E., Swaminathan,H., Copin,R., Lun,D.S., Ernst,J.D.
Abstract:
BACKGROUND: The HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens' epitopes to bind diverse HLA alleles, referred to as an epitope's binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure. METHODS: We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines. RESULTS: We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes. CONCLUSIONS: Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.

Address: Department of Pathology, New York University School of Medicine, New York, New York, USA

3915881d: PLoS One, e72297-e72297(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Beta-amyloid impairs reelin signaling.
Author: Cuchillo-Ibanez,I., Balmaceda,V., Botella-Lopez,A., Rabano,A., Avila,J., Saez-Valero,J.
Abstract:
Reelin is a signaling protein increasingly associated with the pathogenesis of Alzheimer's disease that relevantly modulates tau phosphorylation. We have previously demonstrated that β-amyloid peptide (Aβ) alters reelin expression. We have now attempted to determine whether abnormal reelin triggered by Aβ will result in signaling malfunction, contributing to the pathogenic process. Here, we show that reelin forms induced by β-amyloid are less capable of down-regulating tau phosphorylation via disabled-1 and GSK3β kinase. We also demonstrate that the scaffold protein 14-3-3 that increases tau phosphorylation by modulating GSK3β activity, is up-regulated during defective reelin signaling. Binding of reelin to its receptor, mainly ApoER2 in the brain, relays the signal into the cell. We associate the impaired reelin signaling with inefficiency of reelin in forming active homodimers and decreased ability to bind efficiently to its receptor, ApoER2. More remarkably, reelin from Alzheimer cortex shows a tendency to form large complexes instead of homodimers, the active form for signaling. Our results suggest that reelin expression is altered by Aβ leading to impaired reelin signaling.

Address: Instituto de Neurociencias de Alicante, Universidad Miguel Hernandez-Consejo Superior de Investigaciones Cientificas, Sant Joan d'Alacant, Alicante, Spain

3915882d: PLoS One, e72050-e72050(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Us3 kinase encoded by herpes simplex virus 1 mediates downregulation of cell surface major histocompatibility complex class I and evasion of CD8+ T cells.
Author: Imai,T., Koyanagi,N., Ogawa,R., Shindo,K., Suenaga,T., Sato,A., Arii,J., Kato,A., Kiyono,H., Arase,H., Kawaguchi,Y.
Abstract:
Detection and elimination of virus-infected cells by CD8+ cytotoxic T lymphocytes (CTLs) depends on recognition of virus-derived peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of infected cells. In the present study, we showed that inactivation of the activity of viral kinase Us3 encoded by herpes simplex virus 1 (HSV-1), the etiologic agent of several human diseases and a member of the αherpesvirinae, significantly increased cell surface expression of MHC-I, thereby augmenting CTL recognition of infected cells in vitro. Overexpression of Us3 by itself had no effect on cell surface expression of MHC-I and Us3 was not able to phosphorylate MHC-I in vitro, suggesting that Us3 indirectly downregulated cell surface expression of MHC-I in infected cells. We also showed that inactivation of Us3 kinase activity induced significantly more HSV-1-specific CD8+ T cells in mice. Interestingly, depletion of CD8+ T cells in mice significantly increased replication of a recombinant virus encoding a kinase-dead mutant of Us3, but had no effect on replication of a recombinant virus in which the kinase-dead mutation was repaired. These results indicated that Us3 kinase activity is required for efficient downregulation of cell surface expression of MHC-I and mediates evasion of HSV-1-specific CD8+ T cells. Our results also raised the possibility that evasion of HSV-1-specific CD8+ T cells by HSV-1 Us3-mediated inhibition of MHC-I antigen presentation might in part contribute to viral replication in vivo.

Address: Division of Molecular Virology, Department of Microbiology and Immunology, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan

3915888d: PLoS One, e71463-e71463(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Identification of Trypanosome proteins in plasma from African sleeping sickness patients infected with T. b. rhodesiense.
Author: Eyford,B.A., Ahmad,R., Enyaru,J.C., Carr,S.A., Pearson,T.W.
Abstract:
Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a 'deep-mining" proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification.

Address: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada

3915889d: PLoS One, e71351-e71351(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Impairment of IFN-γ response to synthetic peptides of Mycobacterium tuberculosis in a 7-day whole blood assay.
Author: Gideon,H.P., Hamilton,M.S., Wood,K., Pepper,D., Oni,T., Seldon,R., Banwell,C., Langford,P.R., Wilkinson,R.J., Wilkinson,K.A.
Abstract:
Studies on Mycobacterium tuberculosis (MTB) antigens are of interest in order to improve vaccine efficacy and to define biomarkers for diagnosis and treatment monitoring. The methodologies used for these investigations differ greatly between laboratories and discordant results are common. The IFN-γ response to two well characterized MTB antigens ESAT-6 and CFP-10, in the form of recombinant proteins and synthetic peptides, was evaluated in HIV-1 uninfected persons in both long-term (7 day) and 24 hour, commercially available QuantiFERON TB Gold in Tube (QFT-GIT), whole blood assays. Our findings showed differences in the IFN-γ response between 24 hour and 7 day cultures, with recombinant proteins inducing a significantly higher response than the peptide pools in 7 day whole blood assays. The activity of peptides and recombinant proteins did not differ in 24 hour whole blood or peripheral blood mononuclear cell (PBMC) based assays, nor in the ELISpot assay. Further analysis by SELDI-TOF mass spectrometry showed that the peptides are degraded over the course of 7 days of incubation in whole blood whilst the recombinant proteins remain intact. This study therefore demonstrates that screening antigenic candidates as synthetic peptides in long-term whole blood assays may underestimate immunogenicity.

Address: Clinical Infectious Disease Research Initiative, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa

3915891d: PLoS One, e71320-e71320(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: IFN-ε is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines.
Author: Hermant,P., Francius,C., Clotman,F., Michiels,T.
Abstract:
Type-I interferons (IFNs) form a large family of cytokines that primarily act to control the early development of viral infections. Typical type-I IFN genes, such as those encoding IFN-α or IFN-β are upregulated by viral infection in many cell types. In contrast, the gene encoding IFN-ε was reported to be constitutively expressed by cells of the female reproductive tract and to contribute to the protection against vaginal infections with herpes simplex virus 2 and Chlamydia muridarum. Our data confirm the lack of induction of IFN-ε expression after viral infection and the constitutive expression of IFN-ε by cells of the female but also of the male reproductive organs. Interestingly, when expressed from transfected expression plasmids in 293T, HeLa or Neuro2A cells, the mouse and human IFN-ε precursors were inefficiently processed and secretion of IFN-ε was minimal. Analysis of chimeric constructs produced between IFN-ε and limitin (IFN-zeta) showed that both the signal peptide and the mature moiety of IFN-ε contribute to poor processing of the precursor. Immunofluorescent detection of FLAG-tagged IFN-ε in transfected cells suggested that IFN-ε and chimeric proteins were defective for progression through the secretory pathway. IFN-ε did not, however, act intracellularly and impart an antiviral state to producing cells. Given the constitutive expression of IFN-ε in specialized cells and the poor processing of IFN-ε precursor in fibroblasts and cell lines, we hypothesize that IFN-ε secretion may require a co-factor specifically expressed in cells of the reproductive organs, that might secure the system against aberrant release of this IFN.

Address: Universite catholique de Louvain, de Duve Institute, Brussels, Belgium

3915893d: PLoS One, e71228-e71228(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: HLA-DO as the optimizer of epitope selection for MHC class II antigen presentation.
Author: Poluektov,Y.O., Kim,A., Hartman,I.Z., Sadegh-Nasseri,S.
Abstract:
Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing.

Address: Graduate Program in Immunology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America

3915894d: PLoS One, e70773-e70773(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Gadd153 and NF-κB crosstalk regulates 27-hydroxycholesterol-induced increase in BACE1 and β-amyloid production in human neuroblastoma SH-SY5Y cells.
Author: Marwarha,G., Raza,S., Prasanthi,J.R., Ghribi,O.
Abstract:
β-amyloid (Aβ) peptide, accumulation of which is a culprit for Alzheimer's disease (AD), is derived from the initial cleavage of amyloid precursor protein by the aspartyl protease BACE1. Identification of cellular mechanisms that regulate BACE1 production is of high relevance to the search for potential disease-modifying therapies that inhibit BACE1 to reduce Aβ accumulation and AD progression. In the present study, we show that the cholesterol oxidation product 27-hydroxycholesterol (27-OHC) increases BACE1 and Aβ levels in human neuroblastoma SH-SY5Y cells. This increase in BACE1 involves a crosstalk between the two transcription factors NF-κB and the endoplasmic reticulum stress marker, the growth arrest and DNA damage induced gene-153 (gadd153, also called CHOP). We specifically show that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter and subsequent increase in BACE1 transcription and Aβ production. The NF-κB inhibitor, sc514, significantly attenuated the 27-OHC-induced increase in NF-κB-mediated BACE1 expression and Aβ genesis. We further show that the 27-OHC-induced NF-κB activation and increased NF-κB-mediated BACE1 expression is contingent on the increased activation of gadd153. Silencing gadd153 expression with siRNA alleviated the 27-OHC-induced increase in NF-κB activation, NF-κB binding to the BACE1 promoter, and subsequent increase in BACE1 transcription and Aβ production. We also show that increased levels of BACE1 in the triple transgenic mouse model for AD is preceded by gadd153 and NF-κB activation. In summary, our study demonstrates that gadd153 and NF-κB work in concert to regulate BACE1 expression. Agents that inhibit gadd153 activation and subsequent interaction with NF-κB might be promising targets to reduce BACE1 and Aβ overproduction and may ultimately serve as disease-modifying treatments for AD.

Address: Department of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota, USA

3915895d: PLoS One, e70715-e70715(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Improved bone morphogenetic protein-2 retention in an injectable collagen matrix using bifunctional peptides.
Author: Hamilton,P.T., Jansen,M.S., Ganesan,S., Benson,R.E., Hyde-Deruyscher,R., Beyer,W.F., Gile,J.C., Nair,S.A., Hodges,J.A., Gron,H.
Abstract:
To promote healing of many orthopedic injuries, tissue engineering approaches are being developed that combine growth factors such as Bone Morphogenetic Proteins (BMP) with biomaterial carriers. Although these technologies have shown great promise, they still face limitations. We describe a generalized approach to create target-specific modular peptides that bind growth factors to implantable biomaterials. These bifunctional peptide coatings provide a novel way to modulate biology on the surface of an implant. Using phage display techniques, we have identified peptides that bind with high affinity to BMP-2. The peptides that bind to BMP-2 fall into two different sequence clusters. The first cluster of peptide sequences contains the motif W-X-X-F-X-X-L (where X can be any amino acid) and the second cluster contains the motif F-P-L-K-G. We have synthesized bifunctional peptide linkers that contain BMP-2 and collagen-binding domains. Using a rat ectopic bone formation model, we have injected rhBMP-2 into a collagen matrix with or without a bifunctional BMP-2: collagen peptide (BC-1). The presence of BC-1 significantly increased osteogenic cellular activity, the area of bone formed, and bone maturity at the site of injection. Our results suggest that bifunctional peptides that can simultaneously bind to a growth factor and an implantable biomaterial can be used to control the delivery and release of growth factors at the site of implantation.

Address: Department of Microbiology, North Carolina State University, Raleigh, North Carolina, United States of America

3915896d: PLoS One, e70452-e70452(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Structural guided scaffold phage display libraries as a source of bio-therapeutics.
Author: Man,Y.K., DiCara,D., Chan,N., Vessillier,S., Mather,S.J., Rowe,M.L., Howard,M.J., Marshall,J.F., Nissim,A.
Abstract:
We have developed a structurally-guided scaffold phage display strategy for identification of ligand mimetic bio-therapeutics. As a proof of concept we used the ligand of integrin αvβ6, a tumour cell surface receptor and a major new target for imaging and therapy of many types of solid cancer. NMR structure analysis showed that RGD-helix structures are optimal for αvβ6 ligand-interaction, so we designed novel algorithms to generate human single chain fragment variable (scFv) libraries with synthetic VH-CDR3 encoding RGD-helix hairpins with helices of differing pitch, length and amino acid composition. Study of the lead scFv clones D25scFv and D34scFv and their corresponding VH-CDR3 derived peptides, D25p and D34p, demonstrated: specific binding to recombinant and cellular αvβ6; inhibition of αvβ6-dependent cell and ligand adhesion, αvβ6-dependent cell internalisation; and selective retention by αvβ6-expressing, but not αvβ6-negative, human xenografts. NMR analysis established that both the D25p and D34p retained RGD-helix structures confirming the success of the algorithm. In conclusion, scFv libraries can be engineered based on ligand structural motifs to increase the likelihood of developing powerful bio-therapeutics.

Address: Centre for Tumour Biology, Barts Cancer Institute, Queen Mary's University of London, London, United Kingdom

3915897d: PLoS One, e70399-e70399(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The transmembrane protein of the human endogenous retrovirus--K (HERV-K) modulates cytokine release and gene expression.
Author: Morozov,V.A., Dao Thi,V.L., Denner,J.
Abstract:
Numerous copies of endogenous retroviruses are present in the genome of mammals including man. Although most of them are defective, some, e.g., the human endogenous retroviruses HERV-K, were found to be expressed under certain physiological conditions. For instance, HERV-K is expressed in germ cell tumours and melanomas as well as in the placenta. Most exogenous retroviruses including the human immunodeficiency virus HIV-1 induce severe immunodeficiencies and there is increasing evidence that the transmembrane envelope (TM) proteins of these retroviruses may be involved. We show here that HERV-K particles released from a human teratocarcinoma cell line, a recombinant TM protein and a peptide corresponding to a highly conserved so-called immunosuppressive domain in the TM protein of HERV-K inhibit the proliferation of human immune cells, induce modulation of the expression of numerous cytokines, and modulate the expression of cellular genes as detected by a microarray analysis. The changes in cytokine release and gene expression induced by the TM protein of HERV-K are similar to those found previously induced by the TM protein of HIV-1. These data suggest that the mechanism of immunosuppression may be similar for different retroviruses and that the expression of the TM protein in tumours and in the placenta may suppress immune responses and thus prevent rejection of the tumour and the embryo.

Address: Robert Koch Institute, Berlin, Germany

3915899d: Front.Endocrinol., 98-98(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: IGF-I, IGF-II, and Insulin Stimulate Different Gene Expression Responses through Binding to the IGF-I Receptor.
Author: Versteyhe,S., Klaproth,B., Borup,R., Palsgaard,J., Jensen,M., Gray,S.G., De Meyts,P.
Abstract:
Insulin and the insulin-like growth factors (IGF)-I and -II are closely related peptides important for regulation of metabolism, growth, differentiation, and development. The IGFs exert their main effects through the IGF-I receptor. Although the insulin receptor is the main physiological receptor for insulin, this peptide hormone can also bind at higher concentrations to the IGF-I receptor and exert effects through it. We used microarray gene expression profiling to investigate the gene expression regulated by IGF-I, IGF-II, and insulin after stimulation of the IGF-I receptor. Fibroblasts from mice, knockout for IGF-II and the IGF-II/cation-independent mannose-6-phosphate receptor, and expressing functional IGF-I but no insulin receptors, were stimulated for 4 h with equipotent saturating concentrations of insulin, IGF-I, and IGF-II. Each ligand specifically regulated a group of transcripts that was not regulated by the other two ligands. Many of the functions and pathways these regulated genes were involved in, were consistent with the known biological effects of these ligands. The differences in gene expression might therefore account for some of the different biological effects of insulin, IGF-I, and IGF-II. This work adds to the evidence that not only the affinity of a ligand determines its biological response, but also its nature, even through the same receptor.

Address: Receptor Systems Biology Laboratory, Hagedorn Research Institute, Novo Nordisk A/S , Gentofte , Denmark

3915903d: J.Biol.Chem., 29090-29104(2013)
Link: [ PubMED ] [ DOI ]
Title: Human milk hyaluronan enhances innate defense of the intestinal epithelium.
Author: Hill,D.R., Rho,H.K., Kessler,S.P., Amin,R., Homer,C.R., McDonald,C., Cowman,M.K., De la Motte,C.A.
Abstract:
Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.

Address: From the Department of Molecular Medicine and

3915908d: Nat.Protoc., 1730-1742(2013)
Link: [ PubMED ] [ DOI ]
Title: Analysis of the proteolysis of bioactive peptides using a peptidomics approach.
Author: Kim,Y.G., Lone,A.M., Saghatelian,A.
Abstract:
Identifying the peptidases that inactivate bioactive peptides (e.g., peptide hormones and neuropeptides) in mammals is an important unmet challenge. This protocol describes a recent approach that uses liquid chromatography-mass spectrometry (LC-MS) peptidomics to identify endogenous cleavage sites of a bioactive peptide; it also addresses the subsequent biochemical purification of a candidate peptidase on the basis of these cleavage sites and the validation of the candidate peptidase's role in the physiological regulation of the bioactive peptide by examining a peptidase-knockout mouse. We highlight the successful application of this protocol in the discovery that insulin-degrading enzyme (IDE) regulates physiological calcitonin gene-related peptide (CGRP) levels, and we detail the key stages and steps in this approach. This protocol requires 7 d of work; however, the total time for this protocol is highly variable because of its dependence on the availability of biological reagents such as purified enzymes and knockout mice. The protocol is valuable because it expedites the characterization of mammalian peptidases, such as IDE, which in certain instances can be used to develop novel therapeutics.

Address: Department of Chemical Engineering, Soongsil University, Seoul, South Korea

3915928d: Mol.Vis., 1834-1840(2013)
Link: [ PubMED ] [ OpenAccess]
Title: A novel missense mutation in the FERM domain containing 7 (FRMD7) gene causing X-linked idiopathic congenital nystagmus in a Chinese family.
Author: Liu,Z., Mao,S., Pu,J., Ding,Y., Zhang,B., Ding,M.
Abstract:
PURPOSE: Idiopathic congenital nystagmus (ICN) is a genetically heterogeneous disease. Thus far, the disease gene has been identified as the FERM domain containing 7 (FRMD7) gene. The purpose of this study was to elucidate the clinical and genetic characteristics of a four- generation Chinese family with ICN. METHODS: The clinical data and the genomic DNA of a Chinese ICN family were collected following the provision of informed consent. All coding exons of the FRMD7 gene were amplified by PCR and then sequenced. Af fi nity GST-p21 activated kinase 2 (PAK2) precipitation was used to investigate whether this novel FRMD7 mutant influenced Rac1 signaling activation in the human embryonic kidney 293 T cells (HEK 293T) cells transiently cotransfected with wild-type or mutant FRMD7 and Rac1. RESULTS: A novel missense mutation (c.635T>C) was identified in all affected members. Obligate female carriers were heterozygous in these mutations and the affected males were homozygous, consistent with X-linked inheritance. This mutation is a substitution of proline for leucine. Function analysis showed that this novel mutant influences Rac1 signaling in human HEK 293T cells. CONCLUSIONS: This study widens the mutation spectrum of the FRMD7 gene. This mutant was shown to activate GTPase Rac1 signaling in vitro; however, the quantity of activated Rac1 was obviously decreased compared with the wild type (p<0.05). Taken together, our data strongly support the hypothesis that the identified FRMD7 mutant influences GTPase Rac1 signaling, which regulates neurite development. This mutation may be related to the pathogenesis of X-linked ICN.

Address: Department of Neurology, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China

3915933d: BMC Complement.Altern.Med., 208-208(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Cytotoxicity of Brazilian plant extracts against oral microorganisms of interest to dentistry.
Author: De Oliveira,J.R., De Castro,V.C., Das Gracas Figueiredo Vilela,P., Camargo,S.E., Carvalho,C.A., Jorge,A.O., De Oliveira,L.D.
Abstract:
BACKGROUND: With the emergence of strains resistant to conventional antibiotics, it is important to carry studies using alternative methods to control these microorganisms causing important infections, such as the use of products of plant origin that has demonstrated effective antimicrobial activity besides biocompatibility. Therefore, this study aimed to evaluate the antimicrobial activity of plant extracts of Equisetum arvense L., Glycyrrhiza glabra L., Punica granatum L. and Stryphnodendron barbatimam Mart. against Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Candida albicans, Candida tropicalis, and Candida glabrata, and to analyze the cytotoxicity of these extracts in cultured murine macrophages (RAW 264.7). METHODS: Antimicrobial activity of plant extracts was evaluated by microdilution method based on Clinical and Laboratory Standards Institute (CLSI), M7-A6 and M27-A2 standards. The cytotoxicity of concentrations that eliminated the microorganisms was evaluated by MTT colorimetric method and by quantification of proinflammatory cytokines (IL-1β and TNF-α) using ELISA. RESULTS: In determining the miniμm microbicidal concentration, E. arvense L., P. granatum L., and S. barbatimam Mart. extracts at a concentration of 50 mg/mL and G. glabra L. extract at a concentration of 100 mg/mL, were effective against all microorganisms tested. Regarding cell viability, values were 48% for E. arvense L., 76% for P. granatum L, 86% for S. barbatimam Mart. and 79% for G. glabra L. at the same concentrations. About cytokine production after stimulation with the most effective concentrations of the extracts, there was a significant increase of IL-1β in macrophage cultures treated with S. barbatimam Mart. (3.98 pg/mL) and P. granatum L. (7.72 pg/mL) compared to control (2.20 pg/mL) and a significant decrease of TNF-α was observed in cultures treated with G. glabra L. (4.92 pg/mL), S. barbatimam Mart. (0.85 pg/mL), E. arvense L. (0.83 pg/mL), and P. granatum L. (0.00 pg/mL) when compared to control (41.96 pg/mL). CONCLUSIONS: All plant extracts were effective against the microorganisms tested. The G. glabra L. extract exhibited least cytotoxicity and the E. arvense L. extract was the most cytotoxic.

E-Mail: jroliveira16@hotmail.com
Address: Department of Biosciences and Oral Diagnosis, Laboratory of Microbiology and Immunology, Institute of Science and Technology, Univ Estadual Paulista/UNESP, Av, Engenheiro Francisco Jose Longo, 777-Jardim Sao Dimas, Sao Jose dos Campos, SP, Brazil

3915938d: Anal.Chem., 8585-8593(2013)
Link: [ PubMED ] [ DOI ]
Title: Absolute quantitation of immunoglobulin G and its glycoforms using multiple reaction monitoring.
Author: Hong,Q., Lebrilla,C.B., Miyamoto,S., Ruhaak,L.R.
Abstract:
Studies aimed toward glycan biomarker discovery have focused on glycan characterization by the global profiling of released glycans. Site-specific glycosylation analysis is less developed but may provide new types of biomarkers with higher sensitivity and specificity. Quantitation of peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with specific proteins at distinct sites. We have developed a method using MRM to monitor protein glycosylation normalized to absolute protein concentrations to examine quantitative changes in glycosylation at a site-specific level. This new approach provides information regarding both the absolute amount of protein and the site-specific glycosylation profile and will thus be useful to determine if altered glycosylation profiles in serum/plasma are due to a change in protein glycosylation or a change in protein concentration. The remarkable sensitivity and selectivity of MRM enable the detection of low abundance IgG glycopeptides, even when IgG was digested directly in serum with no cleanup prior to the liquid chromatography. Our results show a low limit of detection of 60 amol and a wide dynamic range of 3 orders magnitude for IgG protein quantitation. The results show that IgG glycopeptides can be analyzed directly from serum (without enrichment) and yield more accurate abundances when normalized to the protein content. This report represents the most comprehensive study so far of the use of multiple reaction monitoring for the quantitation of glycoproteins and their glycosylation patterns in biofluids.

Address: Department of Chemistry, University of California , One Shields Avenue, Davis, California, 95616, United States

3915940d: Proteomics, 2947-2955(2013)
Link: [ PubMED ] [ DOI ]
Title: Increased proteome coverage by combining PAGE and peptide isoelectric focusing: Comparative study of gel-based separation approaches.
Author: Atanassov,I., Urlaub,H.
Abstract:
The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.

Address: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany

3915941d: Proteomics, 2967-2975(2013)
Link: [ PubMED ] [ DOI ]
Title: Improving peptide relative quantification in MALDI-TOF MS for biomarker assessment.
Author: Albalat,A., Stalmach,A., Bitsika,V., Siwy,J., Schanstra,J.P., Petropoulos,A.D., Vlahou,A., Jankowski,J., Persson,F., Rossing,P., Jaskolla,T.W., Mischak,H., Husi,H.
Abstract:
Proteomic profiling by MALDI-TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI-TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI-TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI-TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).

Address: Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, Scotland, UK

3915942d: J.Am.Soc.Mass Spectrom., 1555-1564(2013)
Link: [ PubMED ] [ DOI ]
Title: Fragmentation reactions of methionine-containing protonated octapeptides and fragment ions therefrom: an energy-resolved study.
Author: Harrison,A.G.
Abstract:
The fragmentation reactions of the MH+ ions as well as the b7, a7, and a7* ions derived therefrom have been studied in detail for the octapeptides MAAAAAAA, AAMAAAAA, AAAAMAAA, and AAAAAAMA. Ionization was by electrospray using a QqToF mass spectrometer, which allowed a study of the evolution of the fragmentation channels as a function of the collision energy. Not surprisingly, the product ion mass spectra for the b7 ions are independent of the original precursor sequence, indicating macrocyclization and reopening to the same mixture of protonated oxazolones prior to fragmentation. The results show that this sequence scrambling results in a distinct preference to place the Met residue in the C-terminal position of the protonated oxazolones. The a7 and a7* ions also produce product ion mass spectra independent of the original peptide sequence. The results for the a7 ions indicate that fragmentation occurs primarily from an amide structure analogous to that observed for a4 ions (Bythell et al. in J Am Chem Soc 132:14766-14779, 2010). Clearly, the rearrangement reaction they have proposed applies equally well to an ions as large as a7. The major fragmentation modes of the MH+ ions at low collision energies produce b7, b6, and b5 ions. As the collision energy is increased further fragmentation of these primary products produces, in part, non-direct sequence ions, which become prominent at lower m/z values, particularly for the peptides with the Met residue near the N-terminus.

E-Mail: aharriso@chem.utoronto.ca
Address: Department of Chemistry, University of Toronto, Toronto, ON, M5S 3H6, Canada,

3915943d: J.Am.Soc.Mass Spectrom., 1477-1488(2013)
Link: [ PubMED ] [ DOI ]
Title: Color matters--material ejection and ion yields in UV-MALDI mass spectrometry as a function of laser wavelength and laser fluence.
Author: Soltwisch,J., Jaskolla,T.W., Dreisewerd,K.
Abstract:
The success of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as a widely employed analytical tool in the biomolecular sciences builds strongly on an effective laser-material interaction that is resulting in a soft co-desorption and ionization of matrix and imbedded biomolecules. To obtain a maximized ion yield for the analyte(s) of interest, in general both wavelength and fluence need to be tuned to match the specific optical absorption profile of the used matrix. However, commonly only lasers with fixed emission wavelengths of either 337 or 355 nm are used for MALDI-MS. Here, we employed a wavelength-tunable dye laser and recorded both the neutral material ejection and the MS ion data in a wide wavelength and fluence range between 280 and 377.5 nm. α-Cyano-4-hydroxycinnamic acid (HCCA), 4-chloro-α-cyanocinnamic acid (ClCCA), α-cyano-2,4-difluorocinnamic acid (DiFCCA), and 2,5-dihydroxybenzoic acid (DHB) were investigated as matrices, and several peptides as analytes. Recording of the material ejection was achieved by adopting a photoacoustic approach. Relative ion yields were derived by division of photoacoustic and ion signals. In this way, distinct wavelength/fluence regions can be identified for which maxiμm ion yields were obtained. For the tested matrices, optimal results were achieved for wavelengths corresponding to areas of high optical absorption of the respective matrix and at fluences about a factor of 2-3 above the matrix- and wavelength-dependent ion detection threshold fluences. The material ejection as probed by the photoacoustic method is excellently fitted by the quasithermal model, while a sigmoidal function allows for an empirical description of the ion signal-fluence relationship.

Address: Institute for Hygiene, Westfalische Wilhelms-Universitat Munster, 48149, Munster, Germany

3915950d: Anal.Chem., 8483-8487(2013)
Link: [ PubMED ] [ DOI ]
Title: Hydrophilic polydopamine-coated graphene for metal ion immobilization as a novel immobilized metal ion affinity chromatography platform for phosphoproteome analysis.
Author: Yan,Y., Zheng,Z., Deng,C., Li,Y., Zhang,X., Yang,P.
Abstract:
To discover trace phosphorylated proteins or peptides with great biological significance for in-depth phosphoproteome analysis, it is urgent to develop a novel technique for highly selective and effective enrichment of phosphopeptides. In this work, an IMAC (immobilized metal ion affinity chromatography) material with polydopamine coated on the surface of graphene and functionalized with titanium ions (denoted as Ti(4+)-G@PD) was initially designed and synthesized. The newly prepared Ti(4+)-G@PD with enhanced hydrophilicity and biological compatibility was characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and infrared (IR), and its performance for selective and effective enrichment of phosphopeptide was evaluated with both standard peptide mixtures and human serum.

Address: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University , Shanghai 200433, China

3915957d: J.Biol.Chem., 28418-28427(2013)
Link: [ PubMED ] [ DOI ]
Title: Structural Insight into Golgi Membrane Stacking by GRASP65 and GRASP55 Proteins.
Author: Feng,Y., Yu,W., Li,X., Lin,S., Zhou,Y., Hu,J., Liu,X.
Abstract:
The stacking of Golgi cisternae involves GRASP65 and GRASP55. The oligomerization of the N-terminal GRASP domain of these proteins, which consists of two tandem PDZ domains, is required to tether the Golgi membranes. However, the molecular basis for GRASP assembly is unclear. Here, we determined the crystal structures of the GRASP domain of GRASP65 and GRASP55. The structures reveal similar homotypic interactions: the GRASP domain forms a dimer in which the peptide-binding pockets of the two neighboring PDZ2 domains face each other, and the dimers are further connected by the C-terminal tail of one GRASP domain inserting into the binding pocket of the PDZ1 domain in another dimer. Biochemical analysis suggests that both types of contacts are relatively weak but are needed in combination for GRASP-mediated Golgi stacking. Our results unveil a novel mode of membrane tethering by GRASP proteins and provide insight into the mechanism of Golgi stacking.

Address: From the Department of Biochemistry and Molecular Biology, College of Life Sciences, and State Key Laboratory of Medicinal Chemical Biology and

3915958d: J.Biol.Chem., 27434-27443(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of the C3a Receptor (C3AR1) as the Target of the VGF-derived Peptide TLQP-21 in Rodent Cells.
Author: Hannedouche,S., Beck,V., Leighton-Davies,J., Beibel,M., Roma,G., Oakeley,E.J., Lannoy,V., Bernard,J., Hamon,J., Barbieri,S., Preuss,I., Lasbennes,M.C., Sailer,A.W., Suply,T., Seuwen,K., Parker,C.N., Bassilana,F.
Abstract:
TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.

Address: From Novartis AG, Novartis Campus, CH-4056 Basel, Switzerland and

3915964d: Anal.Biochem., 27-33(2013)
Link: [ PubMED ] [ DOI ]
Title: Evaluation on the effect of different in-gel peptide isoelectric focusing parameters in global proteomic profiling.
Author: Kam,K.T., Liang,X., Wang,J.R., Wong,K.W., Hsiao,W.L., Zhou,H., Jiang,Z.H., Liu,L.
Abstract:
Peptide isoelectric focusing (IEF) is a common technique used in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) proteomic workflow, in which the tryptic peptide is first pre-fractionated based on pI values before being subjected to reverse phase LC-MS analysis. Although this method has been widely used by many research groups, a systemic study on the optimal conditions and fundamental parameters influencing the experimental outcomes has been lacking, including the effect of peptide extraction methods, the extent of pre-fractionation, and the choice of pH range. In this study, we compared the effect of different parameters on the numbers of peptides and proteins identified using two complex mouse proteomes. The results indicated that extraction of peptides from immobilized pH gradient (IPG) strips by sequential elution of increasingly organic solvents provided the highest number of peptide identification. In addition, we showed that approximately 45 more unique proteins were identified for every additional fraction collected during peptide IEF. Although narrow pH ranges provided higher resolution in peptide separation as expected, different pH ranges yielded similar numbers of peptide and protein identification. Overall, we demonstrated that the extraction solvent influenced the numbers of peptide and protein identification and quantitatively demonstrated the advantage of extensive fractionation and the performance of different pH ranges in practice.

Address: State Key Laboratory of Quality Research in Chinese Medicine/Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China

3915966d: J.Mol.Biol., 4479-4495(2013)
Link: [ PubMED ] [ DOI ]
Title: Structural Basis for the Interaction of Human β-Defensin 6 and Its Putative Chemokine Receptor CCR2 and Breast Cancer Microvesicles.
Author: De Paula,V.S., Gomes,N.S., Lima,L.G., Miyamoto,C.A., Monteiro,R.Q., Almeida,F.C., Valente,A.P.
Abstract:
Human β-defensins (hBDs) are believed to function as alarm molecules that stimulate the adaptive immune system when a threat is present. In addition to its antimicrobial activity, defensins present other activities such as chemoattraction of a range of different cell types to the sites of inflammation. We have solved the structure of the hBD6 by NMR spectroscopy that contains a conserved β-defensin domain followed by an extended C-terminus. We use NMR to monitor the interaction of hBD6 with microvesicles shed by breast cancer cell lines and with peptides derived from the extracellular domain of CC chemokine receptor 2 (Nt-CCR2) possessing or not possessing sulfation on Tyr26 and Tyr28. The NMR-derived model of the hBD6/CCR2 complex reveals a contiguous binding surface on hBD6, which comprises amino acid residues of the α-helix and β2-β3 loop. The microvesicle binding surface partially overlaps with the chemokine receptor interface. NMR spin relaxation suggests that free hBD6 and the hBD6/CCR2 complex exhibit microsecond-to-millisecond conformational dynamics encompassing the CCR2 binding site, which might facilitate selection of the molecular configuration optimal for binding. These data offer new insights into the structure-function relation of the hBD6-CCR2 interaction, which is a promising target for the design of novel anticancer agents.

Address: Centro Nacional de Ressonancia Magnetica Nuclear de Macromoleculas, Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

3915968d: Anal.Chem., 8039-8045(2013)
Link: [ PubMED ] [ DOI ]
Title: Integrated sample preparation methodology for proteomics: analysis of native proteins.
Author: Kim,J.H., Inerowicz,D., Hedrick,V., Regnier,F.
Abstract:
An innovative sample preparation strategy is reported for protein identification in complex mixtures based on integration of affinity chromatographic selection and accelerated trypsin digestion using a continuous flow immobilized enzyme reactor (cf-IMER). Affinity selected glycoproteins were released to a cf-IMER column which converted native proteins to peptides in 5 min at elevated temperature. Digestion with the cf-IMER was compared to the traditional 16 h solution-based trypsin digestion of reduced and alkylated proteins. With immobilized antibody selection of Lewis x (Le(x)) glycan bearing glycoproteins from plasma, 66 proteins were identified in total with the two methods while approximately 1/3 of the total proteins and peptides were only observed with the cf-IMER. This suggests that proteomics based on protein identification by reduction and alkylation with solution-based trypsin digestion alone may not be identifying large numbers of proteins or peptides present at detectable levels in samples. Furthermore, except for proteins containing a high content of disulfide bonds, the majority of proteins did not require reduction and alkylation steps for their identification. The validity of the proposed proteolysis was evaluated in several ways by analyses of a model protein and yeast lysates where the reproducibility of quantification was essentially the same with both cf-IMER and solution-based proteolysis.

Address: Department of Chemistry, Purdue University, West Lafayette, Indiana 47906, USA

3915973d: J.Biol.Chem., 29013-29023(2013)
Link: [ PubMED ] [ DOI ]
Title: Autoimmunity against INS-IGF2 Protein Expressed in Human Pancreatic Islets.
Author: Kanatsuna,N., Taneera,J., Vaziri-Sani,F., Wierup,N., Larsson,H.E., Delli,A., Skarstrand,H., Balhuizen,A., Bennet,H., Steiner,D.F., Torn,C., Fex,M., Lernmark,A.
Abstract:
Insulin is a major autoantigen in islet autoimmunity and progression to type 1 diabetes. It has been suggested that the insulin B-chain may be critical to insulin autoimmunity in type 1 diabetes. INS-IGF2 consists of the preproinsulin signal peptide, the insulin B-chain, and eight amino acids of the C-peptide in addition to 138 amino acids from the IGF2 gene. We aimed to determine the expression of INS-IGF2 in human pancreatic islets and autoantibodies in newly diagnosed children with type 1 diabetes and controls. INS-IGF2, expressed primarily in β cells, showed higher levels of expression in islets from normal compared with donors with either type 2 diabetes (p = 0.006) or high HbA1c levels (p < 0.001). INS-IGF2 autoantibody levels were increased in newly diagnosed patients with type 1 diabetes (n = 304) compared with healthy controls (n = 355; p < 0.001). Displacement with cold insulin and INS-IGF2 revealed that more patients than controls had doubly reactive insulin-INS-IGF2 autoantibodies. These data suggest that INS-IGF2, which contains the preproinsulin signal peptide, the B-chain, and eight amino acids of the C-peptide may be an autoantigen in type 1 diabetes. INS-IGF2 and insulin may share autoantibody-binding sites, thus complicating the notion that insulin is the primary autoantigen in type 1 diabetes.

Address: From the Department of Clinical Sciences, Lund University Diabetes Center, Lund University, Skane University Hospital SUS, SE-205 02 Malmo, Sweden and

3915975d: J.Bacteriol., 4678-4688(2013)
Link: [ PubMED ] [ DOI ]
Title: The MerR-Like Regulator BrlR Impairs Pseudomonas aeruginosa Biofilm Tolerance to Colistin by Repressing PhoPQ.
Author: Chambers,J.R., Sauer,K.
Abstract:
While the MerR-like transcriptional regulator BrlR has been demonstrated to contribute to Pseudomonas aeruginosa biofilm tolerance to antimicrobial agents known as multidrug efflux pump substrates, the role of BrlR in resistance to cationic antimicrobial peptides (CAP), which is based on reduced outer membrane susceptibility, is not known. Here, we demonstrate that inactivation of brlR coincided with increased resistance of P. aeruginosa to colistin, while overexpression of brlR resulted in increased susceptibility. brlR expression correlated with reduced transcript abundances of phoP, phoQ, pmrA, pmrB, and arnC. Inactivation of pmrA and pmrB had no effect on the susceptibility of P. aeruginosa biofilms to colistin, while inactivation of phoP and phoQ rendered biofilms more susceptible than the wild type. The susceptibility phenotype of ΔphoP biofilms to colistin was comparable to that of P. aeruginosa biofilms overexpressing brlR. BrlR was found to directly bind to oprH promoter DNA of the oprH-phoPQ operon. BrlR reciprocally contributed to colistin and tobramycin resistance in P. aeruginosa PAO1 and CF clinical isolates, with overexpression of brlR resulting in increased tobramycin MICs and increased tobramycin resistance but decreased colistin MICs and increased colistin susceptibility. The opposite trend was observed upon brlR inactivation. The difference in susceptibility to colistin and tobramycin was eliminated by combination treatment of biofilms with both antibiotics. Our findings establish BrlR as an unusual member of the MerR family, as it not only functions as a multidrug transport activator, but also acts as a repressor of phoPQ expression, thus suppressing colistin resistance.

Address: Department of Biological Sciences, Binghamton University, Binghamton, New York, USA

3915976d: J.Bacteriol., 4709-4715(2013)
Link: [ PubMED ] [ DOI ]
Title: Mapping of the SecA Signal Peptide Binding Site and Dimeric Interface by Using the Substituted Cysteine Accessibility Method.
Author: Bhanu,M.K., Zhao,P., Kendall,D.A.
Abstract:
SecA is an ATPase nanomotor critical for bacterial secretory protein translocation. Secretory proteins carry an amino-terminal signal peptide that is recognized and bound by SecA followed by its transfer across the SecYEG translocon. While this process is crucial for the onset of translocation, exactly where the signal peptide interacts with SecA is unclear. SecA protomers also interact among themselves to form dimers in solution, yet the oligomeric interface and the residues involved in dimerization are unknown. To address these issues, we utilized the substituted cysteine accessibility method (SCAM); we generated a library of 23 monocysteine SecA mutants and probed for the accessibility of each mutant cysteine to maleimide-(polyethylene glycol)2-biotin (MPB), a sulfhydryl-labeling reagent, both in the presence and absence of a signal peptide. Dramatic differences in MPB labeling were observed, with a select few mutants located at the preprotein cross-linking domain (PPXD), the helical wing domain (HWD), and the helical scaffold domain (HSD), indicating that the signal peptide binds at the groove formed between these three domains. The exposure of this binding site is varied under different conditions and could therefore provide an ideal mechanism for preprotein transfer into the translocon. We also identified residues G793, A795, K797, and D798 located at the two-helix finger of the HSD to be involved in dimerization. Adenosine-5'-(γ-thio)-triphosphate (ATPγS) alone and, more extensively, in conjunction with lipids and signal peptides strongly favored dimer dissociation, while ADP supports dimerization. This study provides key insight into the structure-function relationships of SecA preprotein binding and dimer dissociation.

Address: Department of Molecular and Cell Biology

3915979d: Anal.Sci., 805-810(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Determining the concentration of procalcitonin using a magnetic particles-based chemiluminescence assay for the clinical diagnosis of sepsis.
Author: Qi,S., Li,Q., Rao,W., Liu,X., Yin,L., Zhang,H.
Abstract:
Our objective is to develop an assay based on magnetic particles (MPs) to determine the concentration of procalcitonin (PCT) using a chemiluminescence immunoassay (CLIA). Fluorescein isothiocyanate (FITC) and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) were used to label two different anti-procalcitonin (PCT) monoclonal antibodies. The labeled antibodies, the PCT antigen, and the anti-FITC antibody-coated MPs formed a double-sandwiched immunocomplex. The measured relative light units (RLUs) of ABEI in the substrate solution were directly proportional to the amount of PCT present in the samples. The proposed method was linear to 600 ng/mL with a detection limit of 0.03 ng/mL. The coefficient of variation (CV) was <5% and <6% for the intra- and inter-assay precision, respectively. The average recoveries were between 95 and 107%. The linearity-dilution effect gave a linear correlation coefficient of 0.9912. This proposed assay provided an alternative method to quantitatively measure PCT in serum for the diagnosis of sepsis.

Address: Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, Department of Biomedical Engineering, School of Medicine, Shenzhen University, Shenzhen 518060, PR China

3915980d: Appl.Environ.Microbiol., 6293-6300(2013)
Link: [ PubMED ] [ DOI ]
Title: Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method.
Author: Almeida,C., Sousa,J.M., Rocha,R., Cerqueira,L., Fanning,S., Azevedo,N.F., Vieira,M.J.
Abstract:
Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 x 10(-2) to 1 x 10(2) CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.

Address: IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal

3915981d: Anal.Bioanal.Chem., 7679-7686(2013)
Link: [ PubMED ] [ DOI ]
Title: A new label-free approach for the determination of reaction rates in oxidative footprinting experiments.
Author: Pilau,E.J., Iglesias,A.H., Gozzo,F.C.
Abstract:
Hydroxyl radical-mediated oxidative footprinting coupled to mass spectrometric analysis is an attractive technique for protein surface mapping, conformational changes monitoring, and protein-ligand interfaces mapping in solution. In this technique, a protein is oxidized by in situ-generated hydroxy radicals and the site and rate of oxidation can be determined by proteolysis followed by mass spectrometric analysis. Changes in peptide oxidation rate can then be correlated to the changes in solvent exposure, and information about conformational changes or interaction domains can be obtained. The method relies, therefore, on the accurate measurements of peptide oxidation rate. Here, we describe a new label-free method to determine the oxidation rate of peptides that is based on the consumption of the unoxidized peptide instead of measuring the formation of oxidized peptides. The reaction rate thus obtained presents a better linearity and lower variation when compared to the traditional method. The label-free method is also simpler to implement and automation can be achieved through label-free quantitation software.

E-Mail: ejpilau@uem.br
Address: Institute of Chemistry, University of Campinas and Instituto Nacional de Ciencia e Tecnologia de Bioanalitica, CP 6154, 13083-970, Campinas, Sao Paulo, Brazil,

3915984d: Int.J.Mol.Med., 938-944(2013)
Link: [ PubMED ] [ DOI ]
Title: Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization.
Author: Putranto,E.W., Murata,H., Yamamoto,K., Kataoka,K., Yamada,H., Futami,J., Sakaguchi,M., Huh,N.H.
Abstract:
The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid β. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling.

Address: Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 7008558, Japan

3915985d: Plant Cell, 2798-2812(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Parallel proteomic and phosphoproteomic analyses of successive stages of maize leaf development.
Author: Facette,M.R., Shen,Z., Bjornsdottir,F.R., Briggs,S.P., Smith,L.G.
Abstract:
We performed large-scale, quantitative analyses of the maize (Zea mays) leaf proteome and phosphoproteome at four developmental stages. Exploiting the developmental gradient of maize leaves, we analyzed protein and phosphoprotein abundance as maize leaves transition from proliferative cell division to differentiation to cell expansion and compared these developing zones to one another and the mature leaf blade. Comparison of the proteomes and phosphoproteomes suggests a key role for posttranslational regulation in developmental transitions. Analysis of proteins with cell wall- and hormone-related functions illustrates the utility of the data set and provides further insight into maize leaf development. We compare phosphorylation sites identified here to those previously identified in Arabidopsis thaliana. We also discuss instances where comparison of phosphorylated and unmodified peptides from a particular protein indicates tissue-specific phosphorylation. For example, comparison of unmodified and phosphorylated forms of PINFORMED1 (PIN1) suggests a tissue-specific difference in phosphorylation, which correlates with changes in PIN1 polarization in epidermal cells during development. Together, our data provide insights into regulatory processes underlying maize leaf development and provide a community resource cataloging the abundance and phosphorylation status of thousands of maize proteins at four leaf developmental stages.

Address: Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, California 92093

3916000d: Toxicon, 92-100(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular and biochemical characterization of a cytolysin from the Scorpaena plumieri (scorpionfish) venom: Evidence of pore formation on erythrocyte cell membrane.
Author: Gomes,H.L., Andrich,F., Fortes-Dias,C.L., Perales,J., Teixeira-Ferreira,A., Vassallo,D.V., Cruz,J.S., Figueiredo,S.G.
Abstract:
Previously, a potent hemolytic toxin (Sp-CTx - 121 kDa) was isolated from Atlantic Scorpionfish Scorpaena plumieri venom. In the present work, we aimed to elucidate the action mechanisms involved in the hemolytic activity induced by this toxin, but to achieve our goal we faced the need to optimize its purification procedure in order to improve its activity and protein recovery. In this new method, Sp-CTx was purified to homogeneity through a combination of sequential ammonium sulfate precipitation and two chromatographic steps: hydrophobic interaction (Butyl HP) and anion exchange (Synchropak SAX 300). Orbitrap mass spectrometry analysis revealed that the amino acids sequences determined to Sp-CTx peptides are shared by other hemolytic toxins from fish venoms. The hemolytic activity of Sp-CTx upon rabbit erythrocytes was attenuated in the presence of osmotic protectants (polyethylene glycol polymers), and molecules larger than 6 nm in diameter inhibited cell lysis. This result strongly suggests that Sp-CTx may be a pore-forming protein, since it lacks phospholipase A2 activity. All these results contribute to the better understanding of Sp-CTx molecular/cellular actions in envenomation caused by S. plumieri. The results are also in agreement with previous reports of structural and functional similarities among piscine hemolytic toxins.

Address: Programa de Pos-Graduacao em Ciencias Fisiologicas, Centro de Ciencias da Saude, Universidade Federal do Espirito Santo, Vitoria, ES, Brazil

3916001d: J.Proteomics, 375-384(2013)
Link: [ PubMED ] [ DOI ]
Title: Large precursor tolerance database search - A simple approach for estimation of the amount of spectra with precursor mass shifts in proteomic data.
Author: Weng,R.R., Chu,L.J., Shu,H.W., Wu,T.H., Chen,M.C., Chang,Y., Tsai,Y.S., Wilson,M.C., Tsay,Y.G., Goodlett,D.R., Ng,W.V.
Abstract:
Mass measurement and precursor mass assignment are independent processes in proteomic data acquisition. Due to misassignments to C-13 peak, or for other reasons, extensive precursor mass shifts (i.e., deviations of the measured from calculated precursor neutral masses) in LC-MS/MS data obtained with the high-accuracy LTQ-Orbitrap mass spectrometers have been reported in previous studies. Although computational methods for post-acquisition reassignment to monoisotopic mass have been developed to curate the MS/MS spectra prior to database search, a simpler method for estimating the fraction of spectra with precursor mass shift so as to determine whether the data require curation remains desirable. Here, we provide the evidence that an easy approach, which applies a large precursor tolerance (2.1Da or higher) in SEQUEST search against a forward and decoy protein sequence database and then filters the data with PeptideProphet peptide identification probability (p>/=0.9), could detect most of the MS/MS spectra containing inaccurate precursor masses. Furthermore, through the implementation of artificial mass shifts on 4000 randomly selected MS/MS spectra, which originally had accurate precursor mass assigned by the mass spectrometers, we demonstrated that the accuracy of the precursor mass has almost negligible influence on the efficacy and fidelity of peptide identification. BIOLOGICAL SIGNIFICANCE: Integral precursor mass shift is a known problem and thus proteomic data should be handled and analyzed properly to avoid losing important protein identification and/or quantification information. A quick and easy approach for estimating the number of MS/MS spectra with inaccurate precursor mass assignments would be helpful for evaluating the performance of the instrument, determining whether the data requires curation prior to database search or should be searched with specific search parameter(s). Here we demonstrated most of the MS/MS spectra with inaccurate mass assignments (integral or non-integral changes) that could be easily identified by database search with large precursor tolerance windows.

Address: Institute of Biotechnology in Medicine, National Yang Ming University, Taipei, Taiwan, ROC

3916004d: J.Virol.Methods, 706-712(2013)
Link: [ PubMED ] [ DOI ]
Title: Human defined antigenic region on the nucleoprotein of Crimean-Congo hemorrhagic fever virus identified using truncated proteins and a bioinformatics approach.
Author: Burt,F.J., Samudzi,R.R., Randall,C., Pieters,D., Vermeulen,J., Knox,C.M.
Abstract:
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne viral zoonosis widely distributed in Africa, Asia and eastern Europe. In this study, amino acid sequence data for the CCHFV nucleoprotein (NP) was used to identify potential linear epitopic regions which were subsequently included in the design of large and small truncated recombinant NP antigens and peptide libraries. Two truncated recombinant CCHFV NP antigens were prepared based on results of prediction studies to include epitopic regions and exclude hydrophobic regions that could influence protein expression and solubility. Serum samples were collected from acute and convalescent patients. An IgG antibody response was detected in 16/16 samples tested using the large recombinant NP-based ELISA and in 2/16 using the small recombinant NP-based ELISA. A total of 60 peptides covering predicted epitopic regions of the NP were synthesized and peptide NRGGDENPRGPVSR at amino acid position 182-195, reacted with 13/16 human serum samples. In summary, functional assays are required to determine the biological activity of predicted epitopes for development of peptide based assays for antibody detection. Bacterially expressed complete NP antigens have previously been shown to be useful tools for antibody detection. Truncation of the antigen to remove the hydrophobic C terminus had no impact on the ability of the antigen to detect IgG antibody in human sera. The results indicate that the region from amino acids 123 to 396 includes a highly antigenic region of the NP with application in development of antibody detection assays.

Address: Department of Medical Microbiology and Virology, Faculty of Health Sciences, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa

3916006d: Gen.Comp.Endocrinol., 130-140(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification, characterization, and expression profiles of two subtypes of kisspeptin receptors in a scombroid fish (chub mackerel).
Author: Ohga,H., Fujinaga,Y., Selvaraj,S., Kitano,H., Nyuji,M., Yamaguchi,A., Matsuyama,M.
Abstract:
The kisspeptin receptor (Kiss1R) is a cognate receptor for kisspeptin (Kiss), and this Kiss-Kiss1R system has been shown to regulate seasonal reproduction in vertebrates. Our previous study found the chub mackerel (Scomber japonicus) brain expresses both kiss1 and kiss2 and exhibits sexually dimorphic changes during the seasonal reproductive cycle. The present study cloned two subtypes of kissr from the chub mackerel brain, and their signal transduction pathways to Kiss1 and Kiss2 were characterized in a mammalian cell line. Results of identification showed that kissr1 and kissr2 mRNAs encode 369 and 378 deduced amino acids, respectively, and share 52% similarity in amino acid sequences. In vitro functional analysis revealed that chub mackerel Kiss receptor signals are also preferentially transduced via the protein kinase C (PKC) rather than protein kinase A (PKA) pathway. Synthetic chub mackerel Kiss1-15 and Kiss2-12 peptides showed the highest potency for the activation of KissR1 and KissR2, respectively, stronger than their corresponding Kiss-10 peptides. Tissue distribution analyses indicated that both genes are highly expressed in the brain and that only kissr2 mRNA is expressed in the pituitary of both sexes. Unexpectedly, both kissr1 and kissr2 mRNAs were detected only in the testes. Seasonal expression changes showed higher expression levels of both kissr1 and kissr2 mRNAs in the brain of females during the early vitellogenic period; however, no significant differences were found in the brain of males. Pituitary kissr2 mRNA levels showed no significant variations. In the testes, the kissr1 mRNA expression level increased dramatically at spermiation compared with the immature and post-spawning periods. However, kissr2 mRNA levels in the testes did not vary significantly at different testicular stages. These results suggest that both kissr1 and kissr2 likely participate in the seasonal ovarian development of females, and thus in males, we propose a paracrine or autocrine role for kissr1 in testicular development.

Address: Laboratory of Marine Biology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

3916007d: Virus Res., 147-155(2013)
Link: [ PubMED ] [ DOI ]
Title: Evaluation of viral peptide targeting to porcine sialoadhesin using a porcine reproductive and respiratory syndrome virus vaccination-challenge model.
Author: Ooms,K., Van Gorp,H., Botti,S., Van Gaever,T., Delputte,P.L., Nauwynck,H.J.
Abstract:
Targeting antigens to professional antigen presenting cells resident at the sites where effective immune responses are generated is a promising vaccination strategy. As such, targeting sialoadhesin (Sn)-expressing macrophages, abundantly present in spleen and lymph nodes where they appear to be strategically placed for antigen capture and processing, is recently gaining increased attention. Previously, we have shown that humoral immune responses to the model antigen human serum albumin can be enhanced by using a porcine Sn-specific monoclonal antibody to target the model antigen to Sn-expressing macrophages. To date however, no studies have been performed to evaluate whether targeted delivery of a pathogen-derived antigen can enhance the pathogen-specific immune response. Therefore, we selected a linear epitope on glycoprotein 4 of porcine reproductive and respiratory syndrome virus (PRRSV), which is known to be a target of virus-neutralizing antibodies. This paper reports on the targeted delivery of this viral peptide to porcine Sn-expressing macrophages and the evaluation of the subsequent immune response in a vaccination-challenge set-up. Four copies of the selected PRRSV epitope were genetically fused to a previously developed porcine Sn-targeting recombinant antibody or an irrelevant isotype control. Fusion proteins were shown to be efficiently purified from HEK293T cell supernatants and subsequently, only Sn-specific fusion proteins were shown to bind to and to be internalized into Sn-expressing cells. Subsequent immunizations with a single dose of the fusion proteins showed that peptide-specific immune responses and neutralizing antibody responses after PRRSV challenge were enhanced in animals receiving a single 500μg intramuscular dose of the Sn-targeting fusion protein, although correlations between the two read-outs were hard to effectuate. Furthermore, a minor beneficial effect on viral clearance was observed. Together, these data show that viral peptide targeting to porcine Sn-expressing macrophages can improve the anti-viral immune response, although more research will be needed to further explore vaccination potential.

E-Mail: Karen.Ooms@UGent.be
Address: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. Electronic address:

3916012d: Chem.Biol., 1067-1077(2013)
Link: [ PubMED ] [ DOI ]
Title: Capturing linear intermediates and C-terminal variants during maturation of the thiopeptide GE2270.
Author: Tocchetti,A., Maffioli,S., Iorio,M., Alt,S., Mazzei,E., Brunati,C., Sosio,M., Donadio,S.
Abstract:
Thiopeptides are ribosomally synthesized, posttranslationally modified peptides with potent activity against Gram-positives. However, only GE2270 has yielded semisynthetic derivatives under clinical investigations. The pbt gene cluster from the GE2270 producer Planobispora rosea was successfully expressed in the genetically tractable Nonomuraea ATCC39727. Gene deletions established that PbtO, PbtM1, PbtM2, PbtM3, and PbtM4 are involved in regiospecific hydroxylation and methylations of GE2270, leading to the generation of various derivatives with altered decorations. Further deletions established that PbtH and PbtG1 are involved in C-terminal amide and oxazoline formation, respectively. Surprisingly, preventing either step resulted in the accumulation of linear precursors in which the pyridine-generated macrocycle failed to form, and only one of the pyridine-forming serine residues had been dehydrated. Often, these linear precursors present a shortened C terminus but retain the full set of methylation and hydroxylation decorations.

Address: KtedoGen Srl, Via Fantoli 16/15, 20138 Milan, Italy

3916013d: Chem.Biol., 1055-1066(2013)
Link: [ PubMED ] [ DOI ]
Title: Genetic manipulation of the Fusarium fujikuroi fusarin gene cluster yields insight into the complex regulation and fusarin biosynthetic pathway.
Author: Niehaus,E.M., Kleigrewe,K., Wiemann,P., Studt,L., Sieber,C.M., Connolly,L.R., Freitag,M., Guldener,U., Tudzynski,B., Humpf,H.U.
Abstract:
In this work, the biosynthesis and regulation of the polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS)-derived mutagenic mycotoxin fusarin C was studied in the fungus Fusarium fujikuroi. The fusarin gene cluster consists of nine genes (fus1-fus9) that are coexpressed under high-nitrogen and acidic pH conditions. Chromatin immunoprecipitation revealed a correlation between high expression and enrichment of activating H3K9-acetylation marks under inducing conditions. We provide evidence that only four genes are sufficient for the biosynthesis. The combination of genetic engineering with nuclear magnetic resonance and mass-spectrometry-based structure elucidation allowed the discovery of the putative fusarin biosynthetic pathway. Surprisingly, we indicate that PKS/NRPS releases its product with an open ring structure, probably as an alcohol. Our data indicate that 2-pyrrolidone ring closure, oxidation at C-20, and, finally, methylation at C-20 are catalyzed by Fus2, Fus8, and Fus9, respectively.

Address: Institute for Biology and Biotechnology of Plants, Westfalische Wilhelms-Universitat Munster, Schlossplatz 8, Munster 48143, Germany

3916018d: Biomaterials, 8798-8807(2013)
Link: [ PubMED ] [ DOI ]
Title: Therapeutic nanomedicine based on dual-intelligent functionalized gold nanoparticles for cancer imaging and therapy in vivo.
Author: Chen,W.H., Xu,X.D., Jia,H.Z., Lei,Q., Luo,G.F., Cheng,S.X., Zhuo,R.X., Zhang,X.Z.
Abstract:
A novel strategy to construct a therapeutic system based on functionalized AuNPs which can specifically respond to tumor microenvironment was reported. In the therapeutic system, doxorubicin was conjugated to AuNPs via thiol-Au bond by using a peptide substrate, CPLGLAGG, which can be specifically cleaved by the protease. In vivo study shows that after injection of the functionalized AuNPs to the tumor-bearing mice, the over-expressed protease of MMP-2 in tumor tissue and intracellular GSH can lead to the rapid release of the anti-tumor drug (doxorubicin) from the functionalized AuNPs to inhibit tumor growth and realize fluorescently imaging simultaneously. The functionalized AuNPs with tumor-triggered drug release property can further improve the efficacy and reduce side effects significantly.

Address: Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, PR China

3916021d: Anal.Chem., 8787-8795(2013)
Link: [ PubMED ] [ DOI ]
Title: Chemical library screening using a SPR-based inhibition in solution assay: simulations and experimental validation.
Author: Choulier,L., Nomine,Y., Zeder-Lutz,G., Charbonnier,S., Didier,B., Jung,M.L., Altschuh,D.
Abstract:
We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 μM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 μM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 μM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library(R) (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 μM range. We propose that a medium (μM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening.

Address: Biotechnologie et Signalisation Cellulaire, Universite de Strasbourg , CNRS, ESBS, Boulevard Sebastien Brant BP10413, 67412 Illkirch, France

3916023d: J.Liposome Res., 311-317(2013)
Link: [ PubMED ] [ DOI ]
Title: Formulation, characterization and permeability study of nano particles of lipo-endomorphin-1 for oral delivery.
Author: Eskandari,S., Varamini,P., Toth,I.
Abstract:
Three different formulations of a lipid-modified endomorphin-1 peptide (C10LAA-Endo-1) were prepared, characterized, and evaluated for their permeability through Caco-2 cell membranes. Solid lipid nanoparticles (SLN), enteric coated (EC), and the EC-SLN of C10LAA-Endo-1 is a modified structure of endomorphin-1 for oral delivery. Physico-chemical characterization of the formulations showed that among all formulations, EC-[C10LAA-Endo-1] had the lowest particle size and the highest EE% and absolute zeta potential. Release of drug from SLN, EC-SLN and EC-[C10LAA-Endo-1] in acid media was 14.30 (+/-2.7)%, 3.0 (+/-1.0)% and 10.2 (+/-3.0)%, respectively. Release data in buffer media (pH = 7.4) showed that enteric coated formulations released C10LAA-Endo-1 more slowly than uncoated formulations. It was also demonstrated that direct coating of C10LAA-Endo-1 with Eudragit(R) S100 significantly enhanced the permeability of the compound through Caco-2 cell membranes with a 39-fold higher Papp compared to C10LAA-Endo-1. These findings indicated that EC-C10LAA-Endo-1 is a promising candidate to promote the oral delivery of the previously modified endomorphin-1 peptide analogue and is worthy of future animal investigations.

Address: School of Chemistry and Molecular Biosciences (SCMB), University of Queensland , St Lucia, Brisbane, QLD 4072 , Australia

3916024d: Biomacromolecules, 3304-3313(2013)
Link: [ PubMED ] [ DOI ]
Title: Valency-dependent affinity of bioactive hydroxyapatite-binding dendrons.
Author: Tang,W., Ma,Y., Xie,S., Guo,K., Katzenmeyer,B., Wesdemiotis,C., Becker,M.L.
Abstract:
Hydroxyapatite (HA)-coated surfaces are used widely as stationary phase for protein and enzyme purification, coatings for dental and orthopedic implants, and composite materials for tissue engineering substrates. More advanced applications are envisioned, but progress has been slowed by the limited ability to controllably functionalize the surface of HA with biomolecules in a translationally relevant manner. Herein we report the synthesis and characterization of a series of multivalent, HA-binding peptide bioconjugates with variable valency and tether length which afford the ability to precisely tune the desired binding behavior. The respective binding affinities of the multivalent constructs to HA surface were characterized by quartz crystal microbalance with dissipation monitoring (QCM-D) techniques, and the relationship between dendron structure and binding affinity was revealed. Tetravalent constructs of HA-binding peptides show a 100-fold enhancement in binding affinity compared to HA-binding peptide sequences reported previously. Both biotin and bone morphogenic protein-2 (BMP-2) derivative peptide were successfully linked to the focal point as initial demonstrations.

Address: Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States

3916027d: Hum.Gene Ther., 777-785(2013)
Link: [ PubMED ] [ DOI ]
Title: Intravenous adeno-associated virus serotype 8 encoding urocortin-2 provides sustained augmentation of left ventricular function in mice.
Author: Gao,M.H., Lai,N.C., Miyanohara,A., Schilling,J.M., Suarez,J., Tang,T., Guo,T., Tang,R., Parikh,J., Giamouridis,D., Dillmann,W.H., Patel,H.H., Roth,D.M., Dalton,N.D., Hammond,H.K.
Abstract:
Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. Here we ask whether a single intravenous injection of adeno-associated virus type 8 encoding murine urocortin-2 (AAV8.UCn2) could provide long-term elevation in plasma UCn2 levels and increased left ventricular (LV) function. Normal mice received AAV8.UCn2 (5x10(1)(1) genome copies, intravenous). Plasma UCn2 increased 15-fold 6 weeks and >11-fold 7 months after delivery. AAV8 DNA and UCn2 mRNA expression was persistent in LV and liver up to 7 months after a single intravenous injection of AAV8.UCn2. Physiological studies conducted both in situ and ex vivo showed increases in LV +dP/dt and in LV -dP/dt, findings that endured unchanged for 7 months. SERCA2a mRNA and protein expression was increased in LV samples and Ca(2)+ transient studies showed an increased rate of Ca(2)+ decline in cardiac myocytes from mice that had received UCn2 gene transfer. We conclude that a single intravenous injection of AAV8.UCn2 increases plasma UCn2 and increases LV systolic and diastolic function for at least 7 months. The simplicity of intravenous injection of a long-term expression vector encoding a gene with paracrine activity to increase cardiac function is a potentially attractive strategy in clinical settings. Future studies will determine the usefulness of this approach in the treatment of heart failure.

Address: VA San Diego Healthcare System, San Diego, CA 92161, USA

3916035d: J.Microbiol.Biotechnol., 1386-1394(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The influence of the N-terminal region of antimicrobial peptide pleurocidin on fungal apoptosis.
Author: Choi,H., Lee,D.G.
Abstract:
In our previous study, the 25-mer antimicrobial peptide pleurocidin (Ple) had been thought to induce apoptosis in Candida albicans. This study demonstrated that reactive oxygen species (ROS) production was a major cause of Ple-induced apoptosis. Four truncated analogs were synthesized to understand the functional roles in the N- and C-terminal regions of Ple on the apoptosis. Ple, Ple (4-25), Ple (1-22), and Ple (1-19) produced ROS, including hydroxyl radicals, on the order of [Ple > Ple (1-22) > Ple (4-25) > Ple (1-19)], whereas Ple (7-25) did not induce any ROS production. The results suggested that the N-terminal deletion affected the ROS-inducing activities much more than that of the C-terminal deletion, and net hydrophobicity [Ple > Ple (1-22) > Ple (4-25) > Ple (1-19) > Ple (7-25)] was related to ROS generation rather than other primary factors like net charge. Hence, we focused on the N-terminal-truncated peptides, Ple (4-25) and Ple (7-25), and examined other apoptotic features, including mitochondrial membrane depolarization, caspase activation, phosphatidylserine externalization, and DNA and nuclear fragmentation. The results also confirmed the disappearance of apoptotic activity of Ple (7-25) by the truncation of the N-terminal region (1-6) and the specific activity patterns between Ple and analogs. In conclusion, the N-terminal region of Ple played an important role in apoptosis.

Address: School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea

3916039d: J.Virol.Methods, 667-673(2013)
Link: [ PubMED ] [ DOI ]
Title: Development of a peptide ELISA for discrimination between serological responses to equine herpesvirus type 1 and 4.
Author: Lang,A., De Vries,M., Feineis,S., Muller,E., Osterrieder,N., Damiani,A.M.
Abstract:
A peptide-based enzyme-linked immunosorbent assay (ELISA) for discrimination between serological responses to equine herpesvirus type 1 (EHV-1) and 4 (EHV-4) was developed. Three and four peptides for EHV-1 and EHV-4, respectively, were designed and studied initially in the ELISA using sera from foals infected experimentally. The most promising peptide pair, derived from EHV-1 glycoprotein E and EHV-4 glycoprotein G, was evaluated further using acute and convalescent sera from horses infected experimentally and naturally as well as a panel of horse field sera. Ten pre- and post-vaccination serum pairs were similarly tested in the type-specific ELISA. The peptide ELISA was able to identify horses which had been infected with EHV-1 or EHV-4 as derived from the results using acute and convalescent sera collected from natural outbreaks. When applied to a set of field samples, the assay proved robust with respect to determining the EHV-1 and EHV-4 antibody status. Also, the peptide ELISA was able to detect type-specific seroconversion for EHV-1 in vaccinated animals. With further validation, the EHV-1/EHV-4 peptide ELISA described in this study could serve as a reliable and cost-effective alternative to current methods for serological EHV-1 and EHV-4 diagnosis.

Address: Institut fur Virologie, Zentrum fur Infektionsmedizin, Robert von Ostertag-Haus, Freie Universitat Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany

3916042d: J.Proteome Res., 4176-4186(2013)
Link: [ PubMED ] [ DOI ]
Title: Combination of multistep IMAC enrichment with high-pH reverse phase separation for in-depth phosphoproteomic profiling.
Author: Yue,X.S., Hummon,A.B.
Abstract:
Typical mass spectrometric phosphoproteome studies are complicated by the need for large amounts of starting material and extensive sample preparation to ensure sufficient phosphopeptide identifications. In this paper, we present a novel strategy to perform optimized multistep IMAC enrichment from whole cell lysates followed by high-pH reverse phase fractionation (multi-IMAC-HLB; HLB means hydrophilic-lipophilic-balanced reversed-phase cartridge). The peptide-to-IMAC ratio was optimized to maximize IMAC performance, while multistep IMAC enrichment enabled improved phosphopeptide acquisition. The addition of the HLB step further fractionates the IMAC enriched phosphopeptides while desalting the samples, which dramatically reduces the sample manipulation time and sample loss compared to other popular strategies. We compared the phosphopeptide identification results of the multi-IMAC-HLB method with 3 mg of starting material to the well-established SCX-IMAC method with 15 mg of starting material. We identified 8969 unique phosphopeptides with the multi-IMAC-HLB method, compared to 5519 unique phosphopeptides identified with the SCX-IMAC method, an increase of 62.5%. The increase in the numbers of identified phosphopeptides is due to the increase in the ratio of identified phosphopeptides out of all detected peptides, 70.5% with multi-IMAC-HLB method compared to 32.3% with the SCX-IMAC method. Multi-IMAC-HLB is a robust and efficient method for in-depth phosphoproteomic research.

Address: Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556, United States

3916048d: J.Biol.Chem., 28347-28356(2013)
Link: [ PubMED ] [ DOI ]
Title: Structural Basis for the Regulation of the Mitogen-activated Protein (MAP) Kinase p38α by the Dual Specificity Phosphatase 16 MAP Kinase Binding Domain in Solution.
Author: Kumar,G.S., Zettl,H., Page,R., Peti,W.
Abstract:
Mitogen-activated protein kinases (MAPKs) fulfill essential biological functions and are key pharmaceutical targets. Regulation of MAPKs is achieved via a plethora of regulatory proteins including activating MAPKKs and an abundance of deactivating phosphatases. Although all regulatory proteins use an identical interaction site on MAPKs, the common docking and hydrophobic pocket, they use distinct kinase interaction motif (KIM or D-motif) sequences that are present in linear, peptide-like, or well folded protein domains. It has been recently shown that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a unique binding mode to interact with p38α. Here we describe the interaction of the MAPK binding domain of DUSP16 with p38α and show that despite belonging to the same dual specificity phosphatase (DUSP) family, its interaction mode differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an additional helix, α-helix 4, to further engage p38α. This leads to an additional interaction surface on p38α. Together, these structural and energetic differences in p38α engagement highlight the fine-tuning necessary to achieve MAPK specificity and regulation among multiple regulatory proteins.

Address: From the Departments of Molecular Pharmacology, Physiology and Biotechnology

3916050d: Chirality, 628-642(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.
Author: Byrne,C., Miclet,E., Broutin,I., Gallo,D., Pelekanou,V., Kampa,M., Castanas,E., Leclercq,G., Jacquot,Y.
Abstract:
Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation.

Address: Laboratoire des BioMolecules (LBM), CNRS - UMR 7203, Ecole Normale Superieure / Universite Pierre et Marie Curie 24, rue Lhomond, 75231, Paris Cedex 05, France

3916052d: Protein J., 456-466(2013)
Link: [ PubMED ] [ DOI ]
Title: Improved protease stability of the antimicrobial peptide Pin2 substituted with D-amino acids.
Author: Carmona,G., Rodriguez,A., Juarez,D., Corzo,G., Villegas,E.
Abstract:
Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a D-diastereomer of Pandinin 2, D-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of L- and D-Pin2 were to some extent similar, an important reduction in D-Pin2 hemolytic activity (30-40 %) was achieved compared to that of L-Pin2 over human erythrocytes. Furthermore, D-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of L-Pin2. Nevertheless, the antimicrobial activity of D-Pin2 was equally effective as that of L-Pin2 in microdilution assays. Yet, when D- and L-Pin2 were incubated with trypsin, elastase and whole human serum, only D-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, L- and D-Pin2 maintained similar peptide stability. Finally, when L- and D-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, D-Pin2 kept its antibiotic activity while L-Pin2 was not effective.

Address: Laboratorio de Estructura-Funcion e Ingenieria de Proteinas, Centro de Investigacion en Biotecnologia Universidad Autonoma del Estado de Morelos, Av. Universidad 2001, 62609, Cuernavaca, Morelos, Mexico

3916053d: Appl.Biochem.Biotechnol., 1011-1021(2013)
Link: [ PubMED ] [ DOI ]
Title: Proteomic analysis of sarcoplasmic peptides of two related fish species for food authentication.
Author: Barik,S.K., Banerjee,S., Bhattacharjee,S., Das Gupta,S.K., Mohanty,S., Mohanty,B.P.
Abstract:
Detection of species-specific sarcoplasmic peptides can be used as proteomic markers for fish food authentication and identification of species of origin in processed products. In the present study, proteomics technology was employed for differential characterization of sarcoplasmic peptides of two closely related fish species, Sperata seenghala and Sperata aor. Species-specific peptides were searched in white muscle extracts of the two species for identification of unique peptides that might aid in differentiation of the species, under two-dimensional gel electrophoresis platform. A total of 19 proteins were identified by combined matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry, of which nine and two proteins were found to be unique to S. seenghala and S. aor, respectively. One of the proteins, triosephosphate isomerase (TPI) was found to have three isoforms, out of which two were specific to S. aor, and one was specific to S. seenghala. All the three isoforms of TPI were present in the mixed samples of raw protein extracts of S. seenghala and S. aor, an observation that can be exploited to differentiate between the species and detection of deceptive practices of fraudulent substitution of commercially valuable fish species with inferior ones and differential characterization between closely related fish species.

Address: Biochemistry Laboratory, ICAR-Central Inland Fisheries Research Institute, Barrackpore, Kolkata, 700120, India

3916058d: Mol.Biol.Cell, 3123-3132(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences.
Author: Yabuki,T., Morimoto,F., Kida,Y., Sakaguchi,M.
Abstract:
Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10(-8) M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I-like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.

Address: Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo 678-1297, Japan

3916059d: MAbs, 678-689(2013)
Link: [ PubMED ] [ DOI ]
Title: Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency.
Author: Rivera-Hernandez,G., Marin-Argany,M., Blasco-Moreno,B., Bonet,J., Oliva,B., Villegas,S.
Abstract:
Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either meningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ 1-42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded VL domain and the folded VH domain, which suggests the VL domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the VL domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond VL-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest.

Address: Protein Folding and Stability Group

3916060d: Biosci.Biotechnol.Biochem., 1773-1775(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Discriminative phytoalexin accumulation in Lotus japonicus against symbiotic and non-symbiotic microorganisms and related chemical signals.
Author: Masai,M., Arakawa,M., Iwaya,K., Aoki,T., Nakagawa,T., Ayabe,S., Uchiyama,H.
Abstract:
The phytoalexin response of Lotus japonicus seedlings to selected microbes and chemical signals was analyzed. The symbiotic rhizobium induced vestitol production weakly, while non-symbiotic rhizobia and potential pathogens led to increases in its accumulation. Whereas chitin-related molecules were ineffective, a flagellin-derived peptide not of symbiont origin induced phytoalexin production, indicating discriminative antibiotic production by the plant host.

Address: Department of Applied Biological Sciences, Nihon University

3916065d: J.Proteome Res., 4152-4166(2013)
Link: [ PubMED ] [ DOI ]
Title: Candidate serum biomarkers for early intestinal cancer using 15N metabolic labeling and quantitative proteomics in the ApcMin/+ mouse.
Author: Ivancic,M.M., Huttlin,E.L., Chen,X., Pleiman,J.K., Irving,A.A., Hegeman,A.D., Dove,W.F., Sussman,M.R.
Abstract:
Current screening procedures for colorectal cancer are imperfect and highly invasive and result in increased mortality rates due to low compliance. The goal of the experiments reported herein is to identify potential blood-based biomarkers indicative of early stage intestinal cancers using the ApcMin/+ mouse model of intestinal cancer as an experimental system. Serum proteins from tumor-bearing ApcMin/+ mice were quantitatively compared to tumor-free Apc+/+ wild-type mice via in anima metabolic labeling with 14N/15N-labeled Spirulina algae and an LTQ Orbitrap mass spectrometer. Out of 1116 total serum proteins quantified, this study identified 40 that were differentially expressed and correlated with the increase in intestinal neoplasms. A subset of these differentially expressed proteins underwent a secondary quantitative screen using selected reaction monitoring-mass spectrometry with stable isotope-labeled peptides. Using both quantitative techniques, we identified MGAM and COL1A1 as downregulated and ITIH3 and F5 as upregulated in serum. All but COL1A1 were similarly differentially expressed in the mRNA of neoplastic colonic tissues of ApcMin/+ mice compared to normal wild-type tissue. These differentially expressed proteins identified in the ApcMin/+ mouse model have provided a set of candidate biomarkers for future validation screens in humans.

Address: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States

3916066d: Anal.Chem., 8436-8441(2013)
Link: [ PubMED ] [ DOI ]
Title: Fabrication of multifunctional Gd2O3/Au hybrid nanoprobe via a one-step approach for near-infrared fluorescence and magnetic resonance multimodal imaging in vivo.
Author: Sun,S.K., Dong,L.X., Cao,Y., Sun,H.R., Yan,X.P.
Abstract:
Facile fabrication of multimodal imaging probes is highly desired for bioimaging application due to their integrated advantages of several imaging modalities. Here, we report a simple and one-step mild strategy to fabricate a multifunctional Gd2O3/Au hybrid nanoprobe. Bovine serum albumin (BSA) was used as the template in the biomineralization synthesis. The fabricated BSA-Gd2O3/Au nanoprobe showed excellent chemical stability, intense near-infrared (NIR) fluorescence, and good magnetic resonance imaging (MRI) ability. The multimodal imaging potential of the prepared multifunctional nanoprobe was demonstrated by successful NIR fluorescent and magnetic resonance blood pool imaging. Further modification of BSA-Gd2O3/Au with arginine-glycine-aspartic acid peptide c(RGDyK) (RGD) enabled the nanoprobe for targeted tumor imaging in vivo.

Address: State Key Laboratory of Medicinal Chemical Biology (Nankai University), Synergetic Innovation Center of Chemical Science and Engineering (Tianjin), College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China

3916078d: Mol.Med.(N.Y.), 245-252(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Peptides that bind specifically to an antibody from a chronic lymphocytic leukemia clone expressing unmutated immunoglobulin variable region genes.
Author: Liu,Y., Higgins,C.D., Overstreet,C.M., Rai,K.R., Chiorazzi,N., Lai,J.R.
Abstract:
Chronic lymphocytic leukemia (CLL) is a clonal disease of a subset of human B lymphocytes. Although the cause of the disease is unknown, its development and evolution appear to be promoted by signals delivered when B-cell receptors (BCRs) engage (auto)antigens. Here, using a peptide phage display library of enhanced size and diverse composition, we examined the binding specificity of a recombinant monoclonal antibody (mAb) constructed with the heavy chain and light chain variable domains of a CLL BCR that does not exhibit somatic mutations. As determined by testing the peptides identified in the selected peptide phage pool, this CLL-associated unmutated mAb bound a diverse set of sequences, some of which clustered in families based on amino acid sequence. Synthesis of these peptides and characterization of binding with the CLL-associated mAb revealed that mAb-peptide interactions were generally specific. Moreover, the mAb-peptide interactions were of lower affinities (micromolar KD), as measured by surface plasmon resonance, than those observed with a CLL mAb containing somatic mutations (nanomolar KD) and with immunoglobulin heavy chain variable (IGHV)-mutated antibodies selected by environmental antigens. This information may be of value in identifying and targeting B lymphocytes expressing specific BCRs in CLL patients and healthy subjects with monoclonal B lymphocytosis.

Address: The Feinstein Institute for Medical Research, North Shore-LIJ Health System, Manhasset, New York 11030, USA

3916081d: Protein Expr.Purif., 134-146(2013)
Link: [ PubMED ] [ DOI ]
Title: Large scale expression and purification of mouse melanopsin-L in the baculovirus expression system.
Author: Shirzad-Wasei,N., Van Oostrum,J., Bovee-Geurts,P.H., Wasserman,M., Bosman,G.J., Degrip,W.J.
Abstract:
Melanopsin is the mammalian photopigment that primarily mediates non-visual photoregulated physiology. So far, this photopigment is poorly characterized with respect to structure and function. Here, we report large-scale production and purification of the intact long isoform of mouse melanopsin (melanopsin-L) using the baculovirus/insect cell expression system. Exploiting the baculoviral GP67 signal peptide, we obtained expression levels that varied between 10-30pmol/10(6)cells, equivalent to 2-5mg/L. This could be further enhanced using DMSO as a chemical chaperone. LC-MS analysis confirmed that full-length melanopsin-L was expressed and demonstrated that the majority of the expressed protein was N-glycosylated at Asn(30) and Asn(34). Other posttranslational modifications were not yet detected. Purification was achieved exploiting a C-terminal deca-histag, realizing a purification factor of several hundred-fold. The final recovery of purified melanopsin-L averaged 2.5% of the starting material. This was mainly due to low extraction yields, probably since most of the protein was present as the apoprotein. The spectral data we obtained agree with an absorbance maxiμm in the 460-500nm wavelength region and a significant red-shift upon illumination. This is the first report on expression and purification of full length melanopsin-L at a scale that can easily be further amplified.

E-Mail: nazhatshirzad@gmail.com
Address: Department of Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. Electronic address:

3916083d: J.Proteomics, 444-452(2013)
Link: [ PubMED ] [ DOI ]
Title: Novel angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysates of goby (Zosterisessor ophiocephalus) muscle proteins.
Author: Nasri,R., Chataigne,G., Bougatef,A., Chaabouni,M.K., Dhulster,P., Nasri,M., Nedjar-Arroume,N.
Abstract:
In recent years, food protein-derived bioactive peptides have received considerable attention because of their numerous health benefits. Amongst bioactive peptides, those with antihypertensive activity are receiving special attention due to their role in cardiovascular diseases. Goby protein hydrolysates (GPHs) prepared by treatment with five different crude bacterial proteases were found to exhibit varying degrees of angiotensin I-converting enzyme (ACE) inhibitory activity. The hydrolysate generated by the crude protease from Bacillus mojavensis A21, which displayed the highest ACE inhibitory activity, was further fractionated by size exclusion chromatography on a Sephadex G-25 and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses and amino acid sequences of five peptides, in sub-fraction F5-2, which exhibited the highest ACE inhibitory activity, were determined using ESI-MS and ESI-MS/MS, respectively. The structures of these peptides were identified as Ala-Arg-Ser, Val-Val-Ala-Pro-Phe-Ala-His-Gly-Thr, Arg-Ser-Thr-Ala, Phe-Tyr-Pro-Pro, Arg-Cys-Ser-Ala-Gly-Val. Further, the sequences of fifteen peptides in the F5-4 sub-fraction, which exhibited high activity, were determined. Therefore, GPHs have a potential as hypotensive nutraceutical ingredients. BIOLOGICAL SIGNIFICANCE: Peptides find many outlets of application in the biotechnological field, amongst which are pharmaceutical applications. Progression amongst new small molecules deposited like substance medicamentous blows itself. In this context, large pharmaceutical companies invest in peptide research to open therapeutic new prospects. Even if they are used as therapeutic agents for nearly one century in their natural form, the use of peptides remains parsimonious although we experienced a significant development since a few tens of years, in particular thanks to the clarification of the methods of production, chemical in solid or biological phase such as in phage display. Peptides present many advantages compared to traditional drugs that have small molecules, Generation of bioactive peptides by proteolysis of food proteins, using exogenous proteases, is a new and interesting approach for the production and identification of new and potent specific hypotensive agents. From another side, compared with natural peptides isolated from different sources, there is more diversity in structure and mode of action of the derived bioactive peptides. In fact, proteolysis of protein substrates, having different amino acid composition and sequences, by proteases having different specificities may generate numerous specific peptide inhibitors, with different lengths and amino acid sequences. These bioactive peptides have received considerable attention for their effectiveness in both the prevention and the treatment of hypertension.

E-Mail: ryma47@hotmail.fr
Address: Laboratoire de Genie Enzymatique et de Microbiologie, Universite de Sfax, Ecole Nationale d'Ingenieurs de Sfax, B.P. 1173-3038, Sfax, Tunisie. Electronic address:

3916093d: J.Am.Soc.Mass Spectrom., 1565-1572(2013)
Link: [ PubMED ] [ DOI ]
Title: Non-direct sequence ions in the tandem mass spectrometry of protonated peptide amides--an energy-resolved study.
Author: Harrison,A.G., Tasoglu,C., Yalcin,T.
Abstract:
The fragmentation reactions of the MH+ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of bn ions, including significant loss of NH3 from the MH+ ions. Further fragmentation of these bn ions occurs following macrocyclization/ring opening leading in many cases to bn ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies.

E-Mail: AHARRISO@CHEM.UTORONTO.CA
Address: Department of Chemistry, University of Toronto, Toronto, ON, M5S 3H6, Canada,

3916094d: J.Am.Soc.Mass Spectrom., 1710-1721(2013)
Link: [ PubMED ] [ DOI ]
Title: Chemical derivatization of peptide carboxyl groups for highly efficient electron transfer dissociation.
Author: Frey,B.L., Ladror,D.T., Sondalle,S.B., Krusemark,C.J., Jue,A.L., Coon,J.J., Smith,L.M.
Abstract:
The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups-aspartic and glutamic acid side-chains as well as C-termini-were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z > 2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

Address: Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI, 53706, USA

3916095d: Planta, 983-989(2013)
Link: [ PubMED ] [ DOI ]
Title: Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular trichomes.
Author: Sarker,L.S., Demissie,Z.A., Mahmoud,S.S.
Abstract:
The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-eπ-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-eπ-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-eπ-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but-unlike most monoterpene synthases-was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated.

Address: University of British Columbia, Kelowna, BC, Canada

3916097d: Cell Res., 1091-1105(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Genome analysis and signature discovery for diving and sensory properties of the endangered Chinese alligator.
Author: Wan,Q.H., Pan,S.K., Hu,L., Zhu,Y., Xu,P.W., Xia,J.Q., Chen,H., He,G.Y., He,J., Ni,X.W., Hou,H.L., Liao,S.G., Yang,H.Q., Chen,Y., Gao,S.K., Ge,Y.F., Cao,C.C., Li,P.F., Fang,L.M., Liao,L., Zhang,S., Wang,M.Z., Dong,W., Fang,S.G.
Abstract:
Crocodilians are diving reptiles that can hold their breath under water for long periods of time and are crepuscular animals with excellent sensory abilities. They comprise a sister lineage of birds and have no sex chromosome. Here we report the genome sequence of the endangered Chinese alligator (Alligator sinensis) and describe its unique features. The next-generation sequencing generated 314 Gb of raw sequence, yielding a genome size of 2.3 Gb. A total of 22 200 genes were predicted in Alligator sinensis using a de novo, homology- and RNA-based combined model. The genetic basis of long-diving behavior includes duplication of the bicarbonate-binding hemoglobin gene, co-functioning of routine phosphate-binding and special bicarbonate-binding oxygen transport, and positively selected energy metabolism, ammonium bicarbonate excretion and cardiac muscle contraction. Further, we elucidated the robust Alligator sinensis sensory system, including a significantly expanded olfactory receptor repertoire, rapidly evolving nerve-related cellular components and visual perception, and positive selection of the night vision-related opsin and sound detection-associated otopetrin. We also discovered a well-developed immune system with a considerable number of lineage-specific antigen-presentation genes for adaptive immunity as well as expansion of the tripartite motif-containing C-type lectin and butyrophilin genes for innate immunity and expression of antibacterial peptides. Multifluorescence in situ hybridization showed that alligator chromosome 3, which encodes DMRT1, exhibits significant synteny with chicken chromosome Z. Finally, population history analysis indicated population admixture 0.60-1.05 million years ago, when the Qinghai-Tiβn Plateau was uplifted.

Address: The Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, State Conservation Center for Gene Resources of Endangered Wildlife, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China

3916098d: Antimicrob.Agents Chemother., 4903-4910(2013)
Link: [ PubMED ] [ DOI ]
Title: Antimicrobial peptide GL13K is effective in reducing biofilms of Pseudomonas aeruginosa.
Author: Hirt,H., Gorr,S.U.
Abstract:
Human parotid secretory protein (PSP; BPIF2A) is predicted to be structurally similar to bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. Based on the locations of known antimicrobial peptides in the latter two proteins, potential active peptides in the PSP sequence were identified. One such peptide, GL13NH2 (PSP residues 141 to 153) was shown previously to interfere with LPS binding and agglutinate bacteria without bactericidal activity. By introducing three additional positively charged lysine residues, the peptide was converted to the novel bactericidal cationic peptide GL13K (MIC for Pseudomonas aeruginosa, 8 μg/ml [5.6 muM]). We investigated the antibiofilm activity of GL13K against static, monospecies biofilms of P. aeruginosa PAO1. Two-hour exposure of a 24-h biofilm to 64 μg/ml (44.8 μM) GL13K reduced biofilm bacteria by 10(2), and 100 μg/ml (70 μM) GL13K reduced bacteria by 10(3). Similar results could be achieved on 48-h-old biofilms. Lower concentrations of GL13K (32 μg/ml [22.4 muM]) were successful in reducing biofilm cell numbers in combination with tobramycin. This combination treatment also achieved total eradication of the biofilm in a majority (67.5%) of tested samples. An alanine scan of GL13K revealed the importance of the leucine residue in position six of the peptide sequence, where replacement led to a loss of antibiofilm activity, whereas the impact of replacing charged residues was less pronounced. Bacterial metalloproteases were found to partially inactivate GL13K but not a d amino acid version of the peptide.

Address: Department of Diagnostic and Biological Sciences, University of Minnesota School of Dentistry, Minneapolis, Minnesota, USA

3916099d: Antimicrob.Agents Chemother., 5045-5052(2013)
Link: [ PubMED ] [ DOI ]
Title: R-thanatin inhibits growth and biofilm formation of methicillin-resistant Staphylococcus epidermidis in vivo and in vitro.
Author: Hou,Z., Da,F., Liu,B., Xue,X., Xu,X., Zhou,Y., Li,M., Li,Z., Ma,X., Meng,J., Jia,M., Wang,Y., Luo,X.
Abstract:
Staphylococcus epidermidis is one of the most frequent causes of device-associated infections, because it is known to cause biofilms that grow on catheters or other surgical implants. The persistent increasing resistance of S. epidermidis and other coagulase-negative staphylococci (CoNS) has driven the need for newer antibacterial agents with innovative therapeutic strategies. Thanatin is reported to display potent antibiotic activities, especially against extended-spectrum-β-lactamase-producing Escherichia coli. The present study aimed to investigate whether a shorter derivative peptide (R-thanatin) could be used as a novel antibacterial agent. We found that R-thanatin was highly potent in vitro against coagulase-negative staphylococci, such as S. epidermidis, S. haemolyticus, and S. hominis, and inhibited biofilm formation at subinhibitory concentrations. Properties of little toxicity to human red blood cells (hRBCs) and human umbilical vein endothelial cells, a low incidence of resistance, and relatively high stability in plasma were confirmed. Excellent in vivo protective effects were also observed using a methicillin-resistant S. epidermidis (MRSE)-induced urinary tract infection rat model. Electron microscopy and confocal laser-scanning microscopy analyses suggested that R-thanatin disturbed cell division of MRSE severely, which might be the reason for inhibition of MRSE growth. These findings indicate that R-thanatin is active against the growth and biofilm formation of MRSE in vitro and in vivo. R-thanatin might be considered as a specific drug candidate for treating CoNS infections.

Address: Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi'an, China

3916100d: J.Proteomics, 331-337(2013)
Link: [ PubMED ] [ DOI ]
Title: Finding the same needles in the haystack? A comparison of phosphotyrosine peptides enriched by immuno-affinity precipitation and metal-based affinity chromatography.
Author: Di Palma,S., Zoumaro-Djayoon,A., Peng,M., Post,H., Preisinger,C., Munoz,J., Heck,A.J.
Abstract:
Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal-affinity based enrichment (Ti4+-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling. BIOLOGICAL SIGNIFICANCE: Here we evaluated enabling tools for the global analysis of phosphotyrosine phosphorylation. Phosphotyrosine phosphorylation is a key protein modification driving cellular response also involved in disease/cancer molecular pathways.

Address: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

3916101d: Biochimie, 2076-2081(2013)
Link: [ PubMED ] [ DOI ]
Title: Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.
Author: Gasparian,M.E., Bobik,T.V., Kim,Y.V., Ponomarenko,N.A., Dolgikh,D.A., Gabibov,A.G., Kirpichnikov,M.P.
Abstract:
Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 μM and 20 μM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.

E-Mail: marine_gasparian@yahoo.com
Address: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St., 117997 Moscow, Russia. Electronic address:

3916104d: FEBS Lett., 2943-2951(2013)
Link: [ PubMED ] [ DOI ]
Title: The V1/V2 loop of HIV-1 gp120 is necessary for Tat binding and consequent modulation of virus entry.
Author: Cardaci,S., Soster,M., Bussolino,F., Marchio,S.
Abstract:
Preventing cell entry of human immunodeficiency virus 1 (HIV-1) is of interest for the development of innovative therapies. We previously reported a specific interaction between HIV-1 envelope glycoprotein 120 (gp120) and Tat at the cell surface, which enhances virus attachment and entry. We also identified a gp120-mimicking peptide, CT319, that competes with gp120 for Tat binding, thus inhibiting HIV-1 infection. Here we report a molecular dissection of gp120 regions involved in this mechanism. Our findings identify the V1/V2 loop of gp120 as involved in Tat binding, and define this interaction as functionally relevant for HIV-1 entry into host cells.

Address: Department of Oncology, University of Torino, Candiolo, Italy

3916105d: Mol.Biochem.Parasitol., 1-6(2013)
Link: [ PubMED ] [ DOI ]
Title: Characterization of the phytochelatin synthase from the human parasitic nematode Ancylostoma ceylanicum.
Author: Rigouin,C., Vermeire,J.J., Nylin,E., Williams,D.L.
Abstract:
Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependent and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention.

Address: Department of Immunology/Microbiology, Rush University Medical Center, 1735 West Harrison Street, Chicago, IL 60612, United States

3916111d: J.Exp.Biol., 4047-4053(2013)
Link: [ PubMED ] [ DOI ]
Title: Discovery of a novel neurophysin-associated neuropeptide that triggers cardiac stomach contraction and retraction in starfish.
Author: Semmens,D.C., Dane,R.E., Pancholi,M.R., Slade,S.E., Scrivens,J.H., Elphick,M.R.
Abstract:
Feeding in starfish is a remarkable process in which the cardiac stomach is everted over prey and then retracted when prey tissue has been resorbed. Previous studies have revealed that SALMFamide-type neuropeptides trigger cardiac stomach relaxation and eversion in the starfish Asterias rubens. We hypothesized, therefore, that a counteracting neuropeptide system controls cardiac stomach contraction and retraction. Members of the NG peptide family cause muscle contraction in other echinoderms (e.g. NGFFFamide in sea urchins and NGIWYamide in sea cucumbers), so we investigated NG peptides as candidate regulators of cardiac stomach retraction in starfish. Generation and analysis of neural transcriptome sequence data from A. rubens revealed a precursor protein comprising two copies of a novel NG peptide, NGFFYamide, which was confirmed by mass spectrometry. A noteworthy feature of the NGFFYamide precursor is a C-terminal neurophysin domain, indicative of a common ancestry with vasopressin/oxytocin-type neuropeptide precursors. Interestingly, in precursors of other NG peptides the neurophysin domain has been retained (e.g. NGFFFamide) or lost (e.g. NGIWYamide and human neuropeptide S) and its functional significance remains to be determined. Investigation of the pharmacological actions of NGFFYamide in starfish revealed that it is a potent stimulator of cardiac stomach contraction in vitro and that it triggers cardiac stomach retraction in vivo. Thus, discovery of NGFFYamide provides a novel insight into neural regulation of cardiac stomach retraction as well as a rationale for chemically based strategies to control starfish that feed on economically important shellfish (e.g. mussels) or protected marine fauna (e.g. coral).

Address: Queen Mary University of London, School of Biological and Chemical Sciences, Mile End Road, London E1 4NS, UK

3916112d: Genes Dev., 1718-1730(2013)
Link: [ PubMED ] [ DOI ]
Title: Chaperone-mediated autophagy degrades mutant p53.
Author: Vakifahmetoglu-Norberg,H., Kim,M., Xia,H.G., Iwanicki,M.P., Ofengeim,D., Coloff,J.L., Pan,L., Ince,T.A., Kroemer,G., Brugge,J.S., Yuan,J.
Abstract:
Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells.

Address: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

3916113d: Genes Dev., 1640-1649(2013)
Link: [ PubMED ] [ DOI ]
Title: RIP3: a molecular switch for necrosis and inflammation.
Author: Moriwaki,K., Chan,F.K.
Abstract:
The receptor-interacting protein kinase 3 (RIP3/RIPK3) has emerged as a critical regulator of programmed necrosis/necroptosis, an inflammatory form of cell death with important functions in pathogen-induced and sterile inflammation. RIP3 activation is tightly regulated by phosphorylation, ubiquitination, and caspase-mediated cleavage. These post-translational modifications coordinately regulate the assembly of a macromolecular signaling complex termed the necrosome. Recently, several reports indicate that RIP3 can promote inflammation independent of its pronecrotic activity. Here, we review our current understanding of the mechanisms that drive RIP3-dependent necrosis and its role in different inflammatory diseases.

Address: Department of Pathology, Immunology and Microbiology Program, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

3916114d: Wiley Interdiscip.Rev.RNA, 523-533(2013)
Link: [ PubMED ] [ DOI ]
Title: ALREX-elements and introns: two identity elements that promote mRNA nuclear export.
Author: Palazzo,A.F., Mahadevan,K., Tarnawsky,S.P.
Abstract:
The mechanisms that dictate whether a particular mRNA is exported from the nucleus are still poorly defined. However, it has become increasingly clear that these mechanisms act to promote the expression of protein-coding mRNAs over the high levels of spurious transcription that is endemic to most eukaryotic genomes. For example, mRNA processing events that are associated with protein-coding transcripts, such as splicing, act as mRNA identity elements that promote nuclear export of these transcripts. Six years ago, we made the serendipitous discovery that regions within the open reading frame of an mRNA that encode short secretory or mitochondrial-targeting peptides can also act as an mRNA identity element which promotes an alternative mRNA nuclear export (ALREX) pathway. These regions are enriched in protein coding genes and have particular features that can be used to identify this class of protein-coding mRNA. In this article we review our current knowledge of how mRNA export evolved in response to particular events that occurred at the base of the eukaryotic tree. We will then focus on our current understanding of ALREX and compare its features to splicing-dependent export, the main mRNA export pathway in metazoans.

E-Mail: alex.palazzo@utoronto.ca
Address: Department of Biochemistry, University of Toronto, Ontario, Canada

3916115d: Adv.Mater., 5067-5073(2013)
Link: [ PubMED ] [ DOI ]
Title: Caged pt nanoclusters exhibiting corrodibility to exert tumor-inside activation for anticancer chemotherapeutics.
Author: Chien,C.T., Yan,J.Y., Chiu,W.C., Wu,T.H., Liu,C.Y., Lin,S.Y.
Abstract:
We report on caged Pt nanoclusters that are able to exert tumor-inside activation for anticancer chemotherapeutics and to minimize systemic toxicity. By shrinking the Pt size to 1 nm, it possesses corrodibility for dissolution in weakly acidic organelles to release toxic Pt ions. The therapeutic effect in exerting tumor-inside activation is confirmed in vivo by post-modifying a pH-cleavable PEG corona and mixing it with a tumor-homing peptide for tumour suppression.

Address: Center for Nanomedicine Research, National Health Research Institutes, 35 Keyan Road Zhunan, 35053, Taiwan

3916118d: J.Bacteriol., 4496-4505(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of the set of genes, including nonannotated morA, under the direct control of ModE in Escherichia coli.
Author: Kurata,T., Katayama,A., Hiramatsu,M., Kiguchi,Y., Takeuchi,M., Watanabe,T., Ogasawara,H., Ishihama,A., Yamamoto,K.
Abstract:
ModE is the molybdate-sensing transcription regulator that controls the expression of genes related to molybdate homeostasis in Escherichia coli. ModE is activated by binding molybdate and acts as both an activator and a repressor. By genomic systematic evolution of ligands by exponential enrichment (SELEX) screening and promoter reporter assays, we have identified a total of nine operons, including the hitherto identified modA, moaA, dmsA, and napF operons, of which six were activated by ModE and three were repressed. In addition, two promoters were newly identified and direct transcription of novel genes, referred to as morA and morB, located on antisense strands of yghW and torY, respectively. The morA gene encodes a short peptide, MorA, with an unusual initiation codon. Surprisingly, overexpression of the morA 5' untranslated region exhibited an inhibitory influence on colony formation of E. coli K-12.

Address: Department of Frontier Bioscience

3916120d: Nat.Methods, 885-888(2013)
Link: [ PubMED ] [ DOI ]
Title: Adding an unnatural covalent bond to proteins through proximity-enhanced bioreactivity.
Author: Xiang,Z., Ren,H., Hu,Y.S., Coin,I., Wei,J., Cang,H., Wang,L.
Abstract:
Natural proteins often rely on the disulfide bond to covalently link side chains. Here we genetically introduce a new type of covalent bond into proteins by enabling an unnatural amino acid to react with a proximal cysteine. We demonstrate the utility of this bond for enabling irreversible binding between an affibody and its protein substrate, capturing peptide-protein interactions in mammalian cells, and improving the photon output of fluorescent proteins.

Address: The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California, USA

3916121d: J.Am.Soc.Mass Spectrom., 1501-1512(2013)
Link: [ PubMED ] [ DOI ]
Title: Factorial experimental designs elucidate significant variables affecting data acquisition on a quadrupole Orbitrap mass spectrometer.
Author: Randall,S.M., Cardasis,H.L., Muddiman,D.C.
Abstract:
Instrument parameter values for a quadrupole Orbitrap mass spectrometer were optimized for performing global proteomic analyses. Fourteen factors were evaluated for their influence on data-dependent acquisition with an emphasis on both the rate of sequencing and spectral quality by maximizing two individually tested response variables (unique peptides and protein groups). Of the 14 factors, 12 factors were assigned significant contrast values (P < 0.05) for both response variables. Fundamentally, when optimizing parameters, a balance between spectral quality and duty cycle needs to be reached in order to maximize proteome coverage. This is especially true when using a data-dependent approach for sequencing complex proteomes. For example, maxiμm ion injection time, automatic gain control settings, and miniμm threshold settings for triggering MS/MS isolation and activation all heavily influence ion signal, the number of spectra collected, and spectral quality. To better assess the effect these parameters have on data acquisition, all MS/MS data were parsed according to ion abundance by calculating the percent of the AGC target reached for each MS/MS event and then compared with successful peptide-spectrum matches. This proved to be an effective approach for understanding the effect of ion abundance on successful peptide-spectrum matches and establishing miniμm ion abundance thresholds for triggering MS/MS isolation and activation.

Address: W. M. Keck Fourier Transform Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, USA

3916122d: Biol.Pharm.Bull., 1587-1593(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Shuttling protein nucleolin is a microglia receptor for amyloid β peptide 1-42.
Author: Ozawa,D., Nakamura,T., Koike,M., Hirano,K., Miki,Y., Beppu,M.
Abstract:
Amyloid-β peptide 1-42 (Aβ42) plays a key role in the neurotoxicity found in Alzheimer's disease. Mononuclear phagocytes in the brain (microglia), can potentially clear Aβ via phagocytosis. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether this receptor interacts specifically with Aβ type 1-42 and mediates its phagocytosis by microglia. While monomeric and fibril Aβ42 were phagocytosed by mouse microglial EOC2 cells, amyloid β peptide 1-40 (Aβ40) was only weakly phagocytosed. Surface plasmon-resonance analysis revealed that nucleolin strongly associates with Aβ42, but only weakly associates with Aβ40. Immunofluorescence staining of anti-nucleolin antibody revealed that EOC2 cells and rat primary microglia express nucleolin on their cell surfaces. Further, pretreating EOC2 cells with anti-nucleolin antibody, but not immunoglobulin G (IgG), inhibited phagocytosis of monomeric Aβ42 by microglia. Additionally, nucleolin-transfected HEK293 cells phagocytosed monomeric and fibril Aβ42 but not monomeric and fibril Aβ40. Moreover, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited phagocytosis of monomeric and fibril Aβ42, but not monomeric and fibril Aβ40. These results indicate that nucleolin is a receptor that allows microglia to recognize monomeric and fibril Aβ42.

Address: School of Pharmacy, Tokyo University of Pharmacy and Life Sciences

3916123d: Arch.Microbiol., 655-660(2013)
Link: [ PubMED ] [ DOI ]
Title: Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812.
Author: Chen,Y.S., Yu,C.R., Ji,S.H., Liou,M.S., Leong,K.H., Pan,S.F., Wu,H.C., Lin,Y.H., Yu,B., Yanagida,F.
Abstract:
Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 degrees C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH2-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.

E-Mail: yisheng@mail.mcu.edu.tw
Address: Department of Biotechnology, Ming Chuan University, No. 5 De-Ming Rd., Gui-Shan Township, Taoyuan, 333, Taiwan

3916124d: Plant Reprod., 267-285(2013)
Link: [ PubMED ] [ DOI ]
Title: De novo zygotic transcription in wheat (Triticum aestivum L.) includes genes encoding small putative secreted peptides and a protein involved in proteasomal degradation.
Author: Leljak-Levanic,D., Juranic,M., Sprunck,S.
Abstract:
Wheat is one of the world's most important crops, and increasing grain yield is a major challenge for the future. Still, our knowledge about the molecular machineries responsible for early post-fertilization events such as zygotic reprogramming, the initial cell-specification events during embryogenesis, and the intercellular communication between the early embryo and the developing endosperm is very limited. Here, we describe the identification of de novo transcribed genes in the wheat zygote. We used wheat ovaries of defined post-fertilization stages to isolate zygotes and early embryos, and identified genes that are specifically induced in these particular stages. Importantly, we observed that some of the zygotic-induced genes encode proteins with similarity to secreted signaling peptides such as TAPETUM DETERMINANT 1 and EGG APPARATUS 1, and to MATH-BTB proteins which are known substrate-binding adaptors for the Cullin3-based ubiquitin E3 ligase. This suggests that both cell-cell signaling and targeted proteasomal degradation may be important molecular events during zygote formation and the progression of early embryogenesis.

Address: Department of Molecular Biology, Faculty of Science and Mathematics, University of Zagreb, Horvatovac 102a, 10000, Zagreb, Croatia

3916132d: Physiol.Behav., 136-142(2013)
Link: [ PubMED ] [ DOI ]
Title: Specific sequences in N termini of apolipoprotein A-IV modulate its anorectic effect.
Author: Wang,F., Pearson,K.J., Davidson,W.S., Tso,P.
Abstract:
Rodent apoA-IV is expressed predominantly in small intestine and also expressed to a small extent in liver and hypothalamus. ApoA-IV has been shown to inhibit food intake in rats when injected centrally. In the current study, we hypothesize that a specific sequence within rat apoA-IV is responsible for mediating the anorectic effect. We use a bacterial expression system to generate truncation mutants (Δ249-371, Δ117-371 and Δ1-61) of rat apoA-IV and assess the ability of various regions of the molecule to inhibit food intake. The results indicate that a responsible sequence exists within the N-terminal 61 amino acids of rat apoA-IV. Synthetic peptides (1-30 EVTSDQVANVMWDYFTQLSNNAKEAVEQLQ, 1-15 EVTSDQVANVMWDYF and 17-30 QLSNNAKEAVEQLQ) were used to specify the region in between residues 1 and 30. A 14-mer peptide (17-30) encompassing this sequence was capable of reducing food intake in a dose-dependent manner whereas a peptide designed on a more C-terminal region (211-232) of apoA-IV (QEKLNHQMEGLAFQMKKNAEEL) failed to exhibit the dose-dependent anorectic effect. The isolation of this sequence provides a valuable tool for future work directed at identifying apoA-IV binding proteins and is a key step for exploring the potential of therapeutic manipulation of food intake via this pathway.

Address: Departments of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, USA

3916136d: Chem.Biol., 1022-1032(2013)
Link: [ PubMED ] [ DOI ]
Title: Engineering the biosynthesis of the polyketide-nonribosomal peptide collismycin A for generation of analogs with neuroprotective activity.
Author: Garcia,I., Vior,N.M., Gonzalez-Sabin,J., Brana,A.F., Rohr,J., Moris,F., Mendez,C., Salas,J.A.
Abstract:
Collismycin A is a member of the 2,2'-bipyridyl family of natural products that shows cytotoxic activity. Structurally, it belongs to the hybrid polyketides-nonribosomal peptides. After the isolation and characterization of the collismycin A gene cluster, we have used the combination of two different approaches (insertional inactivation and biocatalysis) to increase structural diversity in this natural product class. Twelve collismycin analogs were generated with modifications in the second pyridine ring of collismycin A, thus potentially maintaining biologic activity. None of these analogs showed better cytotoxic activity than the parental collismycin. However, some analogs showed neuroprotective activity and one of them (collismycin H) showed better values for neuroprotection against oxidative stress in a zebrafish model than those of collismycin A. Interestingly, this analog also showed very poor cytotoxic activity, a feature very desirable for a neuroprotectant compound.

Address: Departamento de Biologia Funcional e Instituto Universitario de Oncologia del Principado de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain

3916138d: Nutrition, 1259-1265(2013)
Link: [ PubMED ] [ DOI ]
Title: Octreotide promotes weight loss via suppression of intestinal MTP and apoB48 expression in diet-induced obesity rats.
Author: Huang,W., Liu,R., Ou,Y., Li,X., Qiang,O., Yu,T., Tang,C.W.
Abstract:
OBJECTIVE: The goal of this study was to investigate the effect of octreotide on the expression of intestinal fat absorption-associated apolipoproteinB48 (apoB48), microsomal triglyceride transfer protein (MTP) and apolipoproteinAIV (apoAIV) in a high-fat diet-induced obesity rat model. METHODS: Sprague-Dawley rats were placed into a control or high-fat diet group. Obese rats from the high-fat diet group were further divided into an obese group and an octreotide-treated group. Rats in the octreotide-treated group were subcutaneously injected with octreotide (40 μg/kg body weight) twice daily for 8 d. Body weight, fasting plasma glucose (FPG), fasting serum insulin, triglyceride (TG), total cholesterol (TC), and high density lipoprotein-cholesterol (HDL-C) were measured. Intestinal MTP, apoB48, and apoAIV expression levels were determined by real-time polymerase chain reaction, Western blot, or enzyme-linked immunosorbent assay analysis. RESULTS: We found high-fat diet-induced obesity rats express more apoB, MTP, and apoAIV mRNA as well as apoB48 and MTP protein in the intestine than normal chow-fed rats. This observation occurred along with increased body weight, FPG, TG, TC, fasting serum insulin, and Homeostatic Model Assessment value. Octreotide intervention significantly decreased body weight and blood parameters, and down-regulated expression of apoB mRNA and apoB48 protein, as well as MTP mRNA and proteins. However, apoAIV mRNA was not significantly different between obese and octreotide-treated rats although it was decreased by 47%. CONCLUSION: High-fat diet-induced obesity is associated with increased expression of apoB48, MTP, and apoAIV in the intestine. Octreotide intervention inhibited the overexpression of apoB48 and MTP, and consequently brought about reduced fat absorption and weight loss.

Address: Division of Peptides Related to Human Disease, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Sichuan, P.R. China

3916141d: Curr.Biol., 1670-1676(2013)
Link: [ PubMED ] [ DOI ]
Title: A novel pollen-pistil interaction conferring high-temperature tolerance during reproduction via CLE45 signaling.
Author: Endo,S., Shinohara,H., Matsubayashi,Y., Fukuda,H.
Abstract:
Flowering plants in the reproductive stage are particularly vulnerable to ambient temperature fluctuations. Nevertheless, they maintain seed production under certain levels of exposure to temperature change. The mechanisms underlying this temperature tolerance are largely unknown. Using an in vitro Arabidopsis pollen tube culture, we found that a synthetic CLV3/ESR-related peptide, CLE45, prolonged pollen tube growth. A subsequent screen of Arabidopsis mutants of leucine-rich repeat receptor-like kinase genes identified two candidate receptors for CLE45 peptide, STERILITY-REGULATING KINASE MEMBER1 (SKM1) and SKM2. The double loss-of-function mutant was insensitive to CLE45 peptide in terms of pollen tube growth in vitro. The SKM1 protein actually interacted with CLE45 peptide. CLE45 was preferentially expressed in the stigma in the pistil at 22 degrees C, but upon temperature shift to 30 degrees C, its expression expanded to the transmitting tract, along which pollen tubes elongated. In contrast, both SKM1 and SKM2 were expressed in pollen. Disturbance of CLE45-SKM1/SKM2 signaling transduction by either RNAi suppression of CLE45 expression or introduction of a kinase-dead version of SKM1 into skm1 plants reduced seed production at 30 degrees C, but not at 22 degrees C. Taken together with the finding that CLE45 peptide application alleviated mitochondrial decay during the in vitro pollen tube culture, these results strongly suggest that the pollen-pistil interaction via the CLE45-SKM1/SKM2 signaling pathway sustains pollen performance under higher temperatures, leading to successful seed production.

E-Mail: satoshi@biol.s.u-tokyo.ac.jp
Address: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan. Electronic address:

3916143d: Biomaterials, 8291-8300(2013)
Link: [ PubMED ] [ DOI ]
Title: Nanoparticles-based multi-adjuvant whole cell tumor vaccine for cancer immunotherapy.
Author: Liu,S.Y., Wei,W., Yue,H., Ni,D.Z., Yue,Z.G., Wang,S., Fu,Q., Wang,Y.Q., Ma,G.H., Su,Z.G.
Abstract:
Whole cell tumor vaccine (WCTV), as a potential treatment modality, elicits limited immune responses because of the poor immunogenicity. To address this issue, researchers have attempted to transduce a cytokine adjuvant into tumor cells, but these single-adjuvant WCTVs curtail the high expectations. In present study, we constructed a multi-adjuvant WCTV based on the nanoparticles modified with cell penetrating peptide, which could facilitate the transportation of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 2 (IL-2) into tumor cells. After inactivation, as-designed multi-adjuvant WCTV exhibited programmed promotions on DC recruitment, antigen presentation, and T-cell activation. In vivo evaluations demonstrated the satisfactory effects on tumor growth suppression, metastasis inhibition, and recurrence prevention. Therefore, the nanoparticles-based multi-adjuvant WCTV may serve as a high-performance treatment for anti-tumor immunotherapy.

Address: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China

3916144d: FEBS J., 4876-4887(2013)
Link: [ PubMED ] [ DOI ]
Title: Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models.
Author: Deruaz,M., Bonvin,P., Severin,I.C., Johnson,Z., Krohn,S., Power,C.A., Proudfoot,A.E.
Abstract:
Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.

Address: Merck Serono Geneva Research Centre, Geneva, Switzerland

3916145d: Traffic, 1144-1154(2013)
Link: [ PubMED ] [ DOI ]
Title: Distinctive Conformation of Minor Site-Specific Nuclear Localization Signals Bound to Importin-α.
Author: Chang,C.W., Counago,R.M., Williams,S.J., Boden,M., Kobe,B.
Abstract:
Nuclear localization signals (NLSs) contain one or two clusters of basic residues and are recognized by the import receptor importin-α. There are two NLS-binding sites (major and minor) on importin-α and the major NLS-binding site is considered to be the primary binding site. Here, we used crystallographic and biochemical methods to investigate the binding between importin-α and predicted 'minor site-specific' NLSs: four peptide library-derived peptides, and the NLS from mouse RNA helicase II/Guα. The crystal structures reveal that these atypical NLSs indeed preferentially bind to the minor NLS-binding site. Unlike previously characterized NLSs, the C-terminal residues of these NLSs form an α-helical turn, stabilized by internal H-bond and cation-pi interactions between the aromatic residues from the NLSs and the positively charged residues from importin-α. This helical turn sterically hinders binding at the major NLS-binding site, explaining the minor-site preference. Our data suggest the sequence RXXKR[K/X][F/Y/W]XXAF as the optimal minor NLS-binding site-specific motif, which may help identify novel proteins with atypical NLSs.

Address: School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld, 4072, Australia

3916146d: J.Proteome Res., 3912-3919(2013)
Link: [ PubMED ] [ DOI ]
Title: Hyperplex-MRM: a hybrid multiple reaction monitoring method using mTRAQ/iTRAQ labeling for multiplex absolute quantification of human colorectal cancer biomarker.
Author: Yin,H.R., Zhang,L., Xie,L.Q., Huang,L.Y., Xu,Y., Cai,S.J., Yang,P.Y., Lu,H.J.
Abstract:
Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.

Address: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China

3916156d: Proteomics, 2743-2760(2013)
Link: [ PubMED ] [ DOI ]
Title: Subcellular protein localization (cell envelope) in Phaeobacter inhibens DSM 17395.
Author: Kossmehl,S., Wohlbrand,L., Druppel,K., Feenders,C., Blasius,B., Rabus,R.
Abstract:
Phaeobacter inhibens DSM 17395 is a metabolically versatile, secondary metabolite producing and surface colonizing member of the αproteobacterial Roseobacter clade. Proteins compartmentalized across the Gram-negative cell envelope are expected to be relevant for the habitat success of P. inhibens DSM 17395. Subcellular fractionation was followed by gel- or nano-LC-based separation of proteins and peptides, respectively. Subsequent MS-based identification of in total 1187 proteins allowed allocation to cytoplasm (303 proteins), cytoplasmic membrane (346), periplasm (325), outer membrane (76), and extracellular milieu (22). Multidimensional scaling was used to visualize the spreading of heuristically allocated proteins across the five different compartments. Experimentally inferred subcellular protein localization was compared with PSORTb prediction of protein secretion and membrane localization. Determined subcellular localizations of identified proteins were interpreted to reconstruct the functional traits of the different cell envelope compartments, in particular protein secretion and sorting, direct effector molecule transit, and cell envelope biogenesis. From a proteogenomic perspective, functional prediction of 74 genes (including 17 coding for proteins of hitherto unknown function) could be refined.

Address: Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University Oldenburg, Oldenburg, Germany

3916157d: Biopolymers, 433-437(2013)
Link: [ PubMED ] [ DOI ]
Title: A systematic approach to document cyclotide distribution in plant species from genomic, transcriptomic, and peptidomic analysis.
Author: Gerlach,S.L., Goransson,U., Kaas,Q., Craik,D.J., Mondal,D., Gruber,C.W.
Abstract:
Cyclotides are a large family of plant peptides characterized by their cyclic cystine knot composed of a circular backbone and three disulfide bonds that impart exceptional stability. They, and several acyclic variants, have been isolated from plants within the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Poaceae families. A variety of chemical and genetic approaches have been applied for the discovery and characterization of cyclotides. As investigations of cyclotide expression, distribution, and phylogeny rapidly increase, the authors have proposed the inclusion of information pertaining to plant species that have been analyzed but do not appear to express cyclotides into the CyBase database. CyBase is dedicated to providing web tools and information about cyclic peptides and proteins to the scientific community. Including detailed information about sampling and analysis parameters of plant species that have been investigated but not published elsewhere should assist in the process of selecting species for establishing new cyclotide discovery projects, as well as for detailed reanalysis using alternative technical approaches. In summary, the collection and deposition of all plant species that have been examined (whether cyclotides have been found or not) would help to impart a deeper understanding of cyclotide discovery, evolution, and physiological function.

Address: Department of Pharmacology, School of Medicine, Tulane University, 1430 Tulane Ave, New Orleans, LA, 70112

3916174d: Viruses(Basel), 2531-2545(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Oligopeptide m13 phage display in pathogen research.
Author: Kugler,J., Zantow,J., Meyer,T., Hust,M.
Abstract:
Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

E-Mail: m.hust@tu-bs.de
Address: Technische Universitat Braunschweig, Institut fur Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Spielmannstr. 7, 38106 Braunschweig, Germany

3916175d: Nat.Commun., 2623-2623(2013)
Link: [ PubMED ] [ DOI ]
Title: Crossreactivity of a human autoimmune TCR is dominated by a single TCR loop.
Author: Sethi,D.K., Gordo,S., Schubert,D.A., Wucherpfennig,K.W.
Abstract:
Self-reactive CD4 T cells are thought to have a central role in the pathogenesis of many chronic inflammatory human diseases. Microbial peptides can activate self-reactive T cells, but the structural basis for such crossreactivity is not well understood. The Hy.1B11 T cell receptor (TCR) originates from a patient with multiple sclerosis and recognizes the self-antigen myelin basic protein. Here we report the structural mechanism of TCR crossreactivity with two distinct peptides from human pathogens. The structures show that a single TCR residue (CDR3α F95) makes the majority of contacts with the self-peptide and both microbial peptides (66.7-80.6%) due to a highly tilted TCR-binding topology on the peptide-MHC surface. Further, a neighbouring residue located on the same TCR loop (CDR3α E98) forms an energetically critical interaction with the MHC molecule. These data show how binding by a self-reactive TCR favors crossreactivity between self and microbial antigens.

Address: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA

3916177d: Neuroimmunomodulation, 21-30(2013)
Link: [ PubMED ] [ DOI ]
Title: Contribution of Vasoactive Intestinal Peptide to Immune Homeostasis in Trophoblast-Maternal Leukocyte Interaction under LPS Stimulation.
Author: Fraccaroli,L., Grasso,E., Hauk,V., Cortelezzi,M., Perez Leiros,C., Ramhorst,R.
Abstract:
Background/Aims: The maternal-fetal interface is a unique immunological site that generates an adequate microenvironment during pregnancy, recognizing and eliminating infections and tolerating the trophoblast/placenta unit. For that purpose, trophoblast cells display several tolerogenic mechanisms to allow fetal survival, such as production of the neuropeptide vasoactive intestinal peptide (VIP). Here we investigated the contribution of VIP to maintain homeostasis at the maternal-placental interface under lipopolysaccharide (LPS) stimulation. Methods: We performed cocultures between trophoblast cells (Swan-71 cell line) and maternal leukocytes obtained from fertile women as an in vitro model of maternal-placental interaction, and we focused on the effects of LPS on the modulation of VIP and their receptors (VPAC1 and VPAC2). Results: VIP could prevent the upregulation of IL-6, MCP-1, and nitrite production and maintain the production of IL-10 and TGF-β under LPS (10 μg/ml) stimulation after 48 h of coculture. To gain deeper insight into the mechanisms of how VIP could contribute to a tolerogenic microenvironment even in the presence of LPS, we investigated VIP production by maternal leukocytes and observed a significant increase in the frequency of CD4+VIP+ cells after interaction with Swan-71 cells in the presence of LPS. LPS increased VIP and inducible receptor VPAC2 expression directly on trophoblast cells in a dose- and time-dependent manner. Conclusions: The present results suggest that VIP might act as an additional homeostatic mechanism during early stages at the maternal-placental interface to control exacerbated inflammatory responses such as the ones observed in intrauterine infections.

Address: Immunopharmacology Laboratory, School of Sciences, University of Buenos Aires and IQUIBICEN-CONICET (National Research Council of Science and Technology, CONICET), Buenos Aires, Argentina

3916179d: Int.J.Food Microbiol., 202-207(2013)
Link: [ PubMED ] [ DOI ]
Title: Comparative proteomic analysis of Listeria monocytogenes exposed to enterocin AS-48 in planktonic and sessile states.
Author: Caballero Gomez,N., Abriouel,H., Ennahar,S., Galvez,A.
Abstract:
Enterocin AS-48 is a cyclic peptide of great interest for application in food preservation and sanitation. In the present study, the proteome response of Listeria monocytogenes to purified enterocin AS-48 was studied under two different conditions: planktonic cells and sessile cells grown on polystyrene plates. Ten different proteins were differentially expressed in planktonic L. monocytogenes cells treated with 0.1μg/ml enterocin AS-48 compared to the untreated controls. Overexpressed proteins were related to stress response (DnaK) or carbohydrate transport and metabolism, while underexpressed and unexpressed proteins were related to metabolism (such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate oxidase, glutamate dehydrogenase or glutamate decarboxylase) or stress (GroEL). In the sessile state, L. monocytogenes cells tolerated up to 10μg/ml bacteriocin, and the treated biofilm cells overexpressed a set of 11 proteins, some of which could be related to stress response (DnaK, GroEL), protein synthesis and carbohydrate metabolism, while glyceraldehyde-3-phosphate dehydrogenase was the only unexpressed protein. Some of the overexpressed proteins (such as elongation factor Tu and GroEL) could also be implicated in cell adhesion. These results suggest different cell responses of L. monocytogenes to enterocin AS-48 in the planktonic and in the sessile state, including stress response and cell metabolism proteins. While in the planktonic state the bacterium may tend to compensate for the cytoplasmic cell permeability changes induced by AS-48 by reinforcing carbohydrate transport and metabolism, sessile cells seem to respond by shifting carbohydrate metabolism and reinforcing protein synthesis. Stress response proteins also seem to be important in the response to AS-48, but the stress response seems to be different in planktonic and in sessile cells.

Address: Area de Microbiologia, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaen, 23071 Jaen, Spain

3916180d: Int.J.Food Microbiol., 131-137(2013)
Link: [ PubMED ] [ DOI ]
Title: Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5.
Author: Gonzalez-Gonzalez,C., Gibson,T., Jauregi,P.
Abstract:
In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC50=132μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC50=703μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.

Address: Food and Nutritional Sciences, University of Reading, Whiteknights, RG2 6AP Reading, United Kingdom

3916186d: SpringerPlus, 458-458(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Genomic and expression analysis of a solute carrier protein (CcSLC25a5) gene from Cyprinus carpio Linnaeus.
Author: Jiang,L., Cheng,A., Wang,Y., Zhang,B.
Abstract:
Using the Genefishing method, we identified seven potential regulatory genes involved in the process of scale morphogenesis in fishes. We further characterized a novel solute carrier protein gene (CcSLC), from the common carp which is differentially expressed in mirror carp and Jianli. The ORF encodes a peptide of 298 amino acids with a molecular mass of 31.5 kDa and a theoretical isoelectric point of 7.49. ScanProsite analysis indicated that it is a putative solute carrier protein that contains a substrate binding site. CcSLC was detected in carp embryos by in situ hybridization in the 70%-epiboly, 6-somite, and 14-somite embryonic stages. Gene expression stopped at the long pec stage. However, CcSLC25a5 was re-expressed during the initiation of scale formation in the regions that were scale covered. These findings provide novel insights into the features of early carp embryo and scale development.

Address: College of Fisheries and Life Science, Dalian Ocean University, Dalian, 116023 China

3916187d: Cancer Med., 32-39(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Bisebromoamide, an extract from Lyngbya species, induces apoptosis through ERK and mTOR inhibitions in renal cancer cells.
Author: Suzuki,K., Mizuno,R., Suenaga,K., Teruya,T., Tanaka,N., Kosaka,T., Oya,M.
Abstract:
Advanced renal cell carcinoma (RCC) remains an incurable disease, and newer anticancer drugs are needed. Bisebromoamide, a novel cytotoxic peptide, was isolated from the marine cyanobacterium Lyngbya species at our laboratory in 2009. This compound specifically inhibited the phosphorylation of ERK in platelet-derived growth factor-activated normal rat kidney cells. The aim of this study was to evaluate the effect and elucidate the potential mechanism of Bisebromoamide actions on human RCC cells. Two renal cancer cell lines, 769-P and 786-O, were used. The effects of Bisebromoamide were analyzed employing assays for water-soluble Tetrazolium-1 salts. Apoptosis was determined by flow cytometric TUNEL analysis. Cell-cycle distributions were analyzed by flow cytometry using BrdU/propidium iodide (PI) staining. Kinases of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of Rapamycin (mTOR) pathway and Raf/MEK/ERK pathway were analyzed by Western blotting. After Bisebromoamide treatment for 48 and 72 h, cell viability was significantly decreased in both cell lines at 1 and 10 mumol/L. After treatment with 1 mumol/L Bisebromoamide for 72 h, apoptosis and the increased percentage of cells in the sub-G1 phase were observed in both cell lines. Bisebromoamide inhibited the phosphorylation of ERK and Akt in both cell lines tested. Similar effects were demonstrated for phosphorylation of mTOR and p70 S6. Bisebromoamide is a promising potential agent against RCC due to its ability to inhibit both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways.

Address: Department of Urology, Keio University School of Medicine 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan

3916189d: J.Geriatr.Cardiol., 253-257(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: OptiVol fluid index predicts acute decompensation of heart failure with a high rate of unexplained events.
Author: Yang,X.W., Hua,W., Ding,L.G., Wang,J., Zheng,L.H., Li,C.Q., Liu,Z.M., Chen,K.P., Zhang,S.
Abstract:
BACKGROUND: Intrathoracic impedance monitoring has emerged as a promising new technique for the detection of impending heart failure (HF). Although false positive episodes have been reported in case reports and clinical trials, the efficacy and false positive rate in real-world practice remain unclear. OBJECTIVE: The aim of this study is to investigate the utility and reliability of the OptiVol alert feature in clinical practice. METHODS: We continuously recruited patients who underwent implantable cardioverter-defibrillator (ICD) or cardiac resynchronization therapy with defibrillator (CRT-D) implantation with feature of intrathoracic impedance monitoring system in our center from Sep. 2010 to Oct. 2012. Regular in-office follow-up were required of all patients and the following information was collected at each visit: medical history, device interrogation, N-terminal pro-brain natriuretic peptide (NT-proBNP) measurement and an echocardiogram. Worsening HF was defined as hospitalization or the presentation of signs or symptoms of HF. RESULTS: FORTY THREE PATIENTS (MALE: 76.7%, mean age: 57 +/- 15 years, left ventricular ejection fraction (LVEF): 33% +/- 14%) were included in this observational study. Fifty four alert events and 14 adjudicated worsening HF were detected within 288 +/-163 days follow-up. Eleven (20.4%) alert episodes were associated with acute cardiac decompensation in 9 patients with a positive predictive value of 78.6%. Forty three audible alerts showed no connection to worsening HF. The unexplained alerts rate was 79.6% and 1.27 per person-year. Thirty seven alarm alerts were detected in patients with EF < 45%, among which 9 accompanied with HF, 17 alerts detected in patients with LVEF >/= 45% and 2 associated with HF. There was no significant difference between the two groups (9/37 vs. 2/17; P = 0.47). CONCLUSIONS: Patients with normal or nearly normal left ventricular systolic function also exhibited considerable alert events. The OptiVol fluid index predicted worsening cardiac events with a high unexplained detection rate, and any alert must therefore be analyzed with great caution. Efforts to improve the specificity of this monitoring system represent a significant aspect of future studies.

Address: Center of Arrhythmia Diagnosis and Treatment, Fuwai Cardiovascular Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, No.167 Beilishi Road, Xicheng District, Beijing 100037, China

3916193d: Protein Eng.Des.Sel., 743-753(2013)
Link: [ PubMED ] [ DOI ]
Title: Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer.
Author: Podust,V.N., Sim,B.C., Kothari,D., Henthorn,L., Gu,C., Wang,C.W., McLaughlin,B., Schellenberger,V.
Abstract:
XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimide-thiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability.

Address: Amunix Operating Inc., 500 Ellis Street, Mountain View, CA 94043, USA

3916195d: Acta Reumatol.Port., 39-43(2013)
Link: [ PubMED ]
Title: A seroimmunological profile of erosive hand osteoarthritis.
Author: Vannini,A., Fusconi,M., Aglio,A.C., Tovoli,F., Frisoni,M., Zauli,D.
Abstract:
Objectives: erosive hand osteoarthritis (EHOA) is an inflammatory disorder involving joints of the hands, which may be accompanied by acute phase reactants. The relationship between EHOA and classical osteoarthritis (OA) is still controversial, since some authors consider EHOA as a distinct disease, other as a subset of OA, and some as a border entity between OA and rheumatoid arthritis (RA). Scarce data are available about the seroimmunological profile of the disease, which could aid to identify a possible role of the immune system in EHOA pathogenesis, and could also allow to better differentiate EHOA both from OA and RA. Material and methods: blood was drawn from the following patients: 37 with EHOA, 35 with OA and 45 with RA. All sera were tested for rheumatoid factor, anti-cyclic citrullinated peptide antibodies (anti-CCP), antinuclear antibodies (ANA), anti-extractable nuclear antigens (anti-ENA) and anti-neutrophil cytoplasmic antibodies (ANCA). Results: ANCA were never detected in OA, whereas they were found in 7 (19%) EHOA and 8 (18%) RA patients; the difference between EHOA and OA was statistically significant (p<0.01). Anti-CCP antibodies, which were consistently negative in OA, were positive in 2 EHOA (5%) at a low titre and in 23 (51%) RA patients, usually at a very high titre. The difference between EHOA and OA was not statistically significant, while the number of RA positive patients was significantly higher (p<0.001). Conclusions: our findings suggest that the seroimmunological profile of EHOA is different from that of OA. In EHOA patients ANCA and anti-CCP antibodies might be either markers of inflammation involving neutrophils and/or markers of an underlying autoimmune process.

3916196d: Toxins, 1780-1798(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Molecular Characterization of Lys49 and Asp49 Phospholipases A2 from Snake Venom and Their Antiviral Activities against Dengue virus.
Author: Cecilio,A.B., Caldas,S., Oliveira,R.A., Santos,A.S., Richardson,M., Naumann,G.B., Schneider,F.S., Alvarenga,V.G., Estevao-Costa,M.I., Fuly,A.L., Eble,J.A., Sanchez,E.F.
Abstract:
We report the detailed molecular characterization of two PLA2s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA2s, named BlK-PLA2 and BlD-PLA2, are composed of 121 and 122 amino acids determined by automated sequencing of the native proteins and peptides produced by digestion with trypsin. They contain fourteen cysteines with pIs of 9.05 and 8.18 for BlK- and BlD-PLA2s, and show a high degree of sequence similarity to homologous snake venom PLA2s, but may display different biological effects. Molecular masses of 13,689.220 (Lys49) and 13,978.386 (Asp49) were determined by mass spectrometry. DENV causes a prevalent arboviral disease in humans, and no clinically approved antiviral therapy is currently available to treat DENV infections. The maxiμm non-toxic concentration of the proteins to LLC-MK2 cells determined by MTT assay was 40 μg/mL for Bl-PLA2s (pool) and 20 μg/mL for each isoform. Antiviral effects of Bl-PLA2s were assessed by quantitative Real-Time PCR. Bl-PLA2s were able to reduce DENV-1, DENV-2, and DENV-3 serotypes in LLC-MK2 cells infection. Our data provide further insight into the structural properties and their antiviral activity against DENV, opening up possibilities for biotechnological applications of these Bl-PLA2s as tools of research.

E-Mail: eladio.flores@funed.mg.gov.br
Address: Laboratory of Biotechnology and Health, Research and Development Center, Ezequiel Dias Foundation, Belo Horizonte 30510-010, MG, Brazil

3916198d: Analyst, 7224-7232(2013)
Link: [ PubMED ] [ DOI ]
Title: Reversible hydrazide chemistry-based enrichment for O-GlcNAc-modified peptides and glycopeptides having non-reducing GlcNAc residues.
Author: Nishikaze,T., Kawabata,S., Iwamoto,S., Tanaka,K.
Abstract:
O-Linked N-acetylglucosamine (O-GlcNAc) is an emerging post-translational modification (PTM) of proteins. Analysis of O-GlcNAc modification using mass spectrometry (MS) is often problematic because of the low stoichiometry of the modification. In this study, we developed a new method for enriching O-GlcNAc-modified peptides using reversible hydrazide chemistry. O-GlcNAc-modified peptides were first labeled with N-azidoacetylgalactosamine (GalNAz) using gatactosyltransferase-T1 (Y289L) enzyme. The azide group on the GalNAz residue was then reacted with 3-ethynylbenzaldehyde via copper-catalyzed Huisgen 1,3-cycloaddition "click reaction" to form an aromatic aldehyde group of glycopeptides. Aromatic aldehyde-derivatized glycopeptides were enriched by reversible hydrazone formation with hydrazide resin. Reaction conditions for each step, especially for the click reaction, were optimized to achieve complete reaction without significant side reactions. This method was validated using a tryptic digest of bovine α-crystallin, which is an O-GlcNAc-modified glycoprotein. The developed method was also applied to structure-specific enrichment of N-linked glycopeptides having non-reducing terminal GlcNAc residues. All materials and chemicals required for this method are commercially available and there is no need to prepare any special reagents, facilitating the introduction of this method in any laboratory.

E-Mail: nishikaz@shimadzu.co.jp
Address: Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan

3916200d: PLoS Negl.Trop.Dis., e2497-e2497(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes.
Author: Nascimento,E.J., Mailliard,R.B., Khan,A.M., Sidney,J., Sette,A., Guzman,N., Paulaitis,M., De Melo,A.B., Cordeiro,M.T., Gil,L.V., Lemonnier,F., Rinaldo,C., August,J.T., Marques,E.T.Jr.
Abstract:
Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naive T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design.

Address: Department of Infectious Disease and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America

3916202d: PLoS One, e77375-e77375(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Targeting c-MYC with T-Cells.
Author: Helm,F., Kammertoens,T., Lehmann,F.M., Wilke,A., Bruns,H., Mautner,J., Bornkamm,G.W., Gerbitz,A.
Abstract:
Over-expression of the proto-oncogene c-MYC is frequently observed in a variety of tumors and is a hallmark of Burkitt s lymphoma. The fact that many tumors are oncogene-addicted to c-MYC, renders c-MYC a powerful target for anti-tumor therapy. Using a xenogenic vaccination strategy by immunizing C57BL/6 mice with human c-MYC protein or non-homologous peptides, we show that the human c-MYC protein, despite its high homology between mouse and man, contains several immunogenic epitopes presented in the context of murine H2(b) haplotype. We identified an MHC class II-restricted CD4+ T-cell epitope and therein an MHC class I-restricted CD8+ T-cell epitope (SSPQGSPEPL) that, after prime/boost immunization, protected up to 25% of mice against a lethal lymphoma challenge. Lymphoma-rejecting animals contained MHC multimer-binding CD8+ cell within the peripheral blood and displayed in vivo cytolytic activity with specificity for SSPQGSPEPL. Taken together these data suggest that oncogenic c-MYC can be targeted with specific T-cells.

Address: Department of Immunology, Charite Berlin, Berlin, Germany

3916203d: PLoS One, e77238-e77238(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Protective Immunity against Trichinella spiralis Infection Induced by a Multi-Epitope Vaccine in a Murine Model.
Author: Gu,Y., Wei,J., Yang,J., Huang,J., Yang,X., Zhu,X.
Abstract:
Trichinellosis is one of the most important food-borne parasitic zoonoses throughout the world. Because infected pigs are the major source of human infections, and China is becoming the largest international producer of pork, the development of a transmission-blocking vaccine to prevent swine from being infected is urgently needed for trichinellosis control in China. Our previous studies have demonstrated that specific Trichinella spiralis paramyosin (Ts-Pmy) and Ts-87 antigen could provide protective immunity against T. spiralis infection in immunized mice. Certain protective epitopes of Ts-Pmy and Ts-87 antigen have been identified. To identify more Ts-Pmy protective epitopes, a new monoclonal antibody, termed 8F12, was produced against the N-terminus of Ts-Pmy. This antibody elicited significant protective immunity in mice against T. spiralis infection by passive transfer and was subsequently used to screen a random phage display peptide library to identify recognized epitopes. Seven distinct positive phage clones were identified and their displayed peptides were sequenced. Synthesized epitope peptides conjugated to keyhole limpet hemocyanin were used to immunize mice, four of which exhibited larval reduction (from 18.7% to 26.3%, respectively) in vaccinated mice in comparison to the KLH control. To increase more effective protection, the epitope 8F7 that was found to induce the highest protection in this study was combined with two other previously identified epitopes (YX1 from Ts-Pmy and M7 from Ts-87) to formulate a multi-epitope vaccine. Mice immunized with this multi-epitope vaccine experienced a 35.0% reduction in muscle larvae burden after being challenged with T. spiralis larvae. This protection is significantly higher than that induced by individual-epitope peptides and is associated with high levels of subclasses IgG and IgG1. These results showed that a multi-epitope vaccine induced better protective immunity than an individual epitope and provided a feasible approach for developing a safer and more effective vaccine against trichinellosis.

Address: Department of Parasitology, School of Basic Medical Sciences, Capital Medical University, Beijing, PR China

3916206d: PLoS One, e76907-e76907(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis.
Author: Sisay,S., Pryce,G., Jackson,S.J., Tanner,C., Ross,R.A., Michael,G.J., Selwood,D.L., Giovannoni,G., Baker,D.
Abstract:
Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.

Address: Neuroimmunology, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom

3916208d: PLoS One, e76812-e76812(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus.
Author: Zdzalik,M., Kalinska,M., Wysocka,M., Stec-Niemczyk,J., Cichon,P., Stach,N., Gruba,N., Stennicke,H.R., Jabaiah,A., Markiewicz,M., Kedracka-Krok,S., Wladyka,B., Daugherty,P.S., Lesner,A., Rolka,K., Dubin,A., Potempa,J., Dubin,G.
Abstract:
Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1') with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V) downward arrowS. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 A and 2.1 A. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.

Address: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

3916209d: PLoS One, e75716-e75716(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Adiponectin enhances the responsiveness of the olfactory system.
Author: Loch,D., Heidel,C., Breer,H., Strotmann,J.
Abstract:
The peptide hormone adiponectin is secreted by adipose tissue and the circulating concentration is reversely correlated with body fat mass; it is considered as starvation signal. The observation that mature sensory neurons of the main olfactory epithelium express the adiponectin receptor 1 has led to the concept that adiponectin may affect the responsiveness of the olfactory system. In fact, electroolfactogram recordings from olfactory epithelium incubated with exogenous adiponectin resulted in large amplitudes upon odor stimulation. To determine whether the responsiveness of the olfactory sensory neurons was enhanced, we have monitored the odorant-induced expression of the immediate early gene Egr1. It was found that in an olfactory epithelium incubated with nasally applied adiponectin the number of Egr1 positive cells was significantly higher compared to controls, suggesting that adiponectin rendered the olfactory neurons more responsive to an odorant stimulus. To analyze whether the augmented responsiveness of sensory neurons was strong enough to elicit a higher neuronal activity in the olfactory bulb, the number of activated periglomerular cells of a distinct glomerulus was determined by monitoring the stimulus-induced expression of c-fos. The studies were performed using the transgenic mOR256-17-IRES-tauGFP mice which allowed to visualize the corresponding glomerulus and to stimulate with a known ligand. The data indicate that upon exposure to 2,3-hexanedione in adiponectin-treated mice the number of activated periglomerular neurons was significantly increased compared to controls. The results of this study indicate that adiponectin increases the responsiveness of the olfactory system, probably due to a higher responsiveness of olfactory sensory neurons.

Address: Institute of Physiology, University of Hohenheim, Stuttgart, Germany

3916210d: PLoS One, e75297-e75297(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Snapin, Positive Regulator of Stimulation- Induced Ca2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells.
Author: Kinoshita,S.M., Kogure,A., Taguchi,S., Nolan,G.P.
Abstract:
To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca2+ release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca2+/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4+ T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca2+ signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology.

Address: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America

3916211d: PLoS One, e75119-e75119(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Munc13-Like skMLCK Variants Cannot Mimic the Unique Calmodulin Binding Mode of Munc13 as Evidenced by Chemical Cross-Linking and Mass Spectrometry.
Author: Herbst,S., Maucher,D., Schneider,M., Ihling,C.H., Jahn,O., Sinz,A.
Abstract:
Among the neuronal binding partners of calmodulin (CaM) are Munc13 proteins as essential presynaptic regulators that play a key role in synaptic vesicle priming and are crucial for presynaptic short-term plasticity. Recent NMR structural investigations of a CaM/Munc13-1 peptide complex have revealed an extended structure, which contrasts the compact structures of most classical CaM/target complexes. This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif. Here, we addressed the question whether the 1-5-8-26 CaM binding motif is a Munc13-related feature or whether it can be induced in other CaM targets by altering the motif's core residues. For this purpose, we chose skeletal muscle myosin light chain kinase (skMLCK) with a classical 1-5-8-14 CaM binding motif and constructed three skMLCK peptide variants mimicking Munc13-1, in which the hydrophobic anchor amino acid at position 14 was moved to position 26. Chemical cross-linking between CaM and skMLCK peptide variants combined with high-resolution mass spectrometry yielded insights into the peptides' binding modes. This structural comparison together with complementary binding data from surface plasmon resonance experiments revealed that skMLCK variants with an artificial 1-5-8-26 motif cannot mimic CaM binding of Munc13-1. Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1. We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca2+ concentrations.

Address: Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany

3916215d: J.Infect.Dis., 1553-1560(2013)
Link: [ PubMED ] [ DOI ]
Title: Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo.
Author: Geller,B.L., Marshall-Batty,K., Schnell,F.J., McKnight,M.M., Iversen,P.L., Greenberg,D.E.
Abstract:
Background. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA/RNA analogues that silence expression of specific genes. We studied whether PPMOs targeted to essential genes in Acinetobacter lwoffii and Acinetobacter baumannii are active in vitro and in vivo. Methods. PPMOs were evaluated in vitro using miniμm inhibitory concentration (MIC) and viability assays, and in vivo using murine pulmonary infection models with intranasal PPMO treatment. Results. MICs of PPMOs ranged from 0.1 to 64 μM (approximately 0.6-38 μg/mL). The most effective PPMO tested was (RXR)4-AcpP, which is targeted to acpP. (RXR)4-AcpP reduced viability of A. lwoffii and A. baumannii by >10(3) colony-forming units/mL at 5-8 times MIC. Mice treated with >/=0.25 mg/kg of (RXR)4-AcpP survived longer and had less inflammation and bacterial lung burden than mice treated with a scrambled-sequence PPMO or phosphate-buffered saline. Treatment could be delayed after infection and still increase survival. Conclusions. PPMOs targeted to essential genes of A. lwoffii and A. baumannii were bactericidal and had MICs in a clinically relevant range. (RXR)4-AcpP increased survival of mice infected with A. lwoffii or A. baumannii, even when initial treatment was delayed after infection. PPMOs could be a viable therapeutic approach in dealing with multidrug-resistant Acinetobacter species.

Address: Department of Microbiology, Oregon State University

3916217d: Mol.BioSyst., 2981-2987(2013)
Link: [ PubMED ] [ DOI ]
Title: Cost-effective isobaric tagging for quantitative phosphoproteomics using DiART reagents.
Author: Ramsubramaniam,N., Tao,F., Li,S., Marten,M.R.
Abstract:
We describe the use of an isobaric tagging reagent, Deuterium isobaric Amine Reactive Tag (DiART), for quantitative phosphoproteomic experiments. Using DiART tagged custom mixtures of two phosphorylated peptides from α casein and their non-phosphorylated counterparts, we demonstrate the compatibility of DiART with TiO2 affinity purification of phosphorylated peptides. Comparison of theoretical vs. experimental reporter ion ratios reveals accurate quantification of phosphorylated peptides over a dynamic range of more than 15-fold. Using DiART labelling and TiO2 enrichment (DiART-TiO2) with large quantities of proteins (8 mg) from the cell lysate of model fungus Aspergillus nidulans, we quantified 744 unique phosphopeptides. Overlap of median values of TiO2 enriched phosphopeptides with theoretical values indicates accurate trends. Altogether these findings confirm the feasibility of performing quantitative phosphoproteomic experiments in a cost-effective manner using isobaric tagging reagents, DiART.

E-Mail: marten@umbc.edu
Address: Department of Chemical, Biochemical & Environmental Engineering, UMBC, Engineering Building, Room 314, 1000 Hilltop Circle, Baltimore, MD 21250, USA

3916218d: Sci.Signal., ra92-ra92(2013)
Link: [ PubMED ] [ DOI ]
Title: Distinct temporal patterns of T cell receptor signaling during positive versus negative selection in situ.
Author: Melichar,H.J., Ross,J.O., Herzmark,P., Hogquist,K.A., Robey,E.A.
Abstract:
The recognition by the T cell receptor (TCR) of self-peptides presented by the major histocompatibility complex (MHC) on antigen-presenting cells, such as dendritic cells and thymic epithelial cells, controls T cell fate in the thymus, with weak TCR signals inducing survival (positive selection) and stronger signals inducing death (negative selection). In vitro studies indicate that peptide ligands that induce positive selection stimulate a low, but sustained, pattern of TCR signaling; however, the temporal pattern of TCR signaling in MHC class I-restricted thymocytes (thymocytes that are presented with peptides by MHC class I) in the thymus, under conditions that support positive selection, is unknown. We addressed this question by examining intracellular Ca2+ dynamics and migratory changes in thymocytes undergoing positive and negative selection in thymic slices. Brief, serial signaling events that were separated by migratory periods and low cytosolic Ca2+ concentrations correlated with the positive selection of MHC class I-restricted thymocytes, whereas sustained Ca2+ signaling and the arrest of thymocytes were associated with negative selection. Low-avidity peptides and the presentation of peptides by cortical thymic epithelial cells, rather than dendritic cells, failed to induce strong migratory arrest of thymocytes, which led to transient TCR signaling. Thus, we provide a comparison of positive and negative selection signals in situ and suggest that the absence of strong stop signals distinguishes between positive and negative selection.

Address: 1Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA

3916220d: Mar.Drugs, 3875-3890(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Harnessing the potential of halogenated natural product biosynthesis by mangrove-derived actinomycetes.
Author: Li,X.G., Tang,X.M., Xiao,J., Ma,G.H., Xu,L., Xie,S.J., Xu,M.J., Xiao,X., Xu,J.
Abstract:
Mangrove-derived actinomycetes are promising sources of bioactive natural products. In this study, using homologous screening of the biosynthetic genes and anti-microorganism/tumor assaying, 163 strains of actinomycetes isolated from mangrove sediments were investigated for their potential to produce halogenated metabolites. The FADH2-dependent halogenase genes, identified in PCR-screening, were clustered in distinct clades in the phylogenetic analysis. The coexistence of either polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) as the backbone synthetases in the strains harboring the halogenase indicated that these strains had the potential to produce structurally diversified antibiotics. As a validation, a new enduracidin producer, Streptomyces atrovirens MGR140, was identified and confirmed by gene disruption and HPLC analysis. Moreover, a putative ansamycin biosynthesis gene cluster was detected in Streptomyces albogriseolus MGR072. Our results highlight that combined genome mining is an efficient technique to tap promising sources of halogenated natural products synthesized by mangrove-derived actinomycetes.

E-Mail: minjuanxu@sjtu.edu.cn
Address: College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China

3916221d: Mar.Drugs, 3861-3874(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Chlorella 11-peptide inhibits the production of macrophage-induced adhesion molecules and reduces endothelin-1 expression and endothelial permeability.
Author: Shih,M.F., Chen,L.C., Cherng,J.Y.
Abstract:
The inflammation process in large vessels involves the up-regulation of vascular adhesion molecules such as endothelial cell selectin (E-selectin), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) which are also known as the markers of atherosclerosis. We have reported that Chlorella 11-peptide exhibited effective anti-inflammatory effects. This peptide with an amino sequence Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe was further examined for its potential in preventing atherosclerosis in this study. In particular, the roles of Chlorella 11-peptide in lowering the production of vascular adhesion molecules, monocyte chemoattractant protein (MCP-1) and expression of endothelin-1 (ET-1) from endothelia (SVEC4-10 cells) were studied. The production of E-selectin, ICAM-1, VCAM-1 and MCP-1 in SVEC4-10 cells was measured with ELISA. The mRNA expression of ET-1 was analyzed by RT-PCR and agarose gel. Results showed that Chlorella 11-peptide significantly suppressed the levels of E-selectin, ICAM, VCAM, MCP-1 as well as ET-1 gene expression. The inhibition of ICAM-1 and VCAM-1 production by Chlorella 11-peptide was reversed in the presence of protein kinase A inhibitor (H89) which suggests that the cAMP pathway was involved in the inhibitory cause of the peptide. In addition, this peptide was shown to reduce the extent of increased intercellular permeability induced by combination of 50% of lipopolysaccharide (LPS)-activated RAW 264.7 cells medium and 50% normal SEVC cell culture medium (referred to as 50% RAW-conditioned medium). These data demonstrate that Chlorella 11-peptide is a promising biomolecule in preventing chronic inflammatory-related vascular diseases.

E-Mail: jycherng@yahoo.com
Address: Department of Pharmacy, Chia-Nan University of Pharmacy & Science, 60 Erh-Te Rd, Sec. 1, Tainan 71710, Taiwan

3916224d: Food Chem., 1076-1085(2014)
Link: [ PubMED ] [ DOI ]
Title: Application of taste sensing system for characterisation of enzymatic hydrolysates from shrimp processing by-products.
Author: Cheung,I.W., Li-Chan,E.C.
Abstract:
The objective of this study was to investigate the potential of an instrumental taste-sensing system to distinguish between shrimp processing by-products hydrolysates produced using different proteases and hydrolysis conditions, and the possible association of taste sensor outputs with human gustatory assessment, salt content, and bioactivity. Principal component analysis of taste sensor output data categorised samples according to the proteases used for hydrolysis. High umami sensor outputs were characteristic of bromelain- and Flavourzyme-produced hydrolysates, compared to low saltiness and high bitterness outputs of Alcalase-produced hydrolysates, and high saltiness and low umami outputs of Protamex-produced hydrolysates. Extensively hydrolysed samples showed higher sourness outputs. Saltiness sensor outputs were correlated with conductivity and sodium content, while umami sensor responses were related to gustatory sweetness, bitterness and umami, as well as angiotensin-I converting enzyme inhibitory activity. Further research should explore the dose dependence and sensitivity of each taste sensor to specific amino acids and peptides.

Address: The University of British Columbia, Faculty of Land and Food Systems, Food Nutrition and Health Program, 2205 East Mall, Vancouver, British Columbia V6T 1Z4, Canada

3916226d: Food Chem., 892-902(2014)
Link: [ PubMED ] [ DOI ]
Title: Characterisation of Maillard reaction products derived from LEKFD - A pentapeptide found in β-lactoglobulin sequence, glycated with glucose - By tandem mass spectrometry, molecular orbital calculations and gel filtration chromatography coupled with continuous photodiode array.
Author: Yamaguchi,K., Homma,T., Nomi,Y., Otsuka,Y.
Abstract:
Maillard reaction peptides (MRPs) contribute to taste, aroma, colour, texture and biological activity. However, peptide degradation or the cross-linking of MRPs in the Maillard reaction has not been investigated clearly. A peptide of LEKFD, a part of β-lactoglobulin, was heated at 110 degrees C for 24h with glucose and the reaction products were analysed by HPLC with ODS, ESI-MS, ESI-MS/MS and HPLC with gel-filtration column and DAD detector. In the HPLC fractions, an imminium ion of LEK*FD, a pyrylium ion or a hydroxymethyl furylium ion of LEK*FD, and KFD and EK were detected by ESI-MS. Therefore, those products may be produced by the Maillard reaction. The molecular orbital of glycated LEKFD at the lysine ε-amino residue with Schiff base form was calculated by MOPAC. HPLC with gel-filtration column showed cross-linking and degradation of peptides.

E-Mail: yama-k@fc.jwu.ac.jp
Address: Department of Food and Nutrition, Japan Women's University, Bunkyo-ku, Tokyo 112-8681, Japan. Electronic address:

3916227d: Food Chem., 845-852(2014)
Link: [ PubMED ] [ DOI ]
Title: Susceptibility of milk protein-derived peptides to dipeptidyl peptidase IV (DPP-IV) hydrolysis.
Author: Nongonierma,A.B., Fitzgerald,R.J.
Abstract:
In silico digestion of milk protein-derived peptides with gastrointestinal enzyme activities was used to predict the release of peptides with a Pro residue at position 2 from the N terminus. These peptides are known to act as preferred dipeptidyl peptidase IV (DPP-IV) substrates. Five casein-derived synthetic peptides (Ile-Pro-Ile-Gln-Tyr, Leu-Pro-Leu-Pro-Leu, Tyr-Pro-Tyr-Tyr, Leu-Pro-Tyr-Pro-Tyr and Ile-Pro-Ile) and a casein (CasH), whey (WPH) and lactoferrin hydrolysate (LFH) generated with gastrointestinal enzymes were incubated with DPP-IV at 37 degrees C for 18 or 24h. Peptide breakdown was evident following incubation with DPP-IV. Different modes of DPP-IV inhibition were observed depending on the test compound. Ile-Pro-Ile-Gln-Tyr, Tyr-Pro-Tyr-Tyr and Leu-Pro-Tyr-Pro-Tyr were substrate-, Leu-Pro-Leu-Pro-Leu and CasH were prodrug- while WPH and LFH were true DPP-IV inhibitors. These results are relevant for the bioactivity and bioavailability of functional foods targeting DPP-IV inhibition with potential blood glucose regulatory properties in humans.

Address: Department of Life Sciences and Food for Health Ireland (FHI), University of Limerick, Limerick, Ireland

3916228d: Food Chem., 840-844(2014)
Link: [ PubMED ] [ DOI ]
Title: Proteolytic characterisation in grass carp sausage inoculated with Lactobacillus plantarum and Pediococcus pentosaceus.
Author: Nie,X., Lin,S., Zhang,Q.
Abstract:
The proteolysis in grass carp sausages inoculated with Lactobacillus plantarum ZY40 and Pediococcus pentosaceus GY23 was investigated. As fermentation progressed, sarcoplasmic and myofibrillar proteins in both sausages were obviously degraded, and the proteolytic process was more intense in sausages inoculated with P. pentosaceus GY23. The increases in α-amino nitrogen, trichloroacetic acid (TCA)-soluble peptides and free amino acids were also detected in both sausages. The differences in α-amino nitrogen content and free amino acids concentration were due to the activity of inoculated lactic acid bacteria, while endogenous enzymes contributed to the release of TCA-soluble peptides. Our findings indicate that lactic acid bacteria influence proteolytic characterisation in fermented fish sausage, with strain-dependent activity.

E-Mail: niexiaohua2000@126.com
Address: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China. Electronic address:

3916229d: Food Chem., 632-638(2014)
Link: [ PubMED ] [ DOI ]
Title: Purification and characterization of a gelatinolytic matrix metalloproteinase from the skeletal muscle of grass carp (Ctenopharyngodon idellus).
Author: Wu,J.L., Ge,S.Y., Cai,Z.X., Liu,H., Liu,Y.X., Wang,J.H., Zhang,Q.Q.
Abstract:
A gelatinolytic matrix metalloproteinase (gMMP) from grass carp skeletal muscle was purified by 30-70% ammonium sulphate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, and affinity on gelatin-sepharose. The molecular weight of the proteinase as estimated by SDS-PAGE was 70kDa under non-reducing conditions. The enzyme revealed high activity from 30 to 50 degrees C, and the gelatin hydrolysing activity was investigated at a slightly alkaline pH range using gelatin as substrate. Metalloproteinase inhibitor EDTA completely suppressed the gelatinolytic activity, while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca2+ was essential for the gelatinolytic activity. Further, peptide mass fingerprinting obtained four fragments with 45 amino acid residues, which were highly identical to MMP-2 from fish species. The gMMP could effectively hydrolyse type I collagen even at 4 degrees C, suggesting its involvement in the texture softening of fish muscle during the post-mortem stage.

E-Mail: wujiul590@126.com
Address: Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, China. Electronic address:

3916230d: Food Chem., 473-480(2014)
Link: [ PubMED ] [ DOI ]
Title: Effect of initial protein concentration and pH on in vitro gastric digestion of heated whey proteins.
Author: Zhang,S., Vardhanabhuti,B.
Abstract:
The in vitro digestion of heated whey protein aggregates having different structure and physicochemical properties was evaluated under simulated gastric conditions. Aggregates were formed by heating whey protein isolates (WPI) at 3-9% w/w initial protein concentration and pH 3.0-7.0. Results showed that high protein concentration led to formation of larger WPI aggregates with fewer remaining monomers. Aggregates formed at high protein concentrations showed slower degradation rate compared to those formed at low protein concentration. The effect of initial protein concentration on peptide release pattern was not apparent. Heating pH was a significant factor affecting digestion pattern. At pH above the isoelectric point, the majority of the proteins involved in the aggregation, and aggregates formed at pH 6.0 were more susceptible to pepsin digestion than at pH 7.0. At acidic conditions, only small amount of proteins was involved in the aggregation and heated aggregates were easily digested by pepsin, while the remaining unaggregated proteins were very resistant to gastric digestion. The potential physiological implication of these results on satiety was discussed.

Address: Food Science Program, Division of Food Systems and Bioengineering, University of Missouri, Columbia, MO 65211, USA

3916231d: Food Chem., 66-76(2014)
Link: [ PubMED ] [ DOI ]
Title: Anti-diabetic and antihypertensive activities of two flaxseed protein hydrolysate fractions revealed following their simultaneous separation by electrodialysis with ultrafiltration membranes.
Author: Doyen,A., Udenigwe,C.C., Mitchell,P.L., Marette,A., Aluko,R.E., Bazinet,L.
Abstract:
Flaxseed protein hydrolysate has been fractionated by electrodialysis with two ultrafiltration membranes (20 and 50kDa) stacked in the system for the recovery of two specific cationic peptide fractions (KCl-F1 and KCl-F2). After 360min of treatment, peptide migration increased as a function of time in KCl compartments. Moreover, the use of two different ultrafiltration membrane allowed concentration of the 300-400 and 400-500Da molecular weight range peptides in the KCl-F1 and KCl-F2 fractions, respectively, compared to the initial hydrolysate. After mass spectrometry analysis, higher amounts of low molecular weight peptides were recovered in the KCl-F2 compartment while relatively higher molecular weight peptides were more detected in the KCl-F1 compartment. Amino acid analysis showed that His, Lys and Arg were especially concentrated in the KCl compartments. Finally, glucose-transport assay demonstrated that the KCl-F2 fraction increased glucose uptake while oral administration of KCl-F1 and final FPH decreased systolic blood pressure.

Address: Department of Food Science and Nutrition, Universite Laval, Quebec, Quebec G1V 0A6, Canada

3916232d: J.Chromatogr.A, 154-159(2013)
Link: [ PubMED ] [ DOI ]
Title: Preparation of porous styrenics-based monolithic layers for thin layer chromatography coupled with matrix-assisted laser-desorption/ionization time-of-flight mass spectrometric detection.
Author: Lv,Y., Lin,Z., Tan,T., Svec,F.
Abstract:
Monolithic 50μm thin poly(4-methylstyrene-co-chloromethylstyrene-co-divinylbenzene) layers attached to 6.0cmx3.3cm glass plates have been prepared, using a thermally initiated polymerization process. These layers had a well-defined porous structure with a globular morphology demonstrated with SEM images and exhibited superhydrophobic properties characterized with a water contact angle of 157 degrees . They were then used for thin-layer chromatography of peptides and proteins fluorescently labeled with fluorescamine. The spots of individual separated compounds were visualized using UV light, and their identities were confirmed with a matrix-assisted laser desorption/ionization time of flight mass spectrometry. The presence of chloromethylstyrene units in the polymer enabled hypercrosslinking via a Friedel-Crafts alkylation reaction, and led to monoliths with much larger surface areas, which were suitable for separations of small dye molecules.

Address: Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China

3916234d: BMC Cell Biol., 46-46(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Functional analysis of the rodent CK1tau mutation in the circadian clock of a marine unicellular alga.
Author: Van Ooijen,G., Martin,S.F., Barrios-Llerena,M.E., Hindle,M., Le Bihan,T., O'Neill,J.S., Millar,A.J.
Abstract:
BACKGROUND: Casein Kinase 1 (CK1) is one of few proteins known to affect cellular timekeeping across metazoans, and the naturally occurring CK1tau mutation shortens circadian period in mammals. Functional conservation of a timekeeping function for CK1 in the green lineage was recently identified in the green marine unicell Ostreococcus tauri, in spite of the absence of CK1's transcriptional targets known from other species. The short-period phenotype of CK1tau mutant in mammals depends specifically on increased CK1 activity against PERIOD proteins. To understand how CK1 acts differently upon the algal clock, we analysed the cellular and proteomic effects of CK1tau overexpression in O. tauri. RESULTS: Overexpression of the CK1tau in O. tauri induces period lengthening identical to overexpression of wild-type CK1, in addition to resistance to CK1 inhibitor IC261. Label-free quantitative mass spectrometry of CK1tau overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1tau. Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins. CONCLUSIONS: The identical phenotype for overexpression of wild-type CK1 and CK1tau is in line with the absence of critical targets for rodent CK1tau in O. tauri. Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1tau. These results indicate that the two alleles are functionally indiscriminate in O. tauri, and verify the identified cellular CK1 target proteins in a minimal circadian model organism.

3916246d: PLoS One, e76673-e76673(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Identification of Virulence Properties in Salmonella Typhimurium DT104 Using Caenorhabditis elegans.
Author: Sahu,S.N., Anriany,Y., Grim,C.J., Kim,S., Chang,Z., Joseph,S.W., Cinar,H.N.
Abstract:
Salmonella enterica serover Typhimurium definitive phage type DT104, resistant to multiple antibiotics, is one of the most widespread Salmonella species in human infection worldwide. Although several cohort studies indicate that DT104 carrying the multidrug resistance (MDR) locus on salmonella genomic island 1 is a possible hyper-virulent strain compared to DT104 strains without MDR, or other Salmonella enterica serotypes, existing experimental evidence regarding virulence properties associated with the MDR region is controversial. To address this question, we constructed an isogenic MDR deletion (MDR) mutant strain of DT104, SNS12, by allelic exchange and used Caenorhabditis elegans as a host model to assess differences in virulence between these two strains. SNS12 exhibited decreased virulence in C. elegans, and we observed increased colonization and proliferation of the intestine of C. elegans by DT104. The immune response against MDR-carrying DT104 appears to function through a non-canonical Unfolded Protein Response (UPR) pathway, namely prion-like-(QN-rich)-domain-bearing protein pathway (PQN), in a ced-1 dependent manner in C. elegans. Further, we also demonstrate that genes of the PQN pathway and antimicrobial peptide gene abf-2, are expressed at higher transcriptional levels in worms immediately following exposure to DT104, in comparison with worms exposed to SNS12. Altogether, our results suggest that the MDR region of Salmonella Typhimurium DT104 has a direct role in virulence against Caenorhabditis elegans.

Address: Division of Virulence Assessment, Food and Drug Administration, Laurel, Maryland, United States of America

3916248d: PLoS One, e74389-e74389(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Non-Immunogenicity of Overlapping Gag Peptides Pulsed on Autologous Cells after Vaccination of HIV Infected Individuals.
Author: Kloverpris,H.N., Jackson,A., Handley,A., Hayes,P., Gilmour,J., Riddell,L., Chen,F., Atkins,M., Boffito,M., Walker,B.D., Ackland,J., Sullivan,M., Goulder,P.
Abstract:
BACKGROUND: HIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation. METHODOLOGY: We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach 'OPAL-HIV-Gag(c)'. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6), 24 mg (n = 7), 48 mg (n = 2) or matching placebo (n = 8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS). RESULTS: The OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16). CONCLUSION/SIGNIFICANCE: Despite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required. NAME OF REGISTRY: ClinicalTrials.gov, Registry number: NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search.

Address: Department of Paediatrics, University of Oxford, Oxford, United Kingdom

3916249d: Int.J.Nanomed., 3745-3756(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Preparation and characterization of gadolinium-loaded PLGA particles surface modified with RGDS for the detection of thrombus.
Author: Zhang,Y., Zhou,J., Guo,D., Ao,M., Zheng,Y., Wang,Z.
Abstract:
Thrombotic disease is a leading cause of death and disability worldwide. The development of magnetic resonance molecular imaging provides potential promise for early disease diagnosis. In this study, we explore the preparation and characterization of gadolinium (Gd)-loaded poly (lactic-co-glycolic acid) (PLGA) particles surface modified with the Arg-Gly-Asp-Ser (RGDS) peptide for the detection of thrombus. PLGA was employed as the carrier-delivery system, and a double emulsion solvent-evaporation method (water in oil in water) was used to prepare PLGA particles encapsulating the magnetic resonance contrast agent Gd diethylenetriaminepentaacetic acid (DTPA). To synthesize the Gd-PLGA/chitosan (CS)-RGDS particles, carbodiimide-mediated amide bond formation was used to graft the RGDS peptide to CS to form a CS-RGDS film that coated the surface of the PLGA particles. Blank PLGA, Gd-PLGA, and Gd-PLGA/CS particles were fabricated using the same water in oil in water method. Our results indicated that the RGDS peptide successfully coated the surface of the Gd-PLGA/CS-RGDS particles. The particles had a regular shape, smooth surface, relatively uniform size, and did not aggregate. The high electron density of the Gd-loaded particles and a translucent film around the particles coated with the CS and CS-RGDS films could be observed by transmission electron microscopy. In vitro experiments demonstrated that the Gd-PLGA/CS-RGDS particles could target thrombi and could be imaged using a clinical magnetic resonance scanner. Compared with the Gd-DTPA solution, the longitudinal relaxation time of the Gd-loaded particles was slightly longer, and as the Gd-load concentration increased, the longitudinal relaxation time values decreased. These results suggest the potential of the Gd-PLGA/CS-RGDS particles for the sensitive and specific detection of thrombus at the molecular level.

Address: Department of Radiology, the Second Affiliated Hospital of Chongqing Medical University, Chongqing, People's Republic of China

3916252d: J.Am.Soc.Mass Spectrom., 1534-1542(2013)
Link: [ PubMED ] [ DOI ]
Title: Locating Pb2+ and Zn2+ in zinc finger-like peptides using mass spectrometry.
Author: Banu,L., Blagojevic,V., Bohme,D.K.
Abstract:
The binding preferences of Pb2+and Zn2+ in doubly charged complexes with zinc finger-like 12-residue peptides (Pep), [Mn(Pep-2(n-1)H)]2+ have been explored using tandem mass spectrometry. The peptides were synthesized strategically by blocking the N-terminus with an acetyl group and with four cysteine and/or histidine residues in positions 2, 5, 8, and 11, arranged in different motifs: CCHH, CHCH, and CCCC. The MS(2) spectra of the Pb2+ and Zn2+ complexes show multiple losses of water and a single methane loss and these provide a sensitive method for locating the metal dication and so elucidating its coordination. The elimination of a methane molecule indicated the position of the metal at the Cys2 residue. Whereas lead was observed to preferentially bind to cysteine residues, zinc was found to primarily bind to histidine residues and secondarily to cysteine residues. Preferential binding of lead to cysteine is preserved in the complexes with more than one Pb2+. Key to the mechanism of the loss of water and methane is the metal dication withdrawing electrons from the proximal amidic nitrogen. This acidic nitrogen loses its hydrogen to an amidic oxygen situated four atoms away leading to formation of a five-member ring and the elimination of water.

Address: Department of Chemistry, Centre for Research in Mass Spectrometry, York University, Toronto, Ontario, M3J 1P3, Canada

3916253d: Exp.Mol.Med., e34-e34(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Characterizing affinity epitopes between prion protein and β-amyloid using an epitope mapping immunoassay.
Author: Kang,M., Kim,S.Y., An,S.S., Ju,Y.R.
Abstract:
Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that β-amyloid1-42 oligomer causes neurotoxicity associated with Alzheimer's disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer's disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in β-amyloid. Residues 23-39 and 93-119 in the prion protein were involved in binding to β-amyloid1-40 and 1-42, and monomers of this protein interacted with prion protein residues 93-113 and 123-166. Furthermore, β-amyloid antibodies against the C-terminus detected bound β-amyloid1-42 at residues 23-40, 104-122 and 159-175. β-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to β-amyloid1-40 and 1-42. The 3D structure appears to be necessary for β-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer's disease.

Address: Department of Bionanotechnology, Gachon University, Gyeonggi, Korea

3916254d: Lab Chip, 3853-3861(2013)
Link: [ PubMED ] [ DOI ]
Title: Tailoring of a TiO2 nanotube array-integrated portable microdevice for efficient on-chip enrichment and isotope labeling of serum phosphopeptides.
Author: Min,Q., Chen,X., Zhang,X., Zhu,J.J.
Abstract:
Herein we present a gravity-driven microdevice furnished with tunable TiO2 nanotube arrays (TNAs) inside as the separation medium for consecutive on-chip enrichment and isotope labeling of serum phosphopeptides. The 3D tubular architectures of TNAs dramatically enhanced the affinity towards phosphate-containing molecules and also provided a spacious microenvironment for isotope dimethyl labeling reactions. To maximize the efficiency and capacity of the phosphopeptide enrichment, nanoscale tailoring and microscale fabrication were employed for adjusting the TNAs' pore sizes and the channel patterns. The S-shaped microdevice equipped with interior TNAs anodized at 25 V was utilised for consecutive serum processing, and further differential expression analysis of endogenous phosphopeptides between ovarian cancer patients and healthy women. The phosphorylated fibrinogen peptide A (FPA, AD[pS]GEGDFLAEGGGVR) was found to be down-regulated by about 4 times while its isoform (D[pS]GEGDFLAEGGGV) was 2.4-fold up-regulated in the patient specimens. In principle, this nanostructure-embedded model introduced tailor-made bioseparation materials into the microdevice, undoubtedly facilitating the workflow of sample pretreatment and thus assisting the analysis of disease-associated biomolecules in biomedical research.

E-Mail: jjzhu@nju.edu.cn
Address: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China

3916256d: Analyst, 5757-5761(2013)
Link: [ PubMED ] [ DOI ]
Title: Fabrication of a protease sensor for caspase-3 activity detection based on surface plasmon resonance.
Author: Chen,H., Mei,Q., Hou,Y., Zhu,X., Koh,K., Li,X., Li,G.
Abstract:
Diagnosis of apoptosis is essential to the early detection of therapy efficiency and the evaluation of disease progression. Caspase-3 is supposed to be closely related to cellular apoptosis. We describe here a label-free surface plasmon resonance (SPR) detection of apoptosis based on caspase-3 activity assay through enzyme digestion. An artificial peptide sequence was designed as a substrate of caspase-3 and immobilized on a gold disk through covalent binding. The 4Lys part at the end of the pentadecyl-peptide was designed to form a unique peptide array through electrostatic repulsion. The immobilization of the peptide on the gold surface was carefully characterized by SPR and atomic force microscopy. The catalytic conditions of caspase-3 were optimized with electrochemical impedance spectroscopy. The detection limit of caspase-3 was found at a concentration of 1 pg mL(-1). The activity of caspase-3 in apoptotic cells could also be measured sensitively by the one-step and intuitional SPR response decrease. The fabricated simple and convenient caspase-3 sensor is proposed for application in clinical analysis.

Address: Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, P R China

3916259d: Int.J.Biochem.Cell Biol., 2491-2495(2013)
Link: [ PubMED ] [ DOI ]
Title: The critical role of STIM1-dependent Ca2+ signalling during T-cell development and activation.
Author: Samakai,E., Hooper,R., Soboloff,J.
Abstract:
T lymphocytes are key cellular effectors of adaptive immunity able to recognize a virtually limitless number of antigenic peptides and mount an immune response. Ca2+ signals are crucial to the development and activation of T cells and Stromal Interaction Molecule 1 (STIM1) has been identified as a critical modulator of intracellular Ca2+ levels in T cells. Although the role of STIM1 in T cell activation has been extensively investigated, the role of STIM1 in T cell development has been somewhat controversial. Indeed, deficiencies in STIM1 expression and function lead to both developmental defects associated with the development of autoimmunity yet also interfere with T cell activation leading to severe combined immunodeficiency signifying a multifaceted role of STIM1 in T cell physiology and pathophysiology.

Address: Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, United States

3916260d: Anal.Biochem., 130-137(2013)
Link: [ PubMED ] [ DOI ]
Title: Probing copper/tau protein interactions electrochemically.
Author: Martic,S., Rains,M.K., Kraatz,H.B.
Abstract:
Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid β-peptide (Aβ), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140+/-5mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.

Address: Department of Chemistry, Oakland University, Rochester, NY 48309, USA

3916261d: Biochemistry, 5830-5843(2013)
Link: [ PubMED ] [ DOI ]
Title: Binding of a third metal ion by the human phosphatases PP2Cα and Wip1 is required for phosphatase activity.
Author: Tanoue,K., Jenkins,L.M.M., Durell,S.R., Debnath,S., Sakai,H., Tagad,H.D., Ishida,K., Appella,E., Mazur,S.J.
Abstract:
The PPM phosphatases require millimolar concentrations of Mg(2)+ or Mn(2)+ to activate phosphatase activity in vitro. The human phosphatases PP2Cα (PPM1A) and Wip1 (PPM1D) differ in their physiological function, substrate specificity, and apparent metal affinity. A crystallographic structure of PP2Cα shows only two metal ions in the active site. However, recent structural studies of several bacterial PP2C phosphatases have indicated three metal ions in the active site. Two residues that coordinate the third metal ion are highly conserved, suggesting that human PP2C phosphatases may also bind a third ion. Here, isothermal titration calorimetry analysis of Mg(2)+ binding to PP2Cα distinguished binding of two ions to high affinity sites from the binding of a third ion with a millimolar affinity, similar to the apparent metal affinity required for catalytic activity. Mutational analysis indicated that Asp239 and either Asp146 or Asp243 was required for low-affinity binding of Mg(2)+, but that both Asp146 and Asp239 were required for catalysis. Phosphatase activity assays in the presence of MgCl_2, MnCl_2, or mixtures of the two, demonstrate high phosphatase activity toward a phosphopeptide substrate when Mg(2)+ was bound to the low-affinity site, whether Mg(2)+ or Mn(2)+ ions were bound to the high affinity sites. Mutation of the corresponding putative third metal ion-coordinating residues of Wip1 affected catalytic activity similarly both in vitro and in human cells. These results suggest that phosphatase activity toward phosphopeptide substrates by PP2Cα and Wip1 requires the binding of a Mg(2)+ ion to the low-affinity site.

Address: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States

3916262d: Int.J.Cosmet.Sci., 588-599(2013)
Link: [ PubMED ] [ DOI ]
Title: Rapid screening method to study the reactivity of UV filter substances towards skin proteins by high-performance thin-layer chromatography.
Author: Stiefel,C., Schwack,W.
Abstract:
OBJECTIVE: Most UV filters used in sunscreens and other cosmetic products contain carbonyl groups, which generally are able to react with peptides or free amino acids of the human skin. To estimate their reactivity, we studied different prominent UV filter substances, octocrylene, ethylhexyl salicylate, 4-t-butyl-4'-methoxydibenzoylmethane, ethylhexyl methoxycinnamate, benzophenone-3, hydroxymethylbenzoyl sulphonic acid, octyldimethyl p-aminobenzoic acid, 3-benzylidene camphor, 4-methylbenzylidene camphor, diethylhexyl butamido triazone and ethylhexyl triazone. METHODS: A simple screening method using an amino HPTLC plate as protein model was established. The influence of different reaction conditions like heating and irradiation was determined. RESULTS: The ketones BP-3, HMBS and BM-DBM revealed the highest binding rates after both irradiation and heating. After 1 h of irradiation, 82%, 28% and 96%, respectively, were bonded to the amino phase, while heating resulted in values of 52%, 36% and 16%. For BP-3 and HMBS, even storage in the dark at room temperature resulted in a low binding. Contrarily, for the two camphor derivatives 3-BC and 4-MBC, only irradiation led to a slightly turnover. UV filters with ester groups also showed a different behaviour depending on their main skeleton. While OCR especially reacted under heating with the amino phase, resulting in 36% of bound species after one hour, UV irradiation particularly encouraged a reaction of the other esters. After 1 h irradiation, 15% of EHMC, 38% of EHS and 48% of OD-PABA were bonded to the amino groups of the HPTLC plate, whereas the reactivity of the two triazones, EHT and DEBT, was comparatively low. CONCLUSION: Especially the UV filters BP-3, BM-DBM, HMBS, EHMC or OCR, which are commonly known to cause contact dermatitis, showed a high tendency to form adducts with the amino layer. Thus, the amino plate seems to be a proper tool to screen for skin sensitizers.

Address: Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, Stuttgart, 70599, Germany

3916263d: J.Proteome Res., 4187-4192(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Determination of phosphorylation sites in the DivIVA cytoskeletal protein of Streptomyces coelicolor by targeted LC-MS/MS.
Author: Saalbach,G., Hempel,A.M., Vigouroux,M., Flardh,K., Buttner,M.J., Naldrett,M.J.
Abstract:
The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC-MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC-MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD000095.

E-Mail: gerhard.saalbach@jic.ac.uk
Address: John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom

3916271d: J.Infect.Dis., 1695-1704(2013)
Link: [ PubMED ] [ DOI ]
Title: Heparin-Binding Motifs and Biofilm Formation by Candida albicans.
Author: Green,J.V., Orsborn,K.I., Zhang,M., Tan,Q.K., Greis,K.D., Porollo,A., Andes,D.R., Long Lu,J., Hostetter,M.K.
Abstract:
Candida albicans is a leading pathogen in infections of central venous catheters, which are frequently infused with heparin. Binding of C. albicans to medically relevant concentrations of soluble and plate-bound heparin was demonstrable by confocal microscopy and enzyme-linked immunosorbent assay (ELISA). A sequence-based search identified 34 C. albicans surface proteins containing >/=1 match to linear heparin-binding motifs. The virulence factor Int1 contained the most putative heparin-binding motifs (n = 5); peptides encompassing 2 of 5 motifs bound to heparin-Sepharose. Alanine substitution of lysine residues K805/K806 in 804QKKHQIHK811 (motif 1 of Int1) markedly attenuated biofilm formation in central venous catheters in rats, whereas alanine substitution of K1595/R1596 in 1593FKKRFFKL1600 (motif 4 of Int1) did not impair biofilm formation. Affinity-purified immunoglobulin G (IgG) recognizing motif 1 abolished biofilm formation in central venous catheters; preimmune IgG had no effect. After heparin treatment of C. albicans, soluble peptides from multiple C. albicans surface proteins were detected, such as Eno1, Pgk1, Tdh3, and Ssa1/2 but not Int1, suggesting that heparin changes candidal surface structures and may modify some antigens critical for immune recognition. These studies define a new mechanism of biofilm formation for C. albicans and a novel strategy for inhibiting catheter-associated biofilms.

Address: Division of Pediatric Infectious Diseases

3916275d: Nanoscale, 9110-9117(2013)
Link: [ PubMED ] [ DOI ]
Title: Peptide-based carbon nanotubes for mitochondrial targeting.
Author: Battigelli,A., Russier,J., Venturelli,E., Fabbro,C., Petronilli,V., Bernardi,P., Da Ros,T., Prato,M., Bianco,A.
Abstract:
In the present study, we report the design and synthesis of peptide-based-multi-walled carbon nanotubes (MWCNTs) to target mitochondria. Targeting these intracellular organelles might open the way to develop alternative systems to address diseases related to genetic mutations in mitochondrial (mt)-DNA, by delivering therapeutic oligonucleotides. The first step towards mitochondrial delivery of this type of nucleic acid was to target MWCNTs to mitochondria by covalent functionalization with a well-known endogenous mitochondrial targeting sequence (MTS). The subcellular localization of the conjugates, which were fluorescently labeled, in murine RAW 264.7 macrophages and human HeLa cells was then studied using different microscopy techniques, such as wide-field epifluorescence microscopy, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). The localization of the MTS-MWCNT conjugates into mitochondria was further confirmed by analyzing the isolated organelles using TEM.

E-Mail: prato@units.it
Address: Department of Chemical and Pharmaceutical Sciences, University of Trieste, Piazzale Europa 1, Trieste, 34127, Italy

3916276d: J.Biochem.(Tokyo), 325-332(2013)
Link: [ PubMED ] [ DOI ]
Title: Affinity maturation of a CDR3-grafted VHH using in silico analysis and surface plasmon resonance.
Author: Inoue,H., Suganami,A., Ishida,I., Tamura,Y., Maeda,Y.
Abstract:
A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al., Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971-1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by approximately 20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.

Address: Kyowa Hakko Kirin Co., Ltd., 3-6-6 Asahi-machi, Machida, Tokyo 194-8533

3916277d: J.Biol.Chem., 26635-26648(2013)
Link: [ PubMED ] [ DOI ]
Title: A monoclonal antibody (MCPR3-7) interfering with the activity of proteinase 3 by an allosteric mechanism.
Author: Hinkofer,L.C., Seidel,S.A., Korkmaz,B., Silva,F., Hummel,A.M., Braun,D., Jenne,D.E., Specks,U.
Abstract:
Proteinase 3 (PR3) is an abundant serine protease of neutrophil granules and a major target of autoantibodies (PR3 anti-neutrophil cytoplasmic antibodies) in granulomatosis with polyangiitis. Some of the PR3 synthesized by promyelocytes in the bone marrow escapes the targeting to granules and occurs on the plasma membrane of naive and primed neutrophils. This membrane-associated PR3 antigen may represent pro-PR3, mature PR3, or both forms. To discriminate between mature PR3 and its inactive zymogen, which have different conformations, we generated and identified a monoclonal antibody called MCPR3-7. It bound much better to pro-PR3 than to mature PR3. This monoclonal antibody greatly reduced the catalytic activity of mature PR3 toward extended peptide substrates. Using diverse techniques and multiple recombinant PR3 variants, we characterized its binding properties and found that MCPR3-7 preferentially bound to the so-called activation domain of the zymogen and changed the conformation of mature PR3, resulting in impaired catalysis and inactivation by α1-proteinase inhibitor (α1-antitrypsin). Noncovalent as well as covalent complexation between PR3 and α1-proteinase inhibitor was delayed in the presence of MCPR3-7, but cleavage of certain thioester and paranitroanilide substrates with small residues in the P1 position was not inhibited. We conclude that MCPR3-7 reduces PR3 activity by an allosteric mechanism affecting the S1' pocket and further prime side interactions with substrates. In addition, MCPR3-7 prevents binding of PR3 to cellular membranes. Inhibitory antibodies targeting the activation domain of PR3 could be exploited as highly selective inhibitors of PR3, scavengers, and clearers of the PR3 autoantigen in granulomatosis with polyangiitis.

Address: Institute of Lung Biology and Disease (iLBD), University Hospital, Ludwig Maximilians University and Helmholtz Zentrum Munchen, Member of the German Center for Lung Research (DZL), Max-Lebsche-Platz 31, 81377 Munich, Germany

3916281d: ACS Chem.Biol., 2122-2126(2013)
Link: [ PubMED ] [ DOI ]
Title: MicroRNA-Specific Argonaute 2 Protein Inhibitors.
Author: Schmidt,M.F., Korb,O., Abell,C.
Abstract:
As microRNA silencing processes are mediated by the protein Argonaute 2 and for target RNA binding only a short sequence at the microRNA's 5' end (seed region) is crucial, we report a novel inhibitor class: the microRNA-specific Argonaute 2 protein inhibitors that not only block this short recognition sequence but also bind to the protein's active site. We developed a model for rational drug design, enabling the identification of Argonaute 2 active site binders and their linkage with a peptide nucleic acid sequence (PNA), which addresses the microRNA of interest. The designed inhibitors targeting microRNA-122, a hepatitis C virus drug target, had an IC50 of 100 nM, 10-fold more active than the simple PNA sequence (IC50 of 1 μM), giving evidence that the strategy has potential. Due to their lower molecular weight, these inhibitors may show better pharmacokinetic properties than reported oligonucleotide inhibitors, enabling them for potential therapeutic use.

Address: University Chemical Laboratory, University of Cambridge , Lensfield Road, CB2 1EW Cambridge, United Kingdom

3916283d: Avian Dis.Suppl., 469-473(2013)
Link: [ PubMED ]
Title: Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.
Author: Reddy,S.M., Sun,A., Khan,O.A., Lee,L.F., Lupiani,B.
Abstract:
Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.

Address: College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA

3916288d: J.Am.Soc.Mass Spectrom., 1543-1554(2013)
Link: [ PubMED ] [ DOI ]
Title: Protonated dipeptide losses from b(5) and b(4) ions of side chain hydroxyl group containing pentapeptides.
Author: Atik,A.E., Yalcin,T.
Abstract:
In this study, C-terminal protonated dipeptide eliminations were reported for both b5 and b4 ions of side chain hydroxyl group (-OH) containing pentapeptides. The study utilized the model C-terminal amidated pentapeptides having sequences of XGGFL and AXVYI, where X represents serine (S), threonine (T), glutamic acid (E), aspartic acid (D), or tyrosine (Y) residue. Upon low-energy collision-induced dissociation (CID) of XGGFL (where X = S, T, E, D, and Y) model peptide series, the ions at m/z 279 and 223 were observed as common fragments in all b5 and b4 ion (except b4 ion of YGGFL) mass spectra, respectively. By contrast, peptides, namely SMeGGFL-NH2 and EOMeGGFL-NH2, did not show either the ion at m/z 279 or the ion at m/z 223. It is shown that the side chain hydroxyl group is required for the possible mechanism to take place that furnishes the protonated dipeptide loss from b5 and b4 ions. In addition, the ions at m/z 295 and 281 were detected as common fragments in all b5 and b4 ion (except b4 ion of AYVYI) mass spectra, respectively, for AXVYI model peptide series. The MS(4) experiments exhibited that the fragment ions at m/z 279, 223, 295, and 281 entirely reflect the same fragmentation behavior of [M + H]+ ion generated from commercial dipeptides FL-OH, GF-OH, YI-OH, and VY-OH. These novel eliminations reported here for b5 and b4 ions can be useful in assigning the correct and reliable peptide sequences for high-throughput proteomic studies.

Address: Department of Chemistry, Faculty of Science, Izmir Institute of Technology, Urla-Izmir, Turkey

3916304d: J.Biol.Chem., 26879-26886(2013)
Link: [ PubMED ] [ DOI ]
Title: Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization.
Author: Ahsan,A., Ray,D., Ramanand,S.G., Hegde,A., Whitehead,C., Rehemtulla,A., Morishima,Y., Pratt,W.B., Osawa,Y., Lawrence,T.S., Nyati,M.K.
Abstract:
An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.

Address: From the Departments of Radiation Oncology

3916307d: Biopolymers, 438-452(2013)
Link: [ PubMED ] [ DOI ]
Title: Cyclotide discovery in Gentianales revisited-identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants.
Author: Koehbach,J., Attah,A.F., Berger,A., Hellinger,R., Kutchan,T.M., Carpenter,E.J., Rolf,M., Sonibare,M.A., Moody,J.O., Wong,G.K., Dessein,S., Greger,H., Gruber,C.W.
Abstract:
Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed approximately 300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands.

Address: Center for Physiology and Pharmacology, Medical University of Vienna, Schwarzspanierstrasse 17, 1090, Vienna, Austria

3916309d: Biopolymers, 527-542(2013)
Link: [ PubMED ] [ DOI ]
Title: Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry.
Author: Hegemann,J.D., Zimmermann,M., Zhu,S., Klug,D., Marahiel,M.A.
Abstract:
Lasso peptides are natural products with a unique three dimensional structure resembling a lariat knot. They are from ribosomal origin and are post-translationally modified by two enzymes (B and C), one of which shares little similarity to enzymes outside of lasso peptide biosynthetic gene clusters and as such is a useful target for genome mining. In this study, we demonstrate a B protein-centric genome mining approach through which we were able to identify 102 putative lasso peptide biosynthetic gene clusters from a total of 87 different proteobacterial strains. Ten of these clusters were cloned into the pET41a expression vector, optimized through incorporation of a ribosomal binding site and heterologously expressed in Escherichia coli BL21(DE3). All 12 predicted lasso peptides (namely burhizin, caulonodin I, caulonodin II, caulonodin III, rhodanodin, rubrivinodin, sphingonodin I, sphingonodin II, syanodin I, sphingopyxin I, sphingopyxin II, and zucinodin) were detected by high-resolution Fourier transform mass spectrometry and their proposed primary structure was confirmed through tandem mass spectrometry. High yields (ranging from 0.4 to 5.2 mg/L) were observable for eight of these compounds, while thermostability assays revealed five new representatives of heat labile lasso peptides.

Address: Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse 4 and LOEWE-Center for Synthetic Microbiology, Marburg, D-35032, Germany

3916312d: Dalton Trans., 13495-13501(2013)
Link: [ PubMED ] [ DOI ]
Title: Real time detection of cell cycle regulator cyclin A on living tumor cells with europium emission.
Author: Li,H., Chadbourne,F.L., Lan,R., Chan,C.F., Chan,W.L., Law,G.L., Lee,C.S., Cobb,S.L., Wong,K.L.
Abstract:
Six water-soluble europium complexes (Eu-L1-P(n) and Eu-L2-P(n), n = 1, 2 and 3) with one antenna chromophore, two different linkers (L1 and L2) and three proposed cyclin A specific peptides (P1: -GAKRRLIF-NH2; P2: -GGAKRRLIF-NH2; P3: -Hex- GAKRRLIF-NH2) have been synthesized. With structural information available, comparisons of the cyclin grooves of cyclin A and the six europium complexes have been made, and insights have been gained into the determinants for peptide binding and the foundation of differential binding. Experiment-wise, the linear and two-photon induced photophysical properties of these conjugates were monitored in aqueous solution. Numerous in situ/in vitro biological assays have been carried out, such as responsive emission changes in situ/in vitro, Western blot and cellular uptake. As imaging agents, complexes with peptides P3: -Hex-GAKRRLIF-NH2 showed high selectivity to cyclin A in numerous cancer cells. When it comes to responsive optical signal changes, complex Eu-L2-P3 exhibited a threefold emission enhancement upon binding with cyclin A (100 nM cyclin A, varphi = 8% to 21%, log KB = 5.83, detection limit = 5 nM), and this could be initiated by the shortened distance between the antenna and the lanthanide after they bind/get into cyclin A. It is promising that our compounds (especially compound Eu-L2-P3) could serve as the template for structure-guided efforts to develop potential imaging therapeutics on the basis of selective imaging of CDK2/cyclin A activity.

Address: Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong

3916318d: FASEB J., 4136-4146(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of a novel CD40 ligand for targeted imaging of inflammatory plaques by phage display.
Author: Yu,H., Segers,F., Sliedregt-Bol,K., Bot,I., Woltman,A.M., Boross,P., Verbeek,S., Overkleeft,H., Van der Marel,G.A., Van Kooten,C., Van Berkel,T.J., Biessen,E.A.
Abstract:
The CD40/CD40L dyad is deemed to play a central role in several inflammatory processes, including atherosclerosis. As CD40 is overexpressed in atherosclerotic lesions, it constitutes a promising candidate for targeted imaging approaches. Here we describe the design of a novel, selective peptide ligand for CD40 by phage display. A synthetic peptide corresponding with the phage insert NP31 displayed nanomolar affinity for CD40. Affinity was further enhanced by mutimeric presentation of NP31. An essential 11-mer peptide motif was identified by truncation and alanine scan studies. Enriched phage selectively bound human CD40 and homed to inflammatory joints in a murine model of rheumatoid arthritis. NP31 ablated VEGF and IL-6 transcriptional activation and partially inhibited IL-6 production by CD40L-activated endothelial cells. Notably, NP31 did not only alter the biodistribution profile of a streptavidin scaffold but also markedly increased accumulation of the carrier in atherosclerotic aortic lesions of aged ApoE(-/-) mice in a CD40-dependent manner. This potent and selective peptide ligand has potential for targeted imaging and drug delivery approaches in CD40-dependent inflammatory disorders such as atherosclerosis.-Yu, H., Segers, F., Sliedregt-Bol, K., Bot, I., Woltman, A. M., Boross, P., Verbeek, S., Overkleeft, H., van der Marel, G. A., van Kooten, C., van Berkel, T. J. C., Biessen, E. A. L. Identification of a novel CD40 ligand for targeted imaging of inflammatory plaques by phage display.

E-Mail: erik.biessen@mumc.nl
Address: 2Department of Pathology, University Hospital Maastricht, Debeijelaan 25, 6229 HX Maastricht, The Netherlands

Q. Primary Structure Determination

3915684q: Genome Announc., e00590-e00513(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Complete Genome Sequence for Lactobacillus helveticus CNRZ 32, an Industrial Cheese Starter and Cheese Flavor Adjunct.
Author: Broadbent,J.R., Hughes,J.E., Welker,D.L., Tompkins,T.A., Steele,J.L.
Abstract:
Lactobacillus helveticus is a lactic acid bacterium widely used in the manufacture of cheese and for production of bioactive peptides from milk proteins. We present the complete genome sequence for L. helveticus CNRZ 32, a strain particularly recognized for its ability to reduce bitterness and accelerate flavor development in cheese.

Address: Department of Nutrition, Dietetics, and Food Sciences, Utah State University, Logan, Utah, USA

3915711q: PLoS One, e70782-e70782(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Structure, antimicrobial activities and mode of interaction with membranes of bovel phylloseptins from the painted-belly leaf frog, Phyllomedusa sauvagii.
Author: Raja,Z., Andre,S., Piesse,C., Sereno,D., Nicolas,P., Foulon,T., Oury,B., Ladram,A.
Abstract:
Transcriptomic and peptidomic analysis of skin secretions from the Painted-belly leaf frog Phyllomedusa sauvagii led to the identification of 5 novel phylloseptins (PLS-S2 to -S6) and also of phylloseptin-1 (PSN-1, here renamed PLS-S1), the only member of this family previously isolated in this frog. Synthesis and characterization of these phylloseptins revealed differences in their antimicrobial activities. PLS-S1, -S2, and -S4 (79-95% amino acid sequence identity; net charge = +2) were highly potent and cidal against Gram-positive bacteria, including multidrug resistant S. aureus strains, and killed the promastigote stage of Leishmania infantum, L. braziliensis and L. major. By contrast, PLS-S3 (95% amino acid identity with PLS-S2; net charge = +1) and -S5 (net charge = +2) were found to be almost inactive against bacteria and protozoa. PLS-S6 was not studied as this peptide was closely related to PLS-S1. Differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles combined with circular dichroism spectroscopy and membrane permeabilization assays on bacterial cells indicated that PLS-S1, -S2, and -S4 are structured in an amphipathic α-helix that disrupts the acyl chain packing of anionic lipid bilayers. As a result, regions of two coexisting phases could be formed, one phase rich in peptide and the other lipid-rich. After reaching a threshold peptide concentration, the disruption of lipid packing within the bilayer may lead to local cracks and disintegration of the microbial membrane. Differences in the net charge, α-helical folding propensity, and/or degree of amphipathicity between PLS-S1, -S2 and -S4, and between PLS-S3 and -S5 appear to be responsible for their marked differences in their antimicrobial activities. In addition to the detailed characterization of novel phylloseptins from P. sauvagii, our study provides additional data on the previously isolated PLS-S1 and on the mechanism of action of phylloseptins.

Address: UPMC Univ Paris 06, ER3 Biogenese des Signaux Peptidiques (BIOSIPE), Paris, France

3915898q: PLoS One, e69076-e69076(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Identification and characterization of three novel lipases belonging to families II and V from Anaerovibrio lipolyticus 5ST.
Author: Prive,F., Kaderbhai,N.N., Girdwood,S., Worgan,H.J., Pinloche,E., Scollan,N.D., Huws,S.A., Newbold,C.J.
Abstract:
Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14).

Address: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, Ceredigion, UK

3916222q: Toxins, 1767-1779(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Isolation and Molecular Characterization of Two Lectins from Dwarf Elder (Sambucus ebulus L.) Blossoms Related to the Sam n1 Allergen.
Author: Jimenez,P., Cabrero,P., Basterrechea,J.E., Tejero,J., Cordoba-Diaz,D., Girbes,T.
Abstract:
Sambucus species contain a number of lectins with and without antiribosomal activity. Here, we show that dwarf elder (Sambucus ebulus L.) blossoms express two D-galactose-binding lectins that were isolated and purified by affinity chromatography and gel filtration. These proteins, which we named ebulin blo (A-B toxin) and SELblo (B-B lectin)-blo from blossoms-were subjected to molecular characterization and analysis by MALDI-TOF mass spectrometry and tryptic peptide fingerprinting. Both lectins share a high degree of amino acid sequence homology with Sambucus lectins related to the Sam n1 allergen. Ebulin blo, but not SELblo, was highly toxic by nasal instillation to mice. Overall, our results suggested that both lectins would belong to an allergen family exemplified by Sam n1 and could trigger allergy responses. Furthermore, they raise a concern about ebulin blo toxicity.

Address: Nutrition and Food Science, Faculty of Medicine and CINAD (Center for Research in Nutrition, Food and Dietetics

3916223q: Insect Biochem.Mol.Biol., 1133-1141(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular characterization of the vitellogenin receptor from the tick, Amblyomma hebraeum (Acari: Ixodidae).
Author: Smith,A.D., Kaufman,W.R.
Abstract:
We have identified the full-length cDNA encoding a vitellogenin receptor (VgR) from the African bont tick Amblyomma hebraeum Koch (1844). VgRs are members of the low-density lipoprotein receptor superfamily that promote the uptake of the yolk protein vitellogenin (Vg), from the haemolymph. The AhVgR (GenBank accession No. JX846592) is 5703 bp, and encodes an 1801 aa protein with a 196.5 kDa molecular mass following cleavage of a 22 aa signal peptide. Phylogenetic analysis indicates that AhVgR is highly similar to other tick VgRs. AhVgR is expressed in only the ovary of mated, engorged females, and is absent in all other female tissues and in both fed and unfed males. Unfed, adult females injected with a VgR-dsRNA probe to knock-down VgR expression experienced a significant delay in ovary development and started oviposition significantly later than controls. These results indicate that the expression of AhVgR is important for the uptake of Vg and subsequent maturation of the oocytes.

E-Mail: adsmith@ualberta.ca
Address: Department of Biological Sciences, University of Alberta, CW 405, Biological Sciences Building, Edmonton, Alberta T6G 2E9, Canada. Electronic address:

3916225q: Food Chem., 991-996(2014)
Link: [ PubMED ] [ DOI ]
Title: Novel antioxidative peptides from the protein hydrolysate of oysters (Crassostrea talienwhanensis).
Author: Wang,Q., Li,W., He,Y., Ren,D., Kow,F., Song,L., Yu,X.
Abstract:
The antioxidative activity of hydrolysate peptides from oysters (Crassostrea talienwhanensis) was investigated. After hydrolysis with subtilisin, the yields of the peptides that were soluble in trichloroacetic acid (TCA-soluble) and the antioxidant activities of the resulting hydrolysate were determined using an orthogonal design and a hydroxyl radical scavenging reaction. The hydrolysate was fractionated using Sephadex G-15 gel filtration chromatography, and the two resulting bioactive peptides were subsequently purified by RP-HPLC with a Kromasil C18 (ODS) column. The amino acid sequences were analyzed by nano-ESI-MS/MS. The critical reaction temperature, pH, hydrolysis time and enzyme-to-substrate (E/S) ratio were determined for the optiμm hydrolysis with subtilisin, and the E/S ratio was found to be the most critical reaction condition. The amino acid sequences of the peptides (518 and 440Da) were proline-valine-methionine-glycine-aspartic acid (PVMGA) and glutamine-histidine-glycine-valine (QHGV), respectively. These two novel peptides exhibited high antioxidative actions based on their hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities.

Address: Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, PR China

E. Physiology, Medicine, and Pharmacy

3915417e: Nutr.Hosp.Suppl., 78-87(2013)
Link: [ PubMED ] [ DOI ]
Title: Pathophysiology of diabetes mellitus type 2: beyond the duo "insulin resistance-secretion deficit".
Author: Boada,C.A.C., Martinez-Moreno,J.M.
Abstract:
T2DM involves at least two primary pathogenic mechanisms: (a) a progressive decline in pancreatic islet cell function resulting in reduced insulin secretion and (b) peripheral insulin resistance resulting in a decrease in the metabolic responses to insulin. This dynamic interaction between insulin secretion and insulin resistance is essential to the maintenance of normal glucose tolerance (NGT). The transition from the normal control of glucose metabolism to type 2 diabetes mellitus occurs through the intermediate states of altered metabolism that worsen over time. The first state of the disease is known as prediabetes, and consists of a set of metabolic disorder characterized by a great hyperglycemia, enough to increase of retinopathies, nephropathies and neuropathies incidence. If we advance in the T2DM temporal sequence we found a remarkable change in the pancreatic cells population that form the Langerhans islets, mainly caused by amylin fibers accumulation over these cells from polypeptide hormone called amyloid polypeptide or IAPP. The IAPP hypersecretion and amylin fibers deposition attached to the endoplasmic reticulum stress caused by excessive workload due to biosynthesis overproduction of insulin and IAPP result in ?-cell apoptosis. In addition to these alterations, we must also consider the changes observed in incretins profiles like GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide 1) directly related to glucose homeostasis maintenance. Risk factors that predispose to a healthy individual to develop T2DM are several, but the most important is the obesity. The body mass index (BMI) has been used in numerous epidemiological studies as a powerful indicator of T2DM risk. Lipotoxicity caused by circulating free fatty acids increased, changes in lipoprotein profiles, body fat distribution and glucotoxicity caused by cells over-stimulation are other risk factors to consider in T2DM developing.

E-Mail: carrera.car@gmail.com
Address: Department of Endocrinology, Hospital de Clinicas, Av. Panteon, Urb. San Bernardino, Caracas, Venezuela

3915418e: Nutr.Hosp.Suppl., 35-46(2013)
Link: [ PubMED ] [ DOI ]
Title: Improvement of C peptide zero BMI 24-34 diabetic patients after tailored one anastomosis gastric bypass (BAGUA).
Author: Garciacaballero,M., Martinez-Moreno,J.M., Toval,J.A., Miralles,F., Minguez,A., Osorio,D., Mata,J.M., Reyes-Ortiz,A.
Abstract:
BACKGROUND: Although bariatric surgery proved to be a very effective method in the treatment of patients in whose pancreas still produce insulin (type 2 diabetes), the accompanied metabolic syndrome and their diabetes complications, there is no information on the effect of this type of surgery in BMI24-34 patients when pancreas do not produce insulin at all (type 1, LADA and long term evolution type 2 diabetes among others). PATIENTS AND METHODS: We report preliminary data of a serie of 11 patients all with a C-peptide values below 0.0 ng/ml. They were followed for 6 to 60 months (mean 19 months) after surgery. We studied the changes in glycemic control, evolution of the metabolic syndrome and diabetes complications after one anastomosis gastric bypass (BAGUA). RESULTS: All values relative to glycemic control were improved HbA1c (from 8.9 +/- 0.6 to 6.7 +/- 0.2%), FPG (Fasting Plasma Glucose) [from 222.36 +/- 16.87 to 94 +/- 5 (mg/dl)] as well as the daily insulin requirement of rapid (from 40.6 +/- 12.8 to 0 (U/d) and long-lasting insulin (from 41.27 +/- 7.3 U/day to 15.2 +/- 3.3 U/day). It resolved 100% of the metabolic syndrome diseases as well as severe hypoglycaemia episodes present before surgery and improved some serious complications from diabetes like retinopathy, nephropathy, neuropathy, peripheral vasculopathy and cardiopathy. CONCLUSIONS: Tailored one anastomosis gastric bypass in BMI 24-34 C peptide zero diabetic patients eliminated the use of rapid insulin, reduced to only one injection per day long-lasting insulin and improved the glycemic control. After surgery disappear metabolic syndrome and severe hypoglycaemia episodes and improves significantly retinopathy, neuropathy, nephropathy, peripheral vasculopathy and cardiopathy.

E-Mail: gcaballe@uma.es
Address: Department of Surgery, University Malaga,. University Hospital Parque San Antonio, Malaga, Spain

3915422e: J.Virol., 12102-12109(2013)
Link: [ PubMED ] [ DOI ]
Title: Role of interleukin-2 and herpes simplex virus 1 in central nervous system demyelination in mice.
Author: Mott,K.R., Zandian,M., Allen,S.J., Ghiasi,H.
Abstract:
We have reported previously that ocular infection of different strains of mice with recombinant herpes simplex virus 1 (HSV-1) constitutively expressing interleukin-2 (IL-2) provokes central nervous system (CNS) demyelination and optic neuropathy, as determined by changes in visual evoked cortical potentials and pathological changes in the optic nerve and CNS, whereas recombinant viruses expressing IL-4, γ interferon, IL-12p35, IL-12p40, or IL-12p70 do not induce this neuropathy. The goal of this study was to dissect the mechanism underlying the interplay between the immune system (elevation of IL-2) and an environmental factor (infection with HSV-1) that elicits this pathology. Similar results were obtained upon delivery of IL-2 into the mouse brain using osmotic minipumps or injection of mice with recombinant IL-2 protein, IL-2 DNA, or IL-2 synthetic peptides prior to infection with wild-type (wt) HSV-1 strains McKrae and KOS. The critical role of IL-2 is further supported by our data, indicating that a single mutation at position T27A in IL-2 completely blocks the HSV-1-induced pathology. This study shows a novel model of autoimmunity in which viral infection and enhanced IL-2 cause CNS demyelination.

Address: Center for Neurobiology and Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA

3915427e: Am.J.Physiol.R, 659-668(2013)
Link: [ PubMED ] [ DOI ]
Title: Role of collecting duct endothelin in control of renal function and blood pressure.
Author: Kohan,D.E.
Abstract:
Over 26,000 manuscripts have been published dealing with endothelins since their discovery 25 years ago. These peptides, and particularly endothelin-1 (ET-1), are expressed by, bind to, and act on virtually every cell type in the body, influencing multiple biological functions. Among these actions, the effects of ET-1 on arterial pressure and volume homeostasis have been most extensively studied. While ET-1 modulates arterial pressure through regulation of multiple organ systems, the peptide's actions in the kidney in general, and the collecting duct in particular, are of unique importance. The collecting duct produces large amounts of ET-1 that bind in an autocrine manner to endothelin A and B receptors, causing inhibition of Na+ and water reabsorption; absence of collecting duct ET-1 or its receptors is associated with marked salt-sensitive hypertension. Collecting duct ET-1 production is stimulated by Na+ and water loading through local mechanisms that include sensing of salt and other solute delivery as well as shear stress. Thus the collecting duct ET-1 system exists, at least in part, to detect alterations in, and maintain homeostasis for, extracellular fluid volume. Derangements in collecting duct ET-1 production may contribute to the pathogenesis of genetic hypertension. Blockade of endothelin receptors causes fluid retention due, in large part, to inhibition of the action of ET-1 in the collecting duct; this side effect has substantially limited the clinical utility of this class of drugs. Herein, the biology of the collecting duct ET-1 system is reviewed, with particular emphasis on key issues and questions that need addressing.

Address: Division of Nephrology, University of Utah Health Sciences Center, Salt Lake City, Utah

3915428e: Nanoscale, 9924-9933(2013)
Link: [ PubMED ] [ DOI ]
Title: Octreotide-functionalized and resveratrol-loaded unimolecular micelles for targeted neuroendocrine cancer therapy.
Author: Xu,W., Burke,J.F., Pilla,S., Chen,H., Jaskula-Sztul,R., Gong,S.
Abstract:
Medullary thyroid cancer (MTC) is a neuroendocrine tumor (NET) that is often resistant to standard therapies. Resveratrol suppresses MTC growth in vitro, but it has low bioavailability in vivo due to its poor water solubility and rapid metabolic breakdown, as well as lack of tumor-targeting ability. A novel unimolecular micelle based on a hyperbranched amphiphilic block copolymer was designed, synthesized, and characterized for NET-targeted delivery. The hyperbranched amphiphilic block copolymer consisted of a dendritic Boltorn(R) H40 core, a hydrophobic poly(l-lactide) (PLA) inner shell, and a hydrophilic poly(ethylene glycol) (PEG) outer shell. Octreotide (OCT), a peptide that shows strong binding affinity to somatostatin receptors, which are overexpressed on NET cells, was used as the targeting ligand. Resveratrol was physically encapsulated by the micelle with a drug loading content of 12.1%. The unimolecular micelles exhibited a uniform size distribution and spherical morphology, which were determined by both transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cellular uptake, cellular proliferation, and Western blot analyses demonstrated that the resveratrol-loaded OCT-targeted micelles suppressed growth more effectively than non-targeted micelles. Moreover, resveratrol-loaded NET-targeted micelles affected MTC cells similarly to free resveratrol in vitro, with equal growth suppression and reduction in NET marker production. These results suggest that the H40-based unimolecular micelle may offer a promising approach for targeted NET therapy.

E-Mail: sgong@engr.wisc.edu
Address: Department of Biomedical Engineering, Wisconsin Institutes for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA

3915429e: J.Neurosci., 14107-14116(2013)
Link: [ PubMED ] [ DOI ]
Title: Neuropeptides amplify and focus the monoaminergic inhibition of nociception in Caenorhabditis elegans.
Author: Hapiak,V., Summers,P., Ortega,A., Law,W.J., Stein,A., Komuniecki,R.
Abstract:
Monoamines and neuropeptides interact to modulate most behaviors. To better understand these interactions, we have defined the roles of tyramine (TA), octopamine, and neuropeptides in the inhibition of aversive behavior in Caenorhabditis elegans. TA abolishes the serotonergic sensitization of aversive behavior mediated by the two nociceptive ASH sensory neurons and requires the expression of the adrenergic-like, Gαq-coupled, TA receptor TYRA-3 on inhibitory monoaminergic and peptidergic neurons. For example, TA inhibition requires Gαq and Gαs signaling in the peptidergic ASI sensory neurons, with an array of ASI neuropeptides activating neuropeptide receptors on additional neurons involved in locomotory decision-making. The ASI neuropeptides required for tyraminergic inhibition are distinct from those required for octopaminergic inhibition, suggesting that individual monoamines stimulate the release of different subsets of ASI neuropeptides. Together, these results demonstrate that a complex humoral mix of monoamines is focused by more local, synaptic, neuropeptide release to modulate nociception and highlight the similarities between the tyraminergic/octopaminergic inhibition of nociception in C. elegans and the noradrenergic inhibition of nociception in mammals that also involves inhibitory peptidergic signaling.

Address: Department of Biological Sciences, University of Toledo, Toledo, Ohio 43606-3390, USA

3915430e: J.Neurosci., 13972-13977(2013)
Link: [ PubMED ] [ DOI ]
Title: Block of gap junctions eliminates aberrant activity and restores light responses during retinal degeneration.
Author: Toychiev,A.H., Ivanova,E., Yee,C.W., Sagdullaev,B.T.
Abstract:
Retinal degeneration leads to progressive photoreceptor cell death, resulting in vision loss. Subsequently, inner retinal neurons develop aberrant synaptic activity, compounding visual impairment. In retinal ganglion cells, light responses driven by surviving photoreceptors are obscured by elevated levels of aberrant spiking activity. Here, we demonstrate in rd10 mice that targeting disruptive neuronal circuitry with a gap junction antagonist can significantly reduce excessive spiking. This treatment increases the sensitivity of the degenerated retina to light stimuli driven by residual photoreceptors. Additionally, this enhances signal transmission from inner retinal neurons to ganglion cells, potentially allowing the retinal network to preserve the fidelity of signals either from prosthetic electronic devices, or from cells optogenetically modified to transduce light. Thus, targeting maladaptive changes to the retina allows for treatments to use existing neuronal tissue to restore light sensitivity, and to augment existing strategies to replace lost photoreceptors.

Address: Department of Ophthalmology, Weill Medical College of Cornell University, Burke Medical Research Institute, White Plains, New York 10605, USA

3915433e: Am.J.Clin.Nutr., 1151-1161(2013)
Link: [ PubMED ] [ DOI ]
Title: Hormonal responses and test meal intake among obese teenagers before and after laparoscopic adjustable gastric banding.
Author: Sysko,R., Devlin,M.J., Schebendach,J., Tanofsky-Kraff,M., Zimmerli,E., Korner,J., Yanovski,J.A., Zitsman,J.L., Walsh,B.T.
Abstract:
BACKGROUND: Relatively little is known about changes in eating behavior or hormonal responses to food after bariatric surgery in adolescents. OBJECTIVE: This study compared eating behavior and hormones among adolescents in a bariatric surgery program with those in nonoverweight control adolescents and evaluated changes before and after laparoscopic adjustable gastric banding (LAGB). DESIGN: Fasting leptin, peptide YY (PYY), and ghrelin concentrations were obtained, and postprandial ghrelin and PYY area under the curve (AUC) were assessed after a single-item breakfast. Intake from an ad libitum lunchtime multi-item meal was measured. RESULTS: Compared with controls (n = 9), all presurgical candidates (n = 20) had significantly greater fasting leptin, lower fasting ghrelin, and lower AUC ghrelin but similar PYY and AUC PYY. Preoperative candidates did not differ from controls in total energy consumed during the test meal. Postoperatively, among the 11 participants with data both before and after surgery, BMI (in kg/m(2)) decreased by 3.5 (P < 0.001), significantly less energy was consumed in the test meal, and a smaller number of foods were selected. AUC ghrelin and PYY did not significantly change before or after LAGB. CONCLUSIONS: Few significant short-term changes were observed in appetitive hormones after LAGB. It is unclear whether objective measures of eating behavior will prove useful in evaluating the impact of bariatric surgery on outcomes. This trial was registered at clinicaltrials.gov as CT00764127.

Address: Columbia Center for Eating Disorders, Division of Clinical Therapeutics, New York State Psychiatric Institute, New York, NY (RS, MJD, JS, EZ, BTW)

3915437e: Can.J.Physiol.Pharmacol., 724-732(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of exenatide and metformin in combination on some adipocytokine levels: a comparison with metformin monotherapy.
Author: Derosa,G., Cicero,A.F., Franzetti,I.G., Querci,F., Carbone,A., Ciccarelli,L., D'Angelo,A., Fogari,E., Maffioli,P.
Abstract:
The aim of this study was to evaluate the effects of exenatide on levels of serum adipocytokines and on β-cell function. The study was conducted between 2008 and 2012. After a run-in period with metformin, 174 patients with type-2 diabetes were randomly distributed to either a group receiving exenatide at 10 μg twice daily, or a group receiving the placebo, for 12 months. We evaluated body mass index (BMI), blood pressure, glycemic control, lipid profile, fasting plasma insulin (FPI), HOMA-IR, HOMA-β, fasting plasma proinsulin (FPPr), proinsulin : fasting plasma insulin ratio (Pr/FPI ratio), C-peptide, glucagon, retinol binding protein-4 (RBP-4), visfatin, omentin-1, and microalbuminuria. We used ELISA methods to assess the various parameters. Patients also underwent a combined euglycemic-hyperinsulinemic and hyperglycemic clamp, with subsequent arginine stimulation. After 12 months, a combination of exenatide and metformin produced a better decrease in body mass, BMI, glycemic control, FPI, FPPr, FPPr/FPI ratio, HOMA-IR, and glucagon level. Treatment with exenatide + metformin was superior to the placebo + metformin in increasing HOMA-β, C-peptide, and β-cell function. Significant negative correlations were found between M value, an index of insulin sensitivity, and measured adipocytokines. In conclusion, the combination of exenatide + metformin plays a role in improving some adipocytokine levels, and is better than metformin alone. The significant negative correlation between M value and measured adipocytokines is another confirmation of the positive effects linked to the improvement in insulin sensitivity.

E-Mail: giuseppe.derosa@unipv.it
Address: Department of Internal Medicine and Therapeutics, University of Pavia, Fondazione IRCCS Policlinico San Matteo, Pavia, Piazzale Camillo Golgi 2, 27100 Pavia, Italy

3915438e: Med.J.Aust., 246-249(2013)
Link: [ PubMED ] [ DOI ]
Title: The rationale for combining GLP-1 receptor agonists with basal insulin.
Author: Cohen,N.D., Audehm,R., Pretorius,E., Kaye,J., Chapman,L.H., Colagiuri,S.
Abstract:
* Type 2 diabetes mellitus (T2DM) is progressive; the more intensively it is treated, the greater is the risk of hypoglycaemia and weight gain. Achieving treatment intensification while mitigating these risks presents a challenge to patient management. * Basal insulins provide control of fasting glucose; however, their utility in the control of postprandial glucose excursions is limited. * Glucagon-like peptide-1 (GLP-1) receptor agonists stimulate glucose-medicated insulin secretion, suppress glucagon secretion, delay gastric emptying and decrease appetite. Use of GLP-1 receptor agonists in combination therapy with basal insulin offers an alternative approach to intensification of insulin therapy. * Prospective interventional trials demonstrate that GLP-1 receptor agonists added to basal insulin decrease postprandial glucose levels, lower HbA1c levels, decrease weight and lower basal insulin requirements without increasing the risk of major hypoglycaemic events. * The current clinical data are limited by the lack of any data on the long-term effects of GLP-1 receptor agonists over additional prandial regimens; they may be beneficial or deleterious. * Although cost, gastrointestinal side effects and long-term safety should be taken into account when considering this combination, it appears to be growing in popularity and is likely to be an important therapeutic option for T2DM in the future.

E-Mail: neale.cohen@bakeridi.edu.au
Address: Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia

3915439e: BioMed Res.Int., 729349-729349(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Antibodies against food antigens in patients with autistic spectrum disorders.
Author: De Magistris,L., Picardi,A., Siniscalco,D., Riccio,M.P., Sapone,A., Cariello,R., Abbadessa,S., Medici,N., Lammers,K.M., Schiraldi,C., Iardino,P., Marotta,R., Tolone,C., Fasano,A., Pascotto,A., Bravaccio,C.
Abstract:
PURPOSE: Immune system of some autistic patients could be abnormally triggered by gluten/casein assumption. The prevalence of antibodies to gliadin and milk proteins in autistic children with paired/impaired intestinal permeability and under dietary regimen either regular or restricted is reported. METHODS: 162 ASDs and 44 healthy children were investigated for intestinal permeability, tissue-transglutaminase (tTG), anti-endomysium antibodies (EMA)-IgA, and total mucosal IgA to exclude celiac disease; HLA-DQ2/-DQ8 haplotypes; total systemic antibodies (IgA, IgG, and IgE); specific systemic antibodies: α-gliadin (AGA-IgA and IgG), deamidated-gliadin-peptide (DGP-IgA and IgG), total specific gliadin IgG (all fractions: α, β, γ, and omega), β-lactoglobulin IgG, α-lactalbumin IgG, casein IgG; and milk IgE, casein IgE, gluten IgE,-lactoglobulin IgE, and α-lactalbumin IgE. RESULTS: AGA-IgG and DPG-IgG titers resulted to be higher in ASDs compared to controls and are only partially influenced by diet regimen. Casein IgG titers resulted to be more frequently and significantly higher in ASDs than in controls. Intestinal permeability was increased in 25.6% of ASDs compared to 2.3% of healthy children. Systemic antibodies production was not influenced by paired/impaired intestinal permeability. CONCLUSIONS: Immune system of a subgroup of ASDs is triggered by gluten and casein; this could be related either to AGA, DPG, and Casein IgG elevated production or to impaired intestinal barrier function.

E-Mail: laura.demagistris@unina2.it
Address: CIRANAD, Second University of Naples, via Pansini 5, Building 3, 80131 Naples, Italy

3915440e: BioMed Res.Int., 315848-315848(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Therapeutic effect of exendin-4, a long-acting analogue of glucagon-like peptide-1 receptor agonist, on nerve regeneration after the crush nerve injury.
Author: Yamamoto,K., Amako,M., Yamamoto,Y., Tsuchihara,T., Nukada,H., Yoshihara,Y., Arino,H., Fujita,M., Uenoyama,M., Tachibana,S., Nemoto,K.
Abstract:
Glucagon-like peptide-1 (GLP-1) is glucose-dependent insulinotropic hormone secreted from enteroendocrine L cells. Its long-acting analogue, exendin-4, is equipotent to GLP-1 and is used to treat type 2 diabetes mellitus. In addition, exendin-4 has effects on the central and peripheral nervous system. In this study, we administered repeated intraperitoneal (i.p.) injections of exendin-4 to examine whether exendin-4 is able to facilitate the recovery after the crush nerve injury. Exendin-4 injection was started immediately after crush injury and was repeated every day for subsequent 14 days. Rats subjected to sciatic nerve crush exhibited marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). All these changes, except for the atrophy of TA, were improved significantly by the administration of exendin-4. Functional, electrophysiological, and morphological parameters indicated significant enhancement of nerve regeneration 4 weeks after nerve crush. These results suggest that exendin-4 is feasible for clinical application to treat peripheral nerve injury.

E-Mail: yamagen0610@nifty.com
Address: Department of Orthopedic Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan

3915443e: BioMed Res.Int., 104059-104059(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Cardiovascular and renal effects of bromocriptine in diabetic patients with stage 4 chronic kidney disease.
Author: Mejia-Rodriguez,O., Herrera-Abarca,J.E., Ceballos-Reyes,G., Avila-Diaz,M., Prado-Uribe,C., Belio-Caro,F., Salinas-Gonzalez,A., Vega-Gomez,H., Alvarez-Aguilar,C., Lindholm,B., Garcia-Lopez,E., Paniagua,R.
Abstract:
OBJECTIVE: The objective of this study was to investigate the effect of bromocriptine (BEC) on left ventricular mass index (LVMI) and residual renal function (RRF) in chronic kidney disease (CKD) patients with type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS: A 6-month double-blind randomized controlled trial was conducted in 28 patients with T2D and stage 4 CKD with increased LVMI. Fourteen patients received BEC (2.5 mg, initially 1 tablet with subsequent increase to three times a day) and 14 received a placebo (PBO; initially 1 tablet with subsequent increase to three times a day). Cardiovascular changes were assessed by monitoring 24 h ambulatory blood pressure, two-dimensional-guided M-mode echocardiography, and N-terminal brain natriuretic peptide (NT-proBNP) plasma levels. RRF was evaluated by creatinine clearance and cystatin-C plasma levels. RESULTS: Both BEC and PBO groups decreased blood pressure-but the effect was more pronounced in the BEC group. Average 24 h, diurnal and nocturnal blood pressures, and circadian profile showed improved values compared to the PBO group; LVMI decreased by 14% in BEC and increased by 8% in PBO group. NT-proBNP decreased in BEC (0.54 +/- 0.15 to 0.32 +/- 0.17 pg/mL) and increased in PBO (0.37 +/- 0.15 to 0.64 +/- 0.17 pg/mL). Creatinine clearance did not change in the BEC group and decreased in the PBO group. CONCLUSIONS: BEC resulted in a decrease on blood pressure and LVMI. BEC also prevented the progression of CKD while maintaining the creatinine clearance unchanged.

E-Mail: olivamejia@yahoo.com
Address: Coordinacion de Educacion e Investigacion en Salud, Unidad de Medicina Familiar No 80, Instituto Mexicano del Seguro Social, Morelia, Mich, Mexico

3915444e: Sultan Qaboos Univ.Med.J., 399-403(2013)
Link: [ PubMED ] [ OpenAccess]
Title: The Frequency of Type 2 Diabetes Mellitus among Diabetic Children in El Minia Governorate, Egypt.
Author: Ali,B.A., Abdallah,S.T., Abdallah,A.M., Hussein,M.M.
Abstract:
OBJECTIVES: Type 2 diabetes mellitus (T2DM) in children and adolescents is becoming an increasingly important public health concern throughout the world. This study aimed to estimate the frequency of T2DM among diabetic young people in El-Minia Governorate, Egypt, and to detect its risk factors. METHODS: A total of 210 diabetic patients under 18 years old in Minia Governorate were included in the study and underwent a thorough history-taking, a physical examination and laboratory investigations. RESULTS: T2DM was present in 28 patients (13.3%); it was significantly present in 18 females (64.3%) and 20 (71.4%) of them had a positive family history of DM. T2DM patients had significantly higher BMI and waist circumference centiles for age and sex than those with T1DM. Also, haemoglobin A1c %, serum C-peptide and cholesterol levels were significantly higher in T2DM than T1DM patients. Finally, there were weak significant positive correlations between C-peptide level and both BMI and waist circumference. CONCLUSION: T2DM is no longer a disease of adults but can also occur in children and adolescents. The results suggested that obesity, female gender and a positive family history of DM are risk factors for T2DM. Also, patients with T2DM had poorer glycaemic control and hypercholesterolemia than those with other types of diabetes.

Address: Pediatric department, Faculty of Medicine, Minia University, Egypt

3915448e: Sci.Rep., 2534-2534(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Ligand modified nanoparticles increases cell uptake, alters endocytosis and elevates glioma distribution and internalization.
Author: Gao,H., Yang,Z., Zhang,S., Cao,S., Shen,S., Pang,Z., Jiang,X.
Abstract:
Nanoparticles (NPs) were widely used in drugs/probes delivery for improved disease diagnosis and/or treatment. Targeted delivery to cancer cells is a highly attractive application of NPs. However, few studies have been performed on the targeting mechanisms of these ligand-modified delivery systems. Additional studies are needed to understand the transport of nanoparticles in the cancer site, the interactions between nanoparticles and cancer cells, the intracellular trafficking of nanoparticles within the cancer cells and the subcellular destiny and potential toxicity. Interleukin 13 (IL-13) peptide can specifically bind IL-13Rα2, a receptor that is highly expressed on glioma cells but is expressed at low levels on other normal cells. It was shown that the nanoparticels modification with the IL-13 peptide could improve glioma treatment by selectively increasing cellular uptake, facilitating cell internalization, altering the uptake pathway and increasing glioma localization.

Address: Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education

3915449e: Int.J.Mol.Med., 1011-1020(2013)
Link: [ PubMED ] [ DOI ]
Title: Cardioprotective effects of exenatide against oxidative stress-induced injury.
Author: Chang,G., Zhang,D., Yu,H., Zhang,P., Wang,Y., Zheng,A., Qin,S.
Abstract:
Myocardial ischemia/reperfusion (MI/R) leads to oxidative stress, which may in turn lead to myocardial injury. In the present study, we investigated the effects of exenatide, a glucagon-like peptide-1 (GLP-1) analogue, on oxidative stress-induced injury in vitro and in vivo. In in vitro experiments, H9c2 cells were incubated with exenatide to determine the direct cytoprotective effects of exenatide following exposure to hydrogen peroxide (H2O2). Pre-treatment with exenatide (1 nM), prior to H2O2 exposure, increased cell viability and inhibited H2O2induced reactive oxygen species (ROS) production. Exenatide also decreased the levels of lactate dehydrogenase (LDH) and creatine kinaseMB (CKMB) in the cultured supernatants, as well as those of malondialdehyde (MDA) in the H9c2 cells and increased the total superoxide dismutase (TSOD) levels in the H9c2 cells. In in vivo experiments, an animal model of MI/R was induced by coronary occlusion. Pre-treatment with exenatide (10 μg/kg/day) protected the rat hearts from MI/R-induced injury by decreasing the levels of LDH and CKMB in plasma, increasing the levels of catalase, T-SOD and glutathione peroxidase (GSH-Px) in the heart and decreasing the MDA levels in the rats with MI/R-induced injury. Exenatide also reduced the infarct size and enhanced cardiac function in the rats with MI/R-induced injury. Moreover, pre-treatment with exenatide inhibited cardiomyocyte apoptosis, increased Aktserine473 and Badserine136 phosphorylation and decreased cleaved caspase3 expression in vitro and in vivo; however, these effects were attenuated by the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. Our results suggest that exenatide exerts significant cardioprotective effects against oxidative stress-induced injury in vitro and in vivo. The mechanisms involved may be attributed to the scavenging of oxidative stress products, such as ROS, the increase in the concentrations of antioxidant defense enzymes and the inhibition of cardiomyocyte apoptosis. The antiapoptotic effects of exenatide were, at least in part, associated with the activation of the PI3K/Akt signaling pathway.

Address: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China

3915451e: Oncol.Rep., 2502-2510(2013)
Link: [ PubMED ] [ DOI ]
Title: The cathelicidin-BF Lys16 mutant Cbf-K16 selectively inhibits non-small cell lung cancer proliferation in vitro.
Author: Tian,Y., Wang,H., Li,B., Ke,M., Wang,J., Dou,J., Zhou,C.
Abstract:
The 30-amino acid antimicrobial peptide Cbf-K16 is a cathelicidin-BF (BF-30) Lys16 mutant derived from the snake venom of Bungarus fasciatus. Our previous study found that BF-30 selectively inhibited the proliferation of the metastatic melanoma cell line B16F10 in vitro and in vivo, but had a negligible effect on human lung cells. In the present study, it was demonstrated for the first time that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells in vitro, with low toxicity to normal cells. The half-maximal inhibitory concentrations (IC50) of Cbf-K16 against H460 human non-small cell lung carcinoma cells and mouse Lewis lung cancer cells were only 16.5 and 10.5 μM, respectively, which were much less compared to that of BF-30 (45 and 40.3 μM). Data using a transmission electron microscope (TEM) assay showed that, at 20 and 40 μM, Cbf-K16 induced the rupture of the cytoplasmic membrane, which was consistent with data obtained from lactate dehydrogenase (LDH) release assays. The LDH release increased from 17.8 to 52.9% as the duration and dosage of Cbf-K16 increased. Annexin V- fl uorescein and propidium iodide staining assays indicated that there were no obvious apoptotic effects at the different dosages and times tested. In H460 cells, the rate of genomic DNA binding increased from 51.9 to 86.8% as the concentration of Cbf-K16 increased from 5 to 10 μM. These data indicate that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells via cytoplasmic membrane permeabilization and DNA binding, rather than apoptosis. Although Cbf-K16 displayed significant cytotoxic activity (40 μM) against tumor cells, in splenocytes no significant inhibitory effect was observed and hemolysis was only 5.6%. These results suggest that Cbf-K16 is a low-toxicity anti-lung cancer drug candidate.

Address: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu 210009, P.R. China

3915452e: Parasitol.Res., 3569-3578(2013)
Link: [ PubMED ] [ DOI ]
Title: Characterization of the carbohydrate components of Taenia solium oncosphere proteins and their role in the antigenicity.
Author: Arana,Y., Verastegui,M., Tuero,I., Grandjean,L., Garcia,H.H., Gilman,R.H.
Abstract:
This study examines the carbohydrate composition of Taenia solium whole oncosphere antigens (WOAs), in order to improve the understanding of the antigenicity of the T. solium. Better knowledge of oncosphere antigens is crucial to accurately diagnose previous exposure to T. solium eggs and thus predict the development of neurocysticercosis. A set of seven lectins conjugates with wide carbohydrate specificity were used on parasite fixations and somatic extracts. Lectin fluorescence revealed that D-mannose, D-glucose, D-galactose and N-acetyl-D-galactosamine residues were the most abundant constituents of carbohydrate chains on the surface of T. solium oncosphere. Lectin blotting showed that posttranslational modification with N-glycosylation was abundant while little evidence of O-linked carbohydrates was observed. Chemical oxidation and enzymatic deglycosylation in situ were performed to investigate the immunoreactivity of the carbohydrate moieties. Linearizing or removing the carbohydrate moieties from the protein backbones did not diminish the immunoreactivity of these antigens, suggesting that a substantial part of the host immune response against T. solium oncosphere is directed against the peptide epitopes on the parasite antigens. Finally, using carbohydrate probes, we demonstrated for the first time that the presence of several lectins on the surface of the oncosphere was specific to carbohydrates found in intestinal mucus, suggesting a possible role in initial attachment of the parasite to host cells.

Address: Department of Cellular and Molecular Sciences, Faculty of Sciences and Philosophy, Universidad Peruana Cayetano Heredia, PO Box 5045, Lima, Peru

3915456e: Endocrinology, 4146-4157(2013)
Link: [ PubMED ] [ DOI ]
Title: The Heparin-Binding Domains of IGFBP-2 Mediate Its Inhibitory Effect on Preadipocyte Differentiation and Fat Development in Male Mice.
Author: Xi,G., Solum,M.A., Wai,C., Maile,L.A., Rosen,C.J., Clemmons,D.R.
Abstract:
IGF-binding protein (IGFBP)-2 overexpression confers resistance to high-fat feeding and inhibits the differentiation of preadipocytes in vitro. However, whether administration of IGFBP-2 can regulate adipogenesis in vivo and the domains that mediate this response have not been defined. IGFBP-2 contains 2 heparin-binding domains (HBD), which are localized in the linker region (HBD1) and C-terminal region (HBD2) of IGFBP-2. To determine the relative importance of these domains, we used synthetic peptides as well as mutagenesis. Both HBD1 and HBD2 peptides inhibited preadipocyte differentiation, but the HBD2 peptide was more effective. Selective substitution of charged residues in the HBD1 or HBD2 regions attenuated the ability of the full-length protein to inhibit cell differentiation, but the HBD2 mutant had the greatest reduction. To determine their activities in vivo, pegylated forms of each peptide were administered to IGFBP-2(-/-) mice for 12 weeks. Magnetic resonance imaging scanning showed that only the HBD2 peptide significantly reduced (48 +/- 9%, P < .05) gain in total fat mass. Both inguinal (32 +/- 7%, P < .01) and visceral fat (44 +/- 7%, P < .01) were significantly decreased by HBD2 whereas HBD1 reduced only visceral fat accumulation (24 +/- 5%, P < .05). The HBD2 peptide was more effective peptide in reducing triglyceride content and serum adiponectin, but only the HBD2 peptide increased serum leptin. These findings demonstrate that the HBD2 domain of IGFBP-2 is the primary region that accounts for its ability to inhibit adipogenesis and that a peptide encompassing this region has activity that is comparable with native IGFBP-2.

E-Mail: david_clemmons@med.unc.edu
Address: MD, CB no. 7170, 8024 Burnett Womack, Division of Endocrinology, University of North Carolina, Chapel Hill, NC 27599-7170

3915457e: Development(Camb.), 3915-3926(2013)
Link: [ PubMED ] [ DOI ]
Title: Female-biased dimorphism underlies a female-specific role for post-embryonic Ilp7 neurons in Drosophila fertility.
Author: Castellanos,M.C., Tang,J.C., Allan,D.W.
Abstract:
In Drosophila melanogaster, much of our understanding of sexually dimorphic neuronal development and function comes from the study of male behavior, leaving female behavior less well understood. Here, we identify a post-embryonic population of Insulin-like peptide 7 (Ilp7)-expressing neurons in the posterior ventral nerve cord that innervate the reproductive tracts and exhibit a female bias in their function. They form two distinct dorsal and ventral subsets in females, but only a single dorsal subset in males, signifying a rare example of a female-specific neuronal subset. Female post-embryonic Ilp7 neurons are glutamatergic motoneurons innervating the oviduct and are required for female fertility. In males, they are serotonergic/glutamatergic neuromodulatory neurons innervating the seminal vesicle but are not required for male fertility. In both sexes, these neurons express the sex-differentially spliced fruitless-P1 transcript but not doublesex. The male fruitless-P1 isoform (fruM) was necessary and sufficient for serotonin expression in the shared dorsal Ilp7 subset, but although it was necessary for eliminating female-specific Ilp7 neurons in males, it was not sufficient for their elimination in females. By contrast, sex-specific RNA-splicing by female-specific transformer is necessary for female-type Ilp7 neurons in females and is sufficient for their induction in males. Thus, the emergence of female-biased post-embryonic Ilp7 neurons is mediated in a subset-specific manner by a tra- and fru-dependent mechanism in the shared dorsal subset, and a tra-dependent, fru-independent mechanism in the female-specific subset. These studies provide an important counterpoint to studies of the development and function of male-biased neuronal dimorphism in Drosophila.

Address: Department of Cellular and Physiological Sciences, 2401 Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

3915458e: BMC Med., 188-188(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: A novel serogenetic approach determines the community prevalence of celiac disease and informs improved diagnostic pathways.
Author: Anderson,R.P., Henry,M.J., Taylor,R., Duncan,E.L., Danoy,P., Costa,M.J., Addison,K., Tye-Din,J.A., Kotowicz,M.A., Knight,R.E., Pollock,W., Nicholson,G.C., Toh,B.H., Brown,M.A., Pasco,J.A.
Abstract:
BACKGROUND: Changing perspectives on the natural history of celiac disease (CD), new serology and genetic tests, and amended histological criteria for diagnosis cast doubt on past prevalence estimates for CD. We set out to establish a more accurate prevalence estimate for CD using a novel serogenetic approach. METHODS: The human leukocyte antigen (HLA)-DQ genotype was determined in 356 patients with 'biopsy-confirmed' CD, and in two age-stratified, randomly selected community cohorts of 1,390 women and 1,158 men. Sera were screened for CD-specific serology. RESULTS: Only five 'biopsy-confirmed' patients with CD did not possess the susceptibility alleles HLA-DQ2.5, DQ8, or DQ2.2, and four of these were misdiagnoses. HLA-DQ2.5, DQ8, or DQ2.2 was present in 56% of all women and men in the community cohorts. Transglutaminase (TG)-2 IgA and composite TG2/deamidated gliadin peptide (DGP) IgA/IgG were abnormal in 4.6% and 5.6%, respectively, of the community women and 6.9% and 6.9%, respectively, of the community men, but in the screen-positive group, only 71% and 75%, respectively, of women and 65% and 63%, respectively, of men possessed HLA-DQ2.5, DQ8, or DQ2.2. Medical review was possible for 41% of seropositive women and 50% of seropositive men, and led to biopsy-confirmed CD in 10 women (0.7%) and 6 men (0.5%), but based on relative risk for HLA-DQ2.5, DQ8, or DQ2.2 in all TG2 IgA or TG2/DGP IgA/IgG screen-positive subjects, CD affected 1.3% or 1.9%, respectively, of females and 1.3% or 1.2%, respectively, of men. Serogenetic data from these community cohorts indicated that testing screen positives for HLA-DQ, or carrying out HLA-DQ and further serology, could have reduced unnecessary gastroscopies due to false-positive serology by at least 40% and by over 70%, respectively. CONCLUSIONS: Screening with TG2 IgA serology and requiring biopsy confirmation caused the community prevalence of CD to be substantially underestimated. Testing for HLA-DQ genes and confirmatory serology could reduce the numbers of unnecessary gastroscopies.

3915459e: Mol.Brain, 39-39(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Identification of key structural elements for neuronal calcium sensor-1 function in the regulation of the temperature-dependency of locomotion in C. elegans.
Author: Martin,V.M., Johnson,J.R., Haynes,L.P., Barclay,J.W., Burgoyne,R.D.
Abstract:
BACKGROUND: Intracellular Ca2+ regulates many aspects of neuronal function through Ca2+ binding to EF hand-containing Ca2+ sensors that in turn bind target proteins to regulate their function. Amongst the sensors are the neuronal calcium sensor (NCS) family of proteins that are involved in multiple neuronal signalling pathways. Each NCS protein has specific and overlapping targets and physiological functions and specificity is likely to be determined by structural features within the proteins. Common to the NCS proteins is the exposure of a hydrophobic groove, allowing target binding in the Ca2+-loaded form. Structural analysis of NCS protein complexes with target peptides has indicated common and distinct aspects of target protein interaction. Two key differences between NCS proteins are the size of the hydrophobic groove that is exposed for interaction and the role of their non-conserved C-terminal tails. RESULTS: We characterised the role of NCS-1 in a temperature-dependent locomotion assay in C. elegans and identified a distinct phenotype in the ncs-1 null in which the worms do not show reduced locomotion at actually elevated temperature. Using rescue of this phenotype we showed that NCS-1 functions in AIY neurons. Structure/function analysis introducing single or double mutations within the hydrophobic groove based on information from characterised target complexes established that both N- and C-terminal pockets of the groove are functionally important and that deletion of the C-terminal tail of NCS-1 did not impair its ability to rescue. CONCLUSIONS: The current work has allowed physiological assessment of suggestions from structural studies on the key structural features that underlie the interaction of NCS-1 with its target proteins. The results are consistent with the notion that full length of the hydrophobic groove is required for the regulatory interactions underlying NCS-1 function whereas the C-terminal tail of NCS-1 is not essential. This has allowed discrimination between two potential modes of interaction of NCS-1 with its targets.

3915460e: BMC Neurosci., 90-90(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The duration of sleep promoting efficacy by dual orexin receptor antagonists is dependent upon receptor occupancy threshold.
Author: Gotter,A.L., Winrow,C.J., Brunner,J., Garson,S.L., Fox,S.V., Binns,J., Harrell,C.M., Cui,D., Yee,K.L., Stiteler,M., Stevens,J., Savitz,A., Tannenbaum,P.L., Tye,S.J., McDonald,T., Yao,L., Kuduk,S.D., Uslaner,J., Coleman,P.J., Renger,J.J.
Abstract:
BACKGROUND: Drugs targeting insomnia ideally promote sleep throughout the night, maintain normal sleep architecture, and are devoid of residual effects associated with morning sedation. These features of an ideal compound are not only dependent upon pharmacokinetics, receptor binding kinetics, potency and pharmacodynamic activity, but also upon a compound's mechanism of action. RESULTS: Dual orexin receptor antagonists (DORAs) block the arousal-promoting activity of orexin peptides and, as demonstrated in the current work, exhibit an efficacy signal window dependent upon oscillating levels of endogenous orexin neuropeptide. Sleep efficacy of structurally diverse DORAs in rat and dog was achieved at plasma exposures corresponding to orexin 2 receptor (OX2R) occupancies in the range of 65 to 80%. In rats, the time course of OX2R occupancy was dependent upon receptor binding kinetics and was tightly correlated with the timing of active wake reduction. In rhesus monkeys, direct comparison of DORA-22 with GABA-A modulators at similar sleep-inducing doses revealed that diazepam produced next-day residual sleep and both diazepam and eszopiclone induced next-day cognitive deficits. In stark contrast, DORA-22 did not produce residual effects. Furthermore, DORA-22 evoked only minimal changes in quantitative electroencephalogram (qEEG) activity during the normal resting phase in contrast to GABA-A modulators which induced substantial qEEG changes. CONCLUSION: The higher levels of receptor occupancy necessary for DORA efficacy require a plasma concentration profile sufficient to maintain sleep for the duration of the resting period. DORAs, with a half-life exceeding 8 h in humans, are expected to fulfill this requirement as exposures drop to sub-threshold receptor occupancy levels prior to the wake period, potentially avoiding next-day residual effects at therapeutic doses.

3915470e: Proc.Natl.Acad.Sci.U.S.A., E3723-E3729(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes.
Author: Wigglesworth,K., Lee,K.B., O'Brien,M.J., Peng,J., Matzuk,M.M., Eppig,J.J.
Abstract:
Coordinated regulation of oocyte and ovarian follicular development is essential for fertility. In particular, the progression of meiosis, a germ cell-specific cell division that reduces the number of chromosomes from diploid to haploid, must be arrested until just before ovulation. Follicular somatic cells are well-known to impose this arrest, which is essential for oocyte-follicle developmental synchrony. Follicular somatic cells sustain meiotic arrest via the natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system, and possibly also via high levels of the purine hypoxanthine in the follicular fluid. Upon activation by the ligand NPPC, NPR2, the predominant guanylyl cyclase in follicular somatic cells, produces cyclic guanosine monophosphate (cGMP), which maintains meiotic arrest after transfer to the oocyte via gap junctions. Here we report that both the NPPC/NPR2 system and hypoxanthine require the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme required for the production of guanylyl metabolites and cGMP. Furthermore, oocyte-derived paracrine factors, particularly the growth differentiation factor 9-bone morphogenetic protein 15 heterodimer, promote expression of Impdh and Npr2 and elevate cGMP levels in cumulus cells. Thus, although the somatic compartment of ovarian follicles plays an essential role in the maintenance of oocyte meiotic arrest, as has been known for many years, this function of the somatic cells is surprisingly regulated by signals from the oocyte itself.

Address: The Jackson Laboratory, Bar Harbor, ME 04609

3915471e: Proc.Natl.Acad.Sci.U.S.A., E3526-E3534(2013)
Link: [ PubMED ] [ DOI ]
Title: Natalisin, a tachykinin-like signaling system, regulates sexual activity and fecundity in insects.
Author: Jiang,H., Lkhagva,A., Daubnerova,I., Chae,H.S., Simo,L., Jung,S.H., Yoon,Y.K., Lee,N.R., Seong,J.Y., Zitnan,D., Park,Y., Kim,Y.J.
Abstract:
An arthropod-specific peptidergic system, the neuropeptide designated here as natalisin and its receptor, was identified and investigated in three holometabolous insect species: Drosophila melanogaster, Tribolium castaneum, and Bombyx mori. In all three species, natalisin expression was observed in 3-4 pairs of the brain neurons: the anterior dorso-lateral interneurons, inferior contralateral interneurons, and small pars intercerebralis neurons. In B. mori, natalisin also was expressed in two additional pairs of contralateral interneurons in the subesophageal ganglion. Natalisin-RNAi and the activation or silencing of the neural activities in the natalisin-specific cells in D. melanogaster induced significant defects in the mating behaviors of both males and females. Knockdown of natalisin expression in T. castaneum resulted in significant reduction in the fecundity. The similarity of the natalisin C-terminal motifs to those of vertebrate tachykinins and of tachykinin-related peptides in arthropods led us to identify the natalisin receptor. A G protein-coupled receptor, previously known as tachykinin receptor 86C (also known as the neurokinin K receptor of D. melanogaster), now has been recognized as a bona fide natalisin receptor. Taken together, the taxonomic distribution pattern of the natalisin gene and the phylogeny of the receptor suggest that natalisin is an ancestral sibling of tachykinin that evolved only in the arthropod lineage.

Address: Department of Entomology, Kansas State University, Manhattan, KS 66506

3915473e: Proc.Natl.Acad.Sci.U.S.A., 15061-15066(2013)
Link: [ PubMED ] [ DOI ]
Title: Complement modulates the cutaneous microbiome and inflammatory milieu.
Author: Chehoud,C., Rafail,S., Tyldsley,A.S., Seykora,J.T., Lambris,J.D., Grice,E.A.
Abstract:
The skin is colonized by a plethora of microbes that include commensals and potential pathogens, but it is currently unknown how cutaneous host immune mechanisms influence the composition, diversity, and quantity of the skin microbiota. Here we reveal an interactive role for complement in cutaneous host-microbiome interactions. Inhibiting signaling of the complement component C5a receptor (C5aR) altered the composition and diversity of the skin microbiota as revealed by deep sequencing of the bacterial 16S rRNA gene. In parallel, we demonstrate that C5aR inhibition results in down-regulation of genes encoding cutaneous antimicrobial peptides, pattern recognition receptors, and proinflammatory mediators. Immunohistochemistry of inflammatory cell infiltrates in the skin showed reduced numbers of macrophages and lymphocytes with C5aR inhibition. Further, comparing cutaneous gene expression in germ-free mice vs. conventionally raised mice suggests that the commensal microbiota regulates expression of complement genes in the skin. These findings demonstrate a component of host immunity that impacts colonization of the skin by the commensal microbiota and vice versa, a critical step toward understanding host-microbe immune mutualism of the skin and its implications for health and disease. Additionally, we reveal a role for complement in homeostatic host-microbiome interactions of the skin.

Address: Departments of Dermatology and Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104

3915485e: J.Cell Biol., 747-763(2013)
Link: [ PubMED ] [ DOI ]
Title: JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.
Author: Huntwork-Rodriguez,S., Wang,B., Watkins,T., Ghosh,A.S., Pozniak,C.D., Bustos,D., Newton,K., Kirkpatrick,D.S., Lewcock,J.W.
Abstract:
Neurons are highly polarized cells that often project axons a considerable distance. To respond to axonal damage, neurons must transmit a retrograde signal to the nucleus to enable a transcriptional stress response. Here we describe a mechanism by which this signal is propagated through injury-induced stabilization of dual leucine zipper-bearing kinase (DLK/MAP3K12). After neuronal insult, specific sites throughout the length of DLK underwent phosphorylation by c-Jun N-terminal kinases (JNKs), which have been shown to be downstream targets of DLK pathway activity. These phosphorylation events resulted in increased DLK abundance via reduction of DLK ubiquitination, which was mediated by the E3 ubiquitin ligase PHR1 and the de-ubiquitinating enzyme USP9X. Abundance of DLK in turn controlled the levels of downstream JNK signaling and apoptosis. Through this feedback mechanism, the ubiquitin-proteasome system is able to provide an additional layer of regulation of retrograde stress signaling to generate a global cellular response to localized external insults.

Address: Department of Neuroscience, 2 Department of Microchemical Proteomics, and 3 Department of Physiological Chemistry, Genentech, Inc., South San Francisco, CA 94080, USA

3915486e: Inflamm.Res., 951-959(2013)
Link: [ PubMED ] [ DOI ]
Title: Suppression of Th1 cytokine production by a peptide derived from C4b.
Author: Takeda,Y., Kaneda,K., Jimma,F., Shiobara,N., Saniabadi,A.R., Wakabayashi,I.
Abstract:
OBJECTIVES: The complement system has been proposed to play a significant role in the regulation of T-cell responses. However, the precise mechanism underlying C4-induced immune tolerance remains to be clarified. We recently reported that monomeric C4b inhibits CXCL10 production from blood cells. The purpose of this study was to verify the active site of monomeric C4b. MATERIALS AND METHODS: We investigated the in vitro effects of a C4b-derived peptide (VPAGSARPVAFSVVPTAAA), named HP2 (highly homologous peptide 2), on the IFN-β-induced production of CXCL10 in human blood and the in vivo effects of the administration of HP2 on Th1/2 cytokine production in the spleen in mice. We also tested whether the administration of HP2 influences symptoms of experimentally induced ulcerative colitis in mice. RESULTS: HP2 inhibited CXCL10 production in human blood, and the administration of HP2 significantly suppressed the production of Th1 cytokines, such as IL-2, IFN-γ, and TNF-α, in spleen cells isolated from mice. The administration of HP2 in the mice significantly improved the symptoms of colitis, with down-regulation of colitogenic CD4+CD45RB(high) T cells and up-regulation of CD4+LAP/TGF-β1+ T cells. CONCLUSION: The amino acid sequence described above is suggested to be the active site in C4b for the inhibition of Th1 cytokine production. These results should contribute to the development of new drugs suppressing autoimmune responses.

Address: Department of Environmental and Preventive Medicine, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya, Hyogo, 663-8501, Japan

3915490e: Eur.J.Appl.Physiol., 2737-2747(2013)
Link: [ PubMED ] [ DOI ]
Title: Meal induced gut hormone secretion is altered in aerobically trained compared to sedentary young healthy males.
Author: Lund,M.T., Taudorf,L., Hartmann,B., Helge,J.W., Holst,J.J., Dela,F.
Abstract:
Postprandial insulin release is lower in healthy aerobically trained (T) compared to untrained (UT) individuals. This may be mediated by a lower release of the two incretin hormones [glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP)] in T. The aim of this study was to assess and compare gut hormone response and satiety changes after a liquid meal intake in young, healthy T and UT males. Postprandial gut hormone release and subjective feelings of hunger, satiety, fullness and prospective food consumption were assessed before and frequently for the following 3 h after a 200 ml liquid meal (1,260 kJ and 27, 41 and 32 energy % as protein, carbohydrates and fat, respectively) in ten T and ten UT young, healthy male subjects. The insulin and GIP responses were markedly lower in T than UT and correlated during the first 30 min after the liquid meal. Baseline GLP-1 concentration was higher in T versus UT, but the response in the following 3 h after a liquid meal was similar in T and UT. Satiety measures did not differ between groups throughout the test. It is possible that in aerobically T subjects, a lower GIP release is partly responsible for a lower postprandial incretin stimulated insulin secretion.

E-Mail: michaeltl@sund.ku.dk
Address: Department of Biomedical Sciences, Center for Healthy Aging, Xlab, University of Copenhagen, Blegdamsvej 3b, 2200, Copenhagen, Denmark,

3915491e: Oncogenesis, e66-e66(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Gremlin-1 associates with fibrillin microfibrils in vivo and regulates mesothelioma cell survival through transcription factor slug.
Author: Tamminen,J.A., Parviainen,V., Ronty,M., Wohl,A.P., Murray,L., Joenvaara,S., Varjosalo,M., Lepparanta,O., Ritvos,O., Sengle,G., Renkonen,R., Myllarniemi,M., Koli,K.
Abstract:
Malignant mesothelioma is a form of cancer that is highly resistant to conventional cancer therapy for which no major therapeutic advances have been introduced. Here, we identify gremlin-1, a known bone morphogenetic protein inhibitor crucial for embryonic development, as a potential therapeutic target for mesothelioma. We found high expression levels of gremlin-1 in the mesothelioma tumor tissue, as well as in primary mesothelioma cells cultured from pleural effusion samples. Downregulation of gremlin-1 expression by siRNA-mediated silencing in a mesothelioma cell line inhibited cell proliferation. This was associated with downregulation of the transcription factor slug as well as mesenchymal proteins linked to cancer epithelial-to-mesenchymal transition. Further, resistance to paclitaxel-induced cell death was associated with high gremlin-1 and slug expression. Treatment of gremlin-1-silenced mesothelioma cells with paclitaxel or pemetrexed resulted in efficient loss of cell survival. Finally, our data suggest that concomitant upregulation of fibrillin-2 in mesothelioma provides a mechanism for extracellular localization of gremlin-1 to the tumor microenvironment. This was supported by the demonstration of interactions between gremlin-1, and fibrillin-1 and -2 peptides as well as by colocalization of gremlin-1 to fibrillin microfibrils in cells and tumor tissue samples. Our data suggest that gremlin-1 is also a potential target for overcoming drug resistance in mesothelioma.

Address: 1] Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland [2] Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland

3915492e: Peptides, 9-20(2013)
Link: [ PubMED ] [ DOI ]
Title: New satiety hormone nesfatin-1 protects gastric mucosa against stress-induced injury: Mechanistic roles of prostaglandins, nitric oxide, sensory nerves and vanilloid receptors.
Author: Szlachcic,A., Sliwowski,Z., Krzysiek-Maczka,G., Majka,J., Surmiak,M., Pajdo,R., Drozdowicz,D., Konturek,S.J., Brzozowski,T.
Abstract:
Nesfatin-1 belongs to a family of anorexigenic peptides, which are responsible for satiety and are identified in the neurons and endocrine cells within the gut. These peptides have been implicated in the control of food intake; however, very little is known concerning its contribution to gastric secretion and gastric mucosal integrity. In this study the effects of nesfatin-1 on gastric secretion and gastric lesions induced in rats by 3.5h of water immersion and restraint stress (WRS) were determined. Exogenous nesfatin-1 (5-40μg/kg i.p.) significantly decreased gastric acid secretion and attenuated gastric lesions induced by WRS, and this was accompanied by a significant rise in plasma NUCB2/nefatin-1 levels, the gastric mucosal blood flow (GBF), luminal NO concentration, generation of PGE2 in the gastric mucosa, an overexpression of mRNA for NUBC2 and cNOS, as well as a suppression of iNOS and proinflammatory cytokine IL-1β and TNF-α mRNAs. Nesfatin-1-induced protection was attenuated by suppression of COX-1 and COX-2 activity, the inhibition of NOS with L-NNA, the deactivation of afferent nerves with neurotoxic doses of capsaicin, and the pretreatment with capsazepine to inhibit vanilloid VR1 receptors. This study shows for the first time that nesfatin-1 exerts a potent protective action in the stomach of rats exposed to WRS and these effects depend upon decrease in gastric secretion, hyperemia mediated by COX-PG and NOS-NO systems, the activation of vagal and sensory nerves and vanilloid receptors.

Address: Department of Physiology Jagiellonian University Medical College, Cracow, Poland

3915494e: J.Pharmacol.Exp.Ther., 258-264(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of Intrathecal SNC80, a Δ Receptor Ligand, on Nociceptive Threshold and Dorsal Horn Substance P Release.
Author: Kouchek,M., Takasusuki,T., Terashima,T., Yaksh,T.L., Xu,Q.
Abstract:
Δ-opioid receptors (DOR) are present in the superficial dorsal horn and are believed to regulate the release of small afferent transmitters as evidenced by the effects of spinally delivered δ-opioid preferring peptides. Here we examined the effects of intrathecal SNC80 [+-4-[α(R)-α-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-(methoxyben zyl)-N,N-diethylbenzamide], a selective nonpeptidic DOR agonist, in three preclinical pain models, acute thermal escape, intraplantar carrageenan-tactile allodynia, and intraplantar formalin flinches, and on the evoked release of substance P (SP) from small primary afferents. Rats with chronic intrathecal catheters received intrathecal vehicle or SNC80 (100 or 200 μg). Intrathecal SNC80 did not change acute thermal latencies or carrageenan-induced thermal hyperalgesia. However, SNC80 attenuated carrageenan-induced tactile allodynia and significantly reduced both phase 1 and phase 2 formalin-induced paw flinches, as assessed by an automatic flinch counting device. These effects were abolished by naltrindole (3 mg/kg i.p.), a selective DOR antagonist, but not CTOP (10 μg i.t.), a selective MOR antagonist. Furthermore, intrathecal SNC80 (200 μg) blocked formalin-induced substance P release otherwise evoked in the ispilateral superficial dorsal horn as measured by NK1 receptor internalization. In conclusion, intrathecal SNC80 alleviated pain hypersensitivity after peripheral inflammation in a fashion paralleling its ability to block peptide transmitter release from small peptidergic afferents, which by its pharmacology appears to represent an effect mediated by a spinal DOR.

Address: Department of Anesthesiology, University of California San Diego, La Jolla, California (M.K., T.L.Y., Q.X.)

3915495e: Physiol.Behav., 104-112(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of acute and subchronic AT receptor blockade on cardiovascular, hydromineral and neuroendocrine responses in female rats.
Author: Araujo,I.G., Elias,L.L., Antunes-Rodrigues,J., Reis,L.C., Mecawi,A.S.
Abstract:
Female Wistar rats were ovariectomized (OVX) and separated into two groups that received either estradiol cypionate (EC, 40μg/kg, sc; OVX-EC) or vehicle (corn oil, sc; OVX-oil) for 14 consecutive days. On the 7th day of treatment, a subset of animals from both the OVX-oil and OVX-EC groups was subjected to subchronic losartan (AT1 receptor antagonist) treatment (0.1g/L in drinking water; ~15mg/kg/day) for 7days. Other group of OVX-oil and OVX-EC rats was submitted to an acute losartan injection (100mg/kg, ip) on the 14th day of hormone replacement. In both protocols, the following parameters were measured: I) mean arterial pressure (MAP) and heart rate (HR); II) water and 0.3M saline intake; III) angiotensin II (ANG II), atrial natriuretic peptide (ANP), vasopressin (AVP) and oxytocin (OT) plasma concentrations; and IV) urinary and plasma sodium concentrations. Acute AT1 blockade induced a significant reduction in the MAP in the OVX rats, resulting in increased HR and water intake, which were attenuated by estradiol therapy. Acute AT1 blockade also increased ANG II and OT and reduced ANP plasma concentrations, with no changes in AVP secretion. In addition, acute hypotension was accompanied by a decrease in natriuresis, which was unaltered by estradiol. Subchronic AT1 blockade induced a significant decrease in MAP without changing HR in both groups. Additionally, subchronic losartan treatment induced sodium appetite in OVX rats. Prolonged AT1 blockade increased ANG II and AVP and reduced ANP plasma concentrations. Moreover, it increased natriuresis but did not alter plasma OT concentrations. Finally, estradiol treatment attenuated the increase in salt intake and plasma ANG II concentrations induced by subchronic AT1 blockade. In conclusion, our results suggest differential adaptive responses to the acute or subchronic losartan treatment in OVX and OVX-EC rats.

E-Mail: igaraujo@ufrrj.br
Address: Department of Physiological Sciences, Institute of Biology, Federal Rural University of Rio de Janeiro, BR 465 Km 07, CEP 23897970 Seropedica, Rio de Janeiro, Brazil. Electronic address:

3915498e: Mol.Cancer, 95-95(2013)
Link: [ PubMED ] [ DOI ]
Title: Correlation between centromere protein-F autoantibodies and cancer analyzed by enzyme-linked immunosorbent assay.
Author: Welner,S., Trier,N.H., Frisch,M., Locht,H., Hansen,P.R., Houen,G.
Abstract:
BACKGROUND: Centromere protein-F (CENP-F) is a large nuclear protein of 367 kDa, which is involved in multiple mitosis-related events such as proper assembly of the kinetochores, stabilization of heterochromatin, chromosome alignment and mitotic checkpoint signaling. Several studies have shown a correlation between CENP-F and cancer, e.g. the expression of CENP-F has been described to be upregulated in cancer cells. Furthermore, several studies have described a significant correlation between the expression of autoantibodies to CENP-F and cancer. METHODS: Autoantibodies to CENP-F were detected in a small number of samples during routine indirect immunofluorescence (IIF) analysis for anti-nuclear antibodies (ANA) using HEp-2 cells as substrate. Using overlapping synthetic peptides covering a predicted structural maintenance of chromosomes (SMC) domain, we developed an enzyme-linked immunosorbent assay (ELISA) for detection of CENP-F antibodies. RESULTS: Analyzing the reactivity of the sera positive in IIF for CENP-F antibodies to overlapping CENP-F peptides, we showed that autoantibodies to several peptides correlate with the presence of antibodies to CENP-F and a diagnosis of cancer, as increased CENP-F antibody expression specific for malignant cancer patients to five peptides was found (A9, A12, A14, A16, A27). These antibodies to CENP-F in clinical samples submitted for ANA analysis were found to have a positive predictive value for cancer of 50%. Furthermore, the expression of cancer-correlated CENP-F antibodies seemed to increase as a function of time from diagnosis. CONCLUSION: These results conform to previous findings that approximately 50% of those patients clinically tested for ANA analyses who express CENP-F antibodies are diagnosed with cancer, confirming that these antibodies may function as circulating tumor markers. Thus, a peptide-based CENP-F ELISA focused on the SMC domain may aid in identifying individuals with a potential cancer.

3915499e: Anaesth.Intensive Care, 591-595(2013)
Link:
Title: A preliminary evaluation of plasma b-type natriuretic peptide as a screening test for left ventricular diastolic dysfunction in non-cardiac intensive care.
Author: Sturgess,D.J., Parmar,D., Dulhunty,J.M., Hedge,R., Jarrett,P., Udy,A.
Abstract:
Left ventricular filling and thus diastolic function are frequently monitored and managed in critical care. However, scant data exist regarding possible screening tests for diastolic dysfunction in the intensive care unit (ICU). The present study aimed to evaluate plasma b-type natriuretic peptide (BNP) as a marker of diastolic dysfunction in a single-centre cohort of 'non-cardiac' ICU patients. The ICU is non-cardiac in that it provides mixed medical/surgical services with the exception of cardiology, cardiac surgery and solid organ transplantation. Clinical data were recorded over the first 24 hours of ICU stay for 32 consecutive patients. Transthoracic echocardiogram and blood collection for BNP assay were then performed. Diastolic dysfunction was demonstrated in 34% (n=11). Mean +/- standard deviation BNP values were higher with diastolic dysfunction (238 +/- 195 vs 72 +/- 78 pg/ml; P=0.003). A BNP threshold of >43 pg/ml yielded a sensitivity of 80% and a specificity of 59%; area under the receiver operating characteristic curve was 0.82. BNP correlated independently with E/e' (R=0.425; P=0.015) (E/e': peak early transmitral velocity [E]/early diastolic mitral annular velocity [e']) but not left ventricular ejection fraction (P=0.8), illness severity (Acute Physiological and Chronic Health Evaluation II; P=0.3) or fluid balance (P=0.4). Diastolic dysfunction was common in this cohort of non-cardiac ICU patients and was independently associated with a significantly higher BNP. The potential application as a screening test for diastolic dysfunction is likely to require a threshold lower than previously proposed for heart failure.

E-Mail: d.sturgess@uq.edu.au
Address: Department of Intensive Care, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia

3915508e: PLoS One, e72369-e72369(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Substance p immunoreactivity exhibits frequent colocalization with kisspeptin and neurokinin B in the human infundibular region.
Author: Hrabovszky,E., Borsay,B.A., Racz,K., Herczeg,L., Ciofi,P., Bloom,S.R., Ghatei,M.A., Dhillo,W.S., Liposits,Z.
Abstract:
Neurons synthesizing neurokinin B (NKB) and kisspeptin (KP) in the hypothalamic arcuate nucleus represent important upstream regulators of pulsatile gonadotropin-releasing hormone (GnRH) neurosecretion. In search of neuropeptides co-expressed in analogous neurons of the human infundibular nucleus (Inf), we have carried out immunohistochemical studies of the tachykinin peptide Substance P (SP) in autopsy samples from men (21-78 years) and postmenopausal (53-83 years) women. Significantly higher numbers of SP-immunoreactive (IR) neurons and darker labeling were observed in the Inf of postmenopausal women than in age-matched men. Triple-immunofluorescent studies localized SP immunoreactivity to considerable subsets of KP-IR and NKB-IR axons and perikarya in the infundibular region. In postmenopausal women, 25.1% of NKB-IR and 30.6% of KP-IR perikarya contained SP and 16.5% of all immunolabeled cell bodies were triple-labeled. Triple-, double- and single-labeled SP-IR axons innervated densely the portal capillaries of the infundibular stalk. In quadruple-labeled sections, these axons formed occasional contacts with GnRH-IR axons. Presence of SP in NKB and KP neurons increases the functional complexity of the putative pulse generator network. First, it is possible that SP modulates the effects of KP and NKB in axo-somatic and axo-dendritic afferents to GnRH neurons. Intrinsic SP may also affect the activity and/or neuropeptide release of NKB and KP neurons via autocrine/paracrine actions. In the infundibular stalk, SP may influence the KP and NKB secretory output via additional autocrine/paracrine mechanisms or regulate GnRH neurosecretion directly. Finally, possible co-release of SP with KP and NKB into the portal circulation could underlie further actions on adenohypophysial gonadotrophs.

E-Mail: hrabovszky.erik@koki.hu
Address: Laboratory of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

3915511e: PLoS One, e77505-e77505(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Expression of an engineered heterologous antimicrobial peptide in potato alters plant development and mitigates normal abiotic and biotic responses.
Author: Goyal,R.K., Hancock,R.E., Mattoo,A.K., Misra,S.
Abstract:
Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield.

Address: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada

3915512e: PLoS One, e77326-e77326(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Adequately adapted insulin secretion and decreased hepatic insulin extraction cause elevated insulin concentrations in insulin resistant non-diabetic adrenal incidentaloma patients.
Author: Anderwald,C.H., Tura,A., Gessl,A., Luger,A., Pacini,G., Krebs,M.
Abstract:
BACKGROUND: Insulin-resistance is commonly found in adrenal incidentaloma (AI) patients. However, little is known about β-cell secretion in AI, because comparisons are difficult, since β-cell-function varies with altered insulin-sensitivity. OBJECTIVES: To retrospectively analyze β-cell function in non-diabetic AI, compared to healthy controls (CON). METHODS: AI (n=217, 34%males, 57+/-1years, body-mass-index:27.7+/-0.3kg/m(2)) and CON [n=25, 32%males, 56+/-1years, 26.7+/-0.8kg/m(2)] with comparable anthropometry (p>/=0.31) underwent oral-glucose-tolerance-tests (OGTTs) with glucose, insulin, and C-peptide measurements. 1mg-dexamethasone-suppression-tests were performed in AI. AI were divided according to post-dexamethasone-suppression-test cortisol-thresholds of 1.8 and 5μg/dL into 3subgroups: pDexa<1.8μg/dL, pDexa1.8-5μg/dL and pDexa>5μg/dL. Using mathematical modeling, whole-body insulin-sensitivity [Clamp-like-Index (CLIX)], insulinogenic Index, Disposition Index, Adaptation Index, and hepatic insulin extraction were calculated. RESULTS: CLIX was lower in AI combined (4.9+/-0.2mg.kg(-1).min(-1)), pDexa<1.8μg/dL (4.9+/-0.3) and pDexa1.8-5μg/dL (4.7+/-0.3, p<0.04 vs.CON:6.7+/-0.4). Insulinogenic and Disposition Indexes were 35%-97% higher in AI and each subgroup (p<0.008 vs.CON), whereas C-peptide-derived Adaptation Index, compensating for insulin-resistance, was comparable between AI, subgroups, and CON. Mathematical estimation of insulin-derived (insulinogenic and Disposition) Indexes from associations to insulin-sensitivity in CON revealed that AI-subgroups had ~19%-32% higher insulin-secretion than expectable. These insulin-secretion-index differences negatively (r=-0.45, p<0.001) correlated with hepatic insulin extraction, which was 13-16% lower in AI and subgroups (p<0.003 vs.CON). CONCLUSIONS: AI-patients show insulin-resistance, but adequately adapted insulin secretion with higher insulin concentrations during an OGTT, because of decreased hepatic insulin extraction; this finding affects all AI-patients, regardless of dexamethasone-suppression-test outcome.

Address: Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria

3915522e: Afr.J.Tradit.Complement.Altern.Med., 6-11(2013)
Link: [ PubMED ]
Title: Effect of Four Medicinal Plants on Amyloid-β Induced Neurotoxicity in SH-SY5Y Cells.
Author: Adewusi,E.A., Fouche,G., Steenkamp,V.
Abstract:
Amyloid-β peptide (Aβ) is implicated in the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder. This study was designed to determine the effect of four medicinal plants used to treat neurodegenerative diseases on Aβ-induced cell death. Cytotoxicity of the ethanol extracts of the plants was determined against SH-SY5Y (human neuroblastoma) cells which were untreated, as well as toxically induced with Aβ, using the MTT and neutral red uptake assays. Cell viability was reduced to 16% when exposed to 20 μM Aβ25-35 for 72 h. The methanol extract of the roots of Ziziphus mucronata Willd., Lannea schweinfurthii (Engl.) Engl. and Terminalia sericea Burch. ex DC., were the least toxic to the SH-SY5Ycells at the highest concentration tested (100 μg/ml). All four plants tested were observed to reduce the effects of Aβ-induced neuronal cell death, indicating that they may contain compounds which may be relevant in the prevention of AD progression.

Address: Department of Pharmacology, Faculty of Health Sciences, University of Pretoria, Private Bag X323, Arcadia 0007, South Africa

3915540e: Mol.Med.Rep., 1649-1654(2013)
Link: [ PubMED ] [ DOI ]
Title: Lipopolysaccharide-induced apoptosis in a murine intestinal endocrine cell line by modulation of Bcl-2, Bax and caspase-3.
Author: Lei,L., Wang,J., Zhang,Z., Zhang,H., Chen,H., Cai,D.
Abstract:
Numerous studies have focused on how to modulate the secretion of glucagon-like peptide 1 (GLP-1) due to its marked anti-diabetic function. However, few studies have investigated the apoptosis of enteroendocrine L cells, which secrete GLP-1. The aim of this study was to determine whether lipopolysaccharide (LPS), a gut bacterial product, is capable of inducing apoptosis in the intestinal endocrine cell line STC-1. We found that LPS is capable of reducing the viability of STC-1 cells in a concentration-dependent manner. annexin V/propidium iodide (PI) double staining detected by fluorescence microscopy and flow cytometry revealed a strong apoptosis-inducing ability for LPS in STC-1 cells. Furthermore, western blotting revealed that exposure to LPS significantly decreased the expression of Bcl-2 and increased the expression of Bax and caspase-3. In conclusion, LPS induced the apoptosis of STC-1 cells in a dose-dependent manner, which may be responsible for the reduced secretion of GLP1 in diabetes.

Address: Department of Endocrinology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510280, P.R. China

3915541e: Pharmacol.Rep., 927-936(2013)
Link:
Title: Combination of omeprazole with GLP-1 agonist therapy improves insulin sensitivity and antioxidant activity in liver in type 1 diabetic mice.
Author: Patel,V., Joharapurkar,A., Dhanesha,N., Kshirsagar,S., Detroja,J., Patel,K., Gandhi,T., Patel,K., Bahekar,R., Jain,M.
Abstract:
Background: Combination with suitable pharmacological agents can improve the antiobesity and antidiabetic actions of glucagon like peptide-1 (GLP-1) based therapies. GLP-1 agonist exendin-4 may have insulin-independent effects on amelioration of insulin resistance and hepatic steatosis by virtue of its action on hepatic GLP-1 receptors, and these effects can be improved by combination with proton pump inhibitors. However, it was not assessed whether omeprazole can improve the peripheral actions of exendin-4 in the state of insulin deficiency. Methods: We investigated the effects of combination of omeprazole with GLP-1 agonist exendin-4 in multiple low-dose streptozotocin (STZ)-induced diabetes in C57BL/KsJ mice, a model of type 1 diabetes. Male diabetic mice were treated with exendin-4 and/or omeprazole for a period of 4 weeks. Results: Omeprazole treatment had no significant effect on lowering the blood glucose levels of diabetic mice, when compared to control, although it improved the antihyperglycemic actions of exendin-4. Similarly, serum triglycerides and total cholesterols levels were significantly lower in the combination treated mice compared to either exendin-4 and omeprazole alone. In addition, the combination treatment significantly ameliorated lipid peroxidation and hepatic triglycerides in diabetic mice compared to either exendin-4 and omeprazole alone. The improvement in hepatic insulin sensitivity, as indicated by insulin tolerance test (ITT) and pyruvate tolerance test (IPPTT), was correlated with the expression of nuclear factor erythroid-related factor 2 (Nrf2) and insulin receptor substrate-1 (IRS-1) and the combination treatment significantly improved the insulin sensitivity in comparison to vehicle control. Conclusion: We conclude that combination with omeprazole improves the insulin sensitizing actions of GLP-1 therapy and these effects are partially mediated through the decrease in hepatic steatosis and improvement in antioxidant status in the liver.

E-Mail: amitjoharapurkar@zyduscadila.com
Address: Department of Pharmacology and Toxicology, Zydus Research Centre, Cadila Healthcare Limited, Sarkhej-Bavla N.H. No.8A, Moraiya, Ahmedabad 382210, India

3915542e: Pharmacol.Rep., 884-890(2013)
Link:
Title: Exendin-4 and GLP-1 decreases induced expression of ICAM-1, VCAM-1 and RAGE in human retinal pigment epithelial cells.
Author: Dorecka,M., Siemianowicz,K., Francuz,T., Garczorz,W., Chyra,A., Klych,A., Romaniuk,W.
Abstract:
Background: Advanced glycation end products (AGEs) take part in the development of diabetic retinopathy. Hyperglycemia triggers an inflammatory response in the retina. These mechanisms may lead to an enhanced expression of adhesion molecules (ICAM-1 and VCAM-1) in human retinal pigment epithelium (HRPE). Glucagon-like peptide 1 (GLP-1) functions as an incretin hormone with antidiabetogenic properties. GLP-1 also possesses vasoprotective properties. Methods: The aim of our study was to evaluate the influence of glycated albumin (GlyAlb; 100; 500 and 1000 mg/l) and pro-inflammatory cytokine, TNF-α (2.5 and 10 ng/ml), on expression of RAGE, ICAM-1 and VCAM-1 and to evaluate the influence of GLP-1 (100 nM) and its analogue, exendin-4 (10 nM), on the expression of RAGE, ICAM-1 and VCAM-1 in stimulated HRPE. Results: TNF-α increased RAGE expression in HRPE cells. The addition of GlyAlb (500 and 1000 mg/l) resulted in a decrease of RAGE expression. Both TNF-α and GlyAlb increased the secretion of both adhesion molecules. In cells co-treated with GLP-1 or exendin-4 both incretins decreased RAGE expression in TNF-α treated cells, and in GlyAlb group. The ICAM-1 expression was lowered by exendin-4 and GLP-1 in cells stimulated by TNF-α and GlyAlb. The similar results were obtained for VCAM-1. All observed alterations were statistically significant. Conclusions: The obtained results indicate that both GLP-1 and exendin-4 by decreasing the expression of RAGE in HRPE can make these cells more resistant to circulating AGEs, and decreased expression of circulating VCAM-1 and ICAM-1, can be the result of anti-inflammatory properties of incretins and decreased expression of RAGE.

E-Mail: ksiem@mp.pl
Address: Department of Ophthalmology, Medical University of Silesia, Ceglana 35, PL 40-952 Katowice, Poland

3915543e: Pharmacol.Rep., 836-846(2013)
Link:
Title: Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20.
Author: Kleczkowska,P., Bojnik,E., Lesniak,A., Kosson,P., Van den Eynde,I., Ballet,S., Benyhe,S., Tourwe,D., Lipkowski,A.W.
Abstract:
Background: Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. Methods: The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [35S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. Results: Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [3H]DAMGO and [3H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [35S]GTPγS binding, proving full agonism at both receptor types. In the [35S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the μ and δ receptor subtypes revealed μ-receptor selectivity. Conclusion: The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

E-Mail: andrzej@lipkowski.org
Address: Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, PL 02-106 Warszawa, Poland

3915544e: Pharmacol.Rep., 823-835(2013)
Link:
Title: Synthesis and neuromodulatory effects of TRH-related peptides: inhibitory activity on catecholamine release in vitro.
Author: Brunetti,L., Chiavaroli,A., Cocco,A., Ferrante,C., Ferrucci,A., Luisi,G., Orlando,G., Pinnen,F., Vacca,M.
Abstract:
Background: A detailed comprehension of central mechanisms underlying feeding behavior holds considerable promise for the treatment of alimentary disorders. Methods: In order to elucidate the tight interrelationships occurring at the hypothalamic neuronal endings between aminergic neurotransmitters and co-localized appetite modulators, we initially studied the effects of two anorexigenic peptides structurally related to thyrotropin-releasing hormone (TRH, 1), namely cyclo(His-Pro) (CHP, 2) and pGlu-His-Gly-OH (3), on [3H]-norepinephrine and [3H]-dopamine release from perfused rat hypothalamic synaptosomes. Furthermore, a number of TRH and CHP analogues were synthesized and tested for their ability to influence neurotransmitter release in the selected neuronal model. Results: Peptide 3 showed only a slight inhibitory activity on norepinephrine release, whereas no effect was observed for compound 2. TRH analogue 8, metabolically stabilized by the replacement of pyroglutamate with the pyrohomocysteic acid (pHcs), was found to be inactive. Conversely, a significant inhibitory effect on dopamine and norepinephrine release was observed for the CHP-related diketopiperazines cyclo(Leu-Pro) (11) and cyclo(His-Gly) (14). Conclusions: These results suggest a potential role for cyclo-dipeptides 11 and 14 in the hypothalamic modulation of appetite suppressant circuitry.

E-Mail: mvacca@unich.it
Address: Department of Pharmacy, G. d'Annunzio University, via dei Vestini 31, 66013 Chieti, Italy

3915548e: Can.J.Physiol.Pharmacol., 830-838(2013)
Link: [ PubMED ] [ DOI ]
Title: Aliskiren, exendin-4, and insulin: their impact on endothelin receptor subtype(s) regulation/binding in type 1 diabetic rat hearts.
Author: Al Lafi,S.M., Artinian,S.B., Boutary,S.S., Zwainy,N.S., Bitar,K.M., Bikhazi,A.B.
Abstract:
This study focuses on the impact of aliskiren and (or) glucagon-like peptide-1 analogue on the binding affinity/regulation of endothelin-1 (ET-1) to its receptor subtypes A (ETAR) and B (ETBR) at the level of the coronary endothelium and the cardiomyocytes in a type-1 diabetic rat model. Seven groups were used: (i) normal rats, (ii) rats with induced diabetes, (iii) rats with induced diabetes that were treated with insulin, (iv) rats with induced diabetes that were treated with exendin-4, (v) rats with induced diabetes that were treated with aliskiren, (vi) rats with induced diabetes that were co-treated with insulin plus aliskiren, and (vii) rats with induced diabetes that were co-treated with exendin-4 plus aliskiren. Heart perfusion with [(125)I]-ET-1 was employed to estimate ET-1 binding affinity (tau = 1/K-n) to ETAR and ETBR at the level of the coronary endothelium and the cardiomyocytes. Plasma ET-1 levels were measured using enzyme immunoassay, whereas densities of ETAR and ETBR were detected using Western blot. No significance differences were detected in the tau of ETAR and ETBR between normal and diabetic in cardiomyocytes and the coronary endothelium. Exendin-4 normalized the tau value for ETAR and ETBR on coronary endothelium, while aliskiren normalized it on cardiomyocytes. Furthermore, ETAR and ETBR densities were normalized with monotreatments of aliskiren and exendin-4, compared with up-regulated ETAR and down-regulated ETBR band densities in the diabetic animals. Our data indicate that aliskiren alleviates diabetes-associated hypertrophy in type 1 diabetes mellitus.

Address: a Department of Anatomy, Cell Biology and Physiology, Faculty of Medicine, American University of Beirut, Beirut 11-0236, Lebanon

3915550e: Methods Cell Biol., 391-410(2013)
Link: [ PubMED ] [ DOI ]
Title: Visualization of TCR Nanoclusters via Immunogold Labeling, Freeze-Etching, and Surface Replication.
Author: Fiala,G.J., Rejas,M.T., Schamel,W.W., Van Santen,H.M.
Abstract:
T cells show high sensitivity for antigen, even though their T-cell antigen receptor (TCR) has a low affinity for its ligand, a major histocompatibility complex molecule presenting a short pathogen-derived peptide. Over the past few years, it has become clear that these paradoxical properties rely at least in part on the organization of cell surface-expressed TCRs in TCR nanoclusters. We describe a protocol, comprising immunogold labeling, cell surface replica generation, and electron microscopy (EM) analysis that allows nanoscale resolution of the distribution of TCRs and other cell surface molecules of cells grown in suspension. Unlike most of the light microscopy-based single-molecule resolution techniques, this technique permits visualization of these molecules on cell surfaces that do not adhere to an experimental support. Given the potential of adhesion-induced receptor redistributions, our technique is a relevant complement to the substrate adherence-dependent techniques. Furthermore, it does not rely on introduction of fluorescently labeled recombinant molecules and therefore allows direct analysis of nonmanipulated primary cells.

Address: Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies, Albert Ludwigs University Freiburg, Freiburg, Germany

3915551e: Biol.Open, 960-967(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Central ventilatory and cardiovascular actions of trout gastrin-releasing peptide (GRP) in the unanesthetized trout.
Author: Le Mevel,J.C., Lancien,F., Mimassi,N., Kermorgant,M., Conlon,J.M.
Abstract:
Gastrin-releasing peptide (GRP), a neuropeptide initially isolated from porcine stomach, shares sequence similarity with bombesin. GRP and its receptors are present in the brains and peripheral tissues of several species of teleost fish, but little is known about the ventilatory and cardiovascular effects of this peptide in these vertebrates. The goal of this study was to compare the central and peripheral actions of picomolar doses of trout GRP on ventilatory and cardiovascular variables in the unanesthetized rainbow trout. Compared to vehicle, intracerebroventricular (ICV) injection of GRP (1-50 pmol) significantly elevated the ventilation rate (fV) and the ventilation amplitude (VAMP), and consequently the total ventilation (VTOT). The maxiμm hyperventilatory effect of GRP (VTOT: +225%), observed at a dose of 50 pmol, was mostly due to its stimulatory action on VAMP (+170%) rather than fV (+20%). In addition, ICV GRP (50 pmol) produced a significant increase in mean dorsal aortic blood pressure (P DA) (+35%) and in heart rate (fH) (+25%). Intra-arterial injections of GRP (5-100 pmol) were without sustained effect on the ventilatory variables but produced sporadic and transient increases in ventilatory movement at doses of 50 and 100 pmol. At these doses, GRP elevated P DA by +20% but only the 50 pmol dose significantly increased HR (+15%). In conclusion, our study suggests that endogenous GRP within the brain of the trout may act as a potent neurotransmitter and/or neuromodulator in the regulation of cardio-ventilatory functions. In the periphery, endogenous GRP may act as locally-acting and/or circulating neurohormone with an involvement in vasoregulatory mechanisms.

Address: INSERM UMR1101, Laboratoire de Traitement de l'Information Medicale, Laboratoire de Neurophysiologie, SFR ScInBioS, Faculte de Medecine et des Sciences de la Sante , 22 Avenue Camille Desmoulins, CS 93837, 29238 Brest Cedex 3, CHU de Brest , France

3915555e: PLoS One, e77019-e77019(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Characterization of Lung Cancer by Amide Proton Transfer (APT) Imaging: An In-Vivo Study in an Orthotopic Mouse Model.
Author: Togao,O., Kessinger,C.W., Huang,G., Soesbe,T.C., Sagiyama,K., Dimitrov,I., Sherry,A.D., Gao,J., Takahashi,M.
Abstract:
Amide proton transfer (APT) imaging is one of the chemical exchange saturation transfer (CEST) imaging methods which images the exchange between protons of free tissue water and the amide groups (-NH) of endogenous mobile proteins and peptides. Previous work suggested the ability of APT imaging for characterization of the tumoral grade in the brain tumor. In this study, we tested the feasibility of in-vivo APT imaging of lung tumor and investigated whether the method could differentiate the tumoral types on orthotopic tumor xenografts from two malignant lung cancer cell lines. The results revealed that APT imaging is feasible to quantify lung tumors in the moving lung. The measured APT effect was higher in the tumor which exhibited more active proliferation than the other. The present study demonstrates that APT imaging has the potential to provide a characterization test to differentiate types or grade of lung cancer noninvasively, which may eventually reduce the need invasive needle biopsy or resection for lung cancer.

Address: Advanced Imaging Research Center, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, Texas, United States of America

3915561e: Eur.J.Clin.Microbiol.Infect.Dis., 1377-1391(2013)
Link: [ PubMED ] [ DOI ]
Title: Cryptococcosis: epidemiology, fungal resistance, and new alternatives for treatment.
Author: Gullo,F.P., Rossi,S.A., Sardi,J.de C., Teodoro,V.L.I., Mendes-Giannini,M.J.S., Fusco-Almeida,A.M.
Abstract:
Cryptococcosis is an important systemic mycosis and the third most prevalent disease in human immunodeficiency virus (HIV)-positive individuals. The incidence of cryptococcosis is high among the 25 million people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. In Cryptococcus neoformans, resistance to azoles may be associated with alterations in the target enzyme encoded by the gene ERG11, lanosterol 14α-demethylase. These alterations are obtained through mutations, or by overexpressing the gene encoding. In addition, C. gattii and C. neoformans present a heteroresistance phenotype, which may be related to increased virulence. Other species beyond C. neoformans and C. gattii, such as C. laurentii, have been diagnosed mainly in patients with immunosuppression. Infections of C. albidus have been isolated in cats and marine mammals. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth, which is also related with increased resistance to antifungal agents. Therefore, there is a great need to search for alternative antifungal agents for these fungi. The search for new molecules is currently occurring from nanoparticle drugs of plant peptide origin. This article presents a brief review of the literature regarding the epidemiology of cryptococcosis, as well as fungal resistance and new alternatives for treatment.

Address: Faculty of Pharmaceutical Sciences of Araraquara, Department of Clinical Analysis, Laboratory of Clinical Mycology, Universidade Estadual Paulista (UNESP), R. Expedicionarios do Brasil, 1621, 14801-902, Araraquara, Sao Paulo, Brazil

3915562e: Clinics(Sao Paulo), 1220-1224(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Thymosin ss4 expression in colorectal polyps and adenomas.
Author: Nemolato,S., Cabras,T., Restivo,A., Zorcolo,L., Di Felice,E., Fanni,D., Gerosa,C., Messana,I., Castagnola,M., Faa,G., Casula,G.
Abstract:
OBJECTIVE: Thymosin β 4 (Tβ4) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation. Recently, a role for Tβ4 has been proposed in experimental and human carcinogenesis, including gastrointestinal cancer. This study was aimed at evaluating the relationship between Tβ4 immunoreactivity and the initial steps of carcinogenesis. METHODS: In total, 60 intestinal biopsies, including 10 hyperplastic polyps, 10 sessile serrated adenomas/polyps, 15 colorectal adenomas with low-grade dysplasia, 15 adenomas with high-grade dysplasia, 15 adenocarcinomas and 10 samples of normal colon mucosa, were analyzed for Tβ4 expression by immunohistochemistry. RESULTS: Weak cytoplasmic reactivity for Tβ4 was detected in the normal colon mucosa. No reactivity for Tβ4 was found in hyperplastic and sessile serrated polyps/adenomas. Tβ4 expression was observed in 10/15 colorectal adenocarcinomas. In adenomas with low-grade dysplasia, Tβ4 immunoreactivity was mainly detected in dysplastic glands but was absent in hyperplastic glands. Tβ4 immunoreactivity was characterized by spot-like perinuclear staining. In high-grade dysplastic polyps, immunostaining for Tβ4 appeared diffuse throughout the entire cytoplasm of dysplastic cells. Spot-like perinuclear reactivity was detected in adenocarcinoma tumor cells. CONCLUSIONS: Our study shows for the first time that Tβ4 is expressed during different steps of colon carcinogenesis. The shift of Tβ4 immunolocalization from low-grade to high-grade dysplastic glands suggests a role for Tβ4 in colorectal carcinogenesis. However, the real meaning of Tβ4 reactivity in dysplastic intestinal epithelium remains unknown.

Address: Department of Surgery, University of Cagliari, Cagliari, Italy

3915569e: BMC Anesthesiol., 33-33(2013)
Link: [ PubMED ] [ DOI ]
Title: Xylazine-induced reduction of tissue sensitivity to insulin leads to acute hyperglycemia in diabetic and normoglycemic monkeys.
Author: Xiao,Y.F., Wang,B., Wang,X., Du,F., Benzinou,M., Wang,Y.X.
Abstract:
BACKGROUND: The α2-adrenoceptor agonist xylazine as an anesthetic has been widely used either alone or in combination with other anesthetics, such as ketamine, in veterinary clinic and research. In the last decade xylazine has been used in drug abusers in certain geographic area. This study investigated the effects of xylazine on blood glucose level and insulin secretion in normoglycemic and insulin-dependent diabetic monkeys. METHODS: Both adult cynomolgus (n = 10) and rhesus (n = 8) monkeys with either sex were used in the study. Xylazine (1--2 mg/kg) was administrated intramuscularly. Blood glucose, insulin, glucagon and glucagon-like peptide 1 in overnight-fasted monkeys were measured immediately before and after xylazine administration. The hyperinsulinemic-euglycemic clamp method was used in the study for assessing the potential mechanism of xylazine-induced hyperglycemia. RESULTS: Xylazine administration increased the blood glucose levels from 58 +/- 3 to 108 +/- 12 mg/dL in normoglycemic (n = 5, p < 0.01) and from 158 +/- 9 to 221 +/- 13 mg/dL in insulin-dependent diabetic (n = 5, p < 0.01) monkeys and was not accompanied by any significant changes in blood insulin, glucagon, and glucagon-like peptide-1. Xylazine-induced hyperglycemia occurred within 10 min and reached the peak at 35 min after injection. Xylazine-induced hyperglycemia declined slowly in diabetic animals. The α2-adrenoceptor antagonist yohimbine was administrated to bring down the elevated glucose level to the pre-xylazine one in 4 out of 5 diabetic animals. To assess the potential mechanism, the hyperinsulinemic-euglycemic clamp was used to maintain a nearly saturated and constant insulin level for minimizing endogenous insulin glucoregulation. Xylazine administration decreased glucose infusion rate, from 14.3 +/- 1.4 to 8.3 +/- 0.8 mg/min/kg (n = 6, p < 0.01) in normoglycemic rhesus monkeys, which indicates that the glucose metabolic rate (M rate) was decreased by xylazine. In addition, after clamping blood glucose level in a range of 55 to 75 mg/dL for 40 min with constant glucose infusion, xylazine administration still increased blood glucose concentration. CONCLUSIONS: We conclude that xylazine administration induces hyperglycemia in normoglycemic and insulin-dependent diabetic monkeys potentially via stimulation of α2-adrenoceptors and then reducing tissue sensitivity to insulin and glucose uptake.

3915570e: Anesthesiology, 687-697(2013)
Link: [ PubMED ] [ DOI ]
Title: Glycemia-dependent nuclear factor κ-B activation contributes to mechanical allodynia in rats with chronic postischemia pain.
Author: Ross-Huot,M.C., Laferriere,A., Khorashadi,M., Coderre,T.J.
Abstract:
BACKGROUND: Ischemia-reperfusion injury causes chronic postischemia pain (CPIP), and rats with higher glycemia during ischemia-reperfusion injury exhibit increased allodynia. Glycemia-induced elevation of nuclear factor κB (NFκB) may contribute to increased allodynia. METHODS: Glycemia during a 3-h ischemia-reperfusion injury was manipulated by: normal feeding; or normal feeding with administration of insulin; dextrose; or insulin/dextrose. In these groups, NFκB was measured in ipsilateral hind paw muscle and spinal dorsal horn by enzyme-linked immunosorbent assay (ELISA), and SN50, an NFκB inhibitor, was administered to determine its differential antiallodynic effects depending on glycemia. RESULTS: CPIP fed/insulin rats (12.03 +/- 4.9 g, N = 6) had less allodynia than fed, fed/insulin/dextrose, and fed/dextrose rats (6.29 +/- 3.37 g, N = 7; 4.57 +/- 3.03 g, N = 6; 2.95 +/- 1.10 g, N = 9), respectively. Compared with fed rats (0.209 +/- 0.022 AU, N = 7), NFκB in ipsilateral plantar muscles was significantly lower for fed/insulin rats, and significantly higher for fed/dextrose rats (0.152 +/- 0.053 AU, N = 6; 0.240 +/- 0.057 AU, N = 7, respectively). Furthermore, NFκB in the dorsal horn of fed, fed/insulin/dextrose, and fed/dextrose rats (0.293 +/- 0.049 AU; 0.267 +/- 0.037 AU; 0.315 +/- 0.015 AU, respectively, N = 6 for each) was significantly higher than in fed/insulin animals (0.267 +/- 0.037 AU, N = 6). The antiallodynic SN50 dose-response curves of CPIP rats in the fed/insulin/dextrose, fed/dextrose, and fed conditions exhibited a rightward shift compared with the fed/insulin group. The threshold SN50 dose of CPIP fed/dextrose, fed/insulin/dextrose, and fed rats (328.94 +/- 92.4 ng, 77.80 +/- 44.50 ng, and 24.89 +/- 17.20 ng, respectively) was higher than that for fed/insulin rats (4.06 +/- 7.04 ng). CONCLUSIONS: NFκB was activated in a glycemia-dependent manner in CPIP rats. Hypoglycemic rats were more sensitive to SN50 than rats with higher glycemia. The finding that SN50 reduces mechanical allodynia suggests that NFκB inhibitors might be useful for treating postischemia pain.

Address: Department of Anesthesia, McGill University, Montreal, Quebec, Canada

3915571e: Anesthesiology, 552-561(2013)
Link: [ PubMED ] [ DOI ]
Title: Oxytocin pretreatment attenuates oxytocin-induced contractions in human myometrium in vitro.
Author: Balki,M., Erik-Soussi,M., Kingdom,J., Carvalho,J.C.
Abstract:
BACKGROUND: Oxytocin receptor desensitization has been shown to occur in humans at biomolecular level and in isolated rat myometrium; however, its effect on human myometrial contractility has not been demonstrated. The objective of this in vitro study was to investigate the contractile response of human pregnant myometrium to oxytocin after pretreatment with different concentrations of oxytocin for variable durations. METHODS: Myometrial samples were obtained from 62 women undergoing elective cesarean deliveries under regional anesthesia. The strips were pretreated with oxytocin 10(-10), 10(-8), 10(-5) M, or physiological salt solution (control) for 2, 4, 6, or 12 h, followed by a dose-response testing with oxytocin 10(-10) to 10(-5) M. Amplitude and frequency of contractions, motility index, and area under the curve during the dose-response period were recorded, analyzed with linear regression models, and compared among groups. RESULTS: Pretreatment with oxytocin 10(-5) and 10(-8) M significantly reduced motility index (estimate [standard error]: -0.771 [0.270] square root units, P = 0.005 and -0.697 [0.293], P = 0.02, respectively) and area under the curve (-3.947 [1.909], P = 0.04 and -4.241 [2.189], P = 0.05, respectively) compared with control group, whereas pretreatment with oxytocin 10-(10) M did not significantly attenuate contractions. Increase in duration of oxytocin pretreatment from 2 to 12 h significantly decreased amplitude (type 3 generalized estimating equation analysis: chi-square = 14.0; df = 3; P = 0.003), motility index (chi-square = 9.3; df = 3; P = 0.03), and area under the curve (chi-square = 10.5; df = 3; P = 0.02), but not the frequency of oxytocin-induced contractions. CONCLUSION: Pretreatment with oxytocin decreases oxytocin-induced myometrial contractions in a concentration and time-dependent manner, likely as a function of the oxytocin receptor desensitization phenomenon.

E-Mail: mrinalini.balki@uhn.ca
Address: Department of Anesthesia and Pain Management, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Room 19-104, Toronto, Ontario M5G 1X5, Canada

3915572e: Anesthesiology, 541-551(2013)
Link: [ PubMED ] [ DOI ]
Title: Changes in blood pressure and cardiac output during Cesarean delivery: the effects of oxytocin and carbetocin compared with placebo.
Author: Rosseland,L.A., Hauge,T.H., Grindheim,G., Stubhaug,A., Langesaeter,E.
Abstract:
BACKGROUND: Little is known about maternal hemodynamics after Cesarean delivery. Uterine contractions may increase cardiac output. Oxytocin is the first-line treatment for uterine atony, although the effects of the long-acting oxytocin analogue carbetocin are comparable with that of oxytocin. The authors analyzed the effects of i.v. oxytocin 5 U, carbetocin 100 μg, and placebo on hemodynamics, uterine tone, adverse events, and blood loss after Cesarean delivery. METHODS: This was a randomized, double-blinded, placebocontrolled, parallel-group comparison of carbetocin and oxytocin after elective Cesarean delivery of singletons under spinal anesthesia (n = 76). Continuously measured invasive systolic arterial pressure was the primary outcome measure. RESULTS: The mean systolic arterial pressure decrease was 28 mmHg (95% CI, 22-34) after oxytocin and 26 mmHg (95% CI, 20-31) after carbetocin. The decrease was greatest after 80 (95% CI, 71-89) and 63 s (95% CI, 55-72), respectively (P = 0.006). The differences were nearly undetectable after 2.5 min, although the effect of carbetocin was slightly greater than placebo (P < 0.001). The group differences in systolic arterial pressure decreased over 5 min and were gone at 1 h. Heart rate and cardiac output increased in all three groups. Stroke volume increased after oxytocin and carbetocin but was unchanged after placebo. CONCLUSIONS: The hemodynamic side effects of oxytocin 5 U and carbetocin 100 μg were comparable. The lack of an increase in stroke volume in the placebo group challenges the theory that uterine contraction causes autotransfusion of uterine blood, leading to an increase in preload.

E-Mail: l.a.rosseland@medisin.uio.no
Address: Faculty of Medicine, Institute of Clinical Medicine, University of Oslo, Oslo, Norway

3915574e: Bull.Exp.Biol.Med., 324-326(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of regulatory peptides on the stress-induced changes of lipid metabolism in experimental animals.
Author: Solin,A.V., Korozin,V.I., Lyashev,Y.D.
Abstract:
Experiments on rats showed that restraint stress is associated with an increase in plasma level of nonesterified fatty acids, total cholesterol, triglycerides, VLDL, and LDL. Administration of opioid peptides DSLET and DAGO alleviated stress-induced shifts in lipid metabolism. The concentrations of nonesterified fatty acids, total cholesterol, and triglycerides decreased and HDL content increased under these conditions. Treatment with dynorphin A (1-13) prevented a significant increase in the concentration of nonesterified fatty acids in blood plasma, but did not affect the content of triglycerides and total cholesterol. Hepatocyte growth factor had minor influence on the analyzed parameters. The observed effects can be related to the stress-limiting effect of opioids, in particular, attenuation of catecholamine influence on the lipid tissue and generation of LPO products that inhibit cholesterol-degrading enzyme.

E-Mail: medps@yandex.ru
Address: Department of Patophysiology, Kursk State Medical University, Ministry of Health and Social Development of the Russian Federation, Russia

3915576e: Oncol.Lett., 407-414(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Atrial natriuretic peptide modulates the proliferation of human gastric cancer cells via KCNQ1 expression.
Author: Zhang,J., Zhao,Z., Zu,C., Hu,H., Shen,H., Zhang,M., Wang,J.
Abstract:
Atrial natriuretic peptide (ANP) and brain NP (BNP) belong to the NP family that regulates mammalian blood volume and blood pressure. ANP signaling through NP receptor A (NPR-A)/cyclic guanosine 3'5'-monophosphate (cGMP)/ cGMP-dependent protein kinase (PKG) activates various downstream effectors involved in cell growth, apoptosis, proliferation and inflammation. Evidence has shown the critical role of plasma K+ channels in the regulation of tumor cell proliferation. However, the role of ANP in the proliferation of gastric cancer cells is not clear. In the present study, the expression of NPR-A in the human gastric cancer cell line, AGS, and the effect of ANP on the proliferation of AGS cells were investigated using western blotting, immunofluorescence, qPCR and patch clamp assays. The K+ current was also analyzed in the effect of ANP on the proliferation of AGS cells. NPR-A was expressed in the human gastric cancer AGS cell line. Lower concentrations of ANP promoted the proliferation of the AGS cells, although higher concentrations decreased their proliferation. Significant increases in the levels of cGMP activity were observed in the AGS cells treated with 10-10, 10-9 and 10-8 M ANP compared with the controls, but no significant differences were observed in the 10-7 and 10-6 M ANP groups. The patch clamp results showed that 10-9 M ANP significantly increased the tetraethylammonium (TEA)- and 293B-sensitive K+ current, while 10-6 M ANP significantly decreased the TEA- and 293B-sensitive K+ current. The results showed that 10-10 and 10-9 M ANP significantly upregulated the expression of potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) at the protein and mRNA levels, although 10-7 and 10-6 M ANP significantly downregulated the expression of KCNQ1. The data indicated that lower and higher concentrations of ANP have opposite effects on the proliferation of AGS cells through cGMP-dependent or -independent pathways. KCNQ1 upregulation and downregulation by lower and higher concentrations of ANP, respectively, have separate effects on the promotion and inhibition of proliferation.

Address: Department of Surgical Oncology, First Affiliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China

3915579e: Exp.Ther.Med., 995-999(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Role of the NT-proBNP level in the diagnosis of pediatric heart failure and investigation of novel combined diagnostic criteria.
Author: Lin,C.W., Zeng,X.L., Jiang,S.H., Wu,T., Wang,J.P., Zhang,J.F., Ou,Y.H.
Abstract:
The aim of this study was to investigate the modified Ross criteria score and the diagnostic cut-off level for plasmatic amino-terminal pro-brain natriuretic peptide (NT-proBNP) in the diagnosis of pediatric heart failure, by analyzing the receiver operating characteristic (ROC) curve. The plasma NT-proBNP level was measured in 80 children diagnosed with heart failure according to the modified Ross criteria, 80 children with non-cardiogenic dyspnea and 80 healthy children. The NT-proBNP levels were then compared using an F-test. The cut-off score for heart failure in the modified Ross criteria and the diagnostic cut-off level for plasmatic NT-proBNP in pediatric heart failure were determined by ROC curve analysis. The results demonstrated that the NT-proBNP level was markedly increased in 76 of the 80 children with heart failure, and the correlation with the modified Ross criteria was 95%. Based on ROC curve analysis, the diagnosis of pediatric heart failure was most accurate when the modified Ross criteria score was >/=4 and the plasmatic NT-proBNP level was >/=598 ng/l. The NT-proBNP level was normal (0-300 ng/l) in the children with non-cardiogenic dyspnea and the healthy children. Significant differences were observed in the comparison of the three groups (P<0.01). In conclusion, a NT-proBNP level of >/=598 ng/l, combined with a modified Ross criteria score >/=4, is highly diagnostic of heart failure in children.

Address: Department of Pediatrics, The Women and Children's Health Care Hospital of Shunde, Shunde, Foshan, Guangdong 528300, P.R. China

3915580e: Exp.Ther.Med., 786-790(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Conjugates of cytochrome and antennapedia peptide activate apoptosis and inhibit proliferation of HeLa cancer cells.
Author: Imesch,P., Scheiner,D., Szabo,E., Fink,D., Fedier,A.
Abstract:
Polycationic cell-penetrating peptides (CPPs) deliver macromolecules into cells without losing the functional properties of the cargoed macromolecule. The aim of this study was to determine whether exogenous cytochrome c is delivered to HeLa cervical carcinoma cells by the CPP antennapedia (Antp) and activates apoptosis. HeLa cervical carcinoma cells were treated with conjugated Antp-SMCC-cytochrome c (cytochrome c chemically conjugated to Antp) or with non-conjugated Antp and cytochrome c. Sensitivity to the treatments was determined by the clonogenic assay (proliferation) and by immunoblot analysis (apoptosis activation). We report that conjugated Antp-SMCC-cytochrome c activated apoptosis in HeLa cells as demonstrated by poly (ADP-ribose) polymerase 1 (PARP-1) cleavage and inhibited their proliferation. The Antp-SMCC-cytochrome c-induced apoptosis was inhibited by z-VAD-fmk, a pan-caspase inhibitor peptide. Unconjugated Antp or cytochrome c demonstrated no inhibitory effect on survival and proliferation. Our results suggest that chemical coupling of cytochrome c to CPPs may present a possible strategy for delivering cytochrome c into cells and for activating apoptosis.

Address: Department of Gynecology, University Hospital Zurich, Zurich CH-8091, Switzerland

3915581e: Exp.Ther.Med., 727-730(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Application of adenosine stress echocardiography in the prognosis of acute myocardial infarction following percutaneous coronary interventional therapy.
Author: Ren,L., Liu,Y., Lin,J., Ye,H., Wang,P., Liu,Y.
Abstract:
The aim of this study was to investigate the application of adenosine stress echocardiography (ASE) in the prognosis of acute myocardial infarction (AMI) following percutaneous coronary intervention (PCI). A total of 79 patients with AMI who underwent PCI were selected for the study. ASE testing was performed within one week following the PCI. Subsequent to the ASE, the patients with >/=5% increases in the left ventricular ejection fraction (LVEF) levels were included in the improved LVEF group, while patients with <5% increases in LVEF levels were included in the unimproved LVEF group. A follow-up study was performed during the 24 months subsequent to the ASE. The incidence of major adverse cardiovascular events (MACEs) was observed and compared between the two groups and logistic regression analysis was applied to identify the risk factors for clinical prognosis. There were no significant differences in Killip classification, LVEF, left ventricular end-diastolic diameter or blood plasma B-type natriuretic peptide concentration between the two groups following PCI. The incidence of MACEs in the improved LVEF group was significantly lower than that in unimproved LVEF group (14.29 versus 43.24%, respectively; P<0.05). Logistic regression analysis identified LVEF increases of <5% and segment improvements of </=3 as the risk factors for the clinical prognosis of AMI following treatment with PCI. Therefore, ASE is an effective method of assessing the clinical effect of PCI treatment, which may be utilized to predict the incidence of MACEs following PCI.

Address: Department of Cardiology, Beijing Shijitan Hospital Affiliated to Capital Medical University, Beijing 100038, P.R. China

3915582e: Exp.Ther.Med., 715-720(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Correlation between gastrointestinal hormones and anxiety-depressive states in irritable bowel syndrome.
Author: Han,B.
Abstract:
The aim of this study was to investigate the mechanism(s) of action of gastrointestinal hormones in the pathogenesis of irritable bowel syndrome (IBS), and the correlation between gastrointestinal hormones and psychological factors. Patients with IBS were divided into IBS with normal emotional state ratings and IBS in anxiety-depressive states groups. The two groups were then subdivided into IBS-constipation predominant (IBS-C) and IBS-diarrhea predominant (IBS-D) groups. Non-IBS patients with normal depression and anxiety ratings were recruited as controls. The serum concentrations of somatostatin (SS) and vasoactive intestinal peptide (VIP) were measured by radioimmunoassay, and the expression of SS and VIP in the colonic mucosa was detected by immunohistochemistry and radioimmunoassay. The anxiety-depression scores of patients with IBS were significantly different from those of the control group (P<0.05). The expression levels of SS and VIP in the serum and colonic mucosa of the patients with IBS were higher compared with those of the control group. Furthermore, the expression level of SS in the IBS-C group demonstrated a significantly larger increase than that in the IBS-D group (P<0.05); however, there was no significant difference in the expression of VIP between the IBS-C and IBS-D groups (P>0.05). In addition, the expression levels of SS and VIP in the IBS groups with normal emotional state ratings were notably different from those in the IBS groups in anxiety-depressive states (P<0.05). Anxiety-depressive states may lead to changes in the secretion of SS and VIP, and subsequently to changes in gastrointestinal motility and function.

Address: Department of Gastroenterology, Xingtai People's Hospital, Xingtai, Hebei 054031, P.R. China

3915583e: Exp.Ther.Med., 679-683(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Expression of the nociceptin/orphanin FQ receptor in the intestinal mucosa of IBS patients.
Author: Li,L., Dong,L., Wang,S.
Abstract:
Nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide (NOP) receptor play important roles in regulating gastrointestinal function. To assess whether the NOP receptor is implicated in the etiopathogenesis of irritable bowel syndrome (IBS), we measured the levels of NOP receptor mRNA and protein in the jejunal and colonic tissues of healthy subjects and patients with diarrhea-predominant IBS (D-IBS) and constipation-predominant IBS (C-IBS). Mucosal biopsies were obtained from the jejunum and colon of patients diagnosed with D-IBS and C-IBS by the Rome III criteria and from healthy control subjects. The expression of NOP receptor mRNA was measured quantitatively using quantitative PCR (qPCR) and NOP protein expression was assayed immunohistochemically using a rabbit monoclonal antibody to OFQ. NOP receptor mRNA was detected in the jejunum and colon of healthy subjects and was more highly expressed in the jejunum than in the colon. Expression was lower in the jejunum and colon of patients with D-IBS; however, it was similar in patients with C-IBS and healthy subjects. The numbers of OFQ-positive cells in the jejunum and colon were similar among the three groups. The NOP receptor may be involved in the regulation of intestinal movement in healthy individuals. Its involvement in the pathophysiology of IBS may depend on whether the IBS is constipation- or diarrhea-predominant.

Address: Department of Gastroenterology, The Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China

3915587e: Einstein(Sao Paulo), 338-344(2013)
Link: [ PubMED ]
Title: Assessing prognosis of pulmonary embolism using tissue-Doppler echocardiography and brain natriuretic peptide.
Author: Rodrigues,A.C., Cordovil,A., Monaco,C.G., Guimaraes,L.A., Oliveira,W.A., Fischer,C.H., Lira-Filho,E.B., Vieira,M.L., Morhy,S.S.
Abstract:
OBJECTIVE: To assess prognosis of pulmonary thromboembolism using tissue Doppler echocardiography and brain natriuretic peptide. METHODS: Patients aged over 18 years were evaluated within 24 hours of confirmed diagnosis (chest tomography/pulmonary scintigraphy) of pulmonary embolism using two-dimensional echocardiography and tissue Doppler for right ventricular systolic (s') velocities, strain, tissue tracking and myocardial performance index. Plasma brain natriuretic peptide was also obtained within 24 hour. The influence of echocardiographic and clinical variables on mortality was examined (up to 12 months) using Cox regression analysis. RESULTS: Out of 118 patients, 100 patients were included in the study (60 males, aged 55+/-17 years). Right ventricular dysfunction was observed in 28% using two-dimensional echocardiography. Tissue Doppler right ventricular variables (s' velocities, tissue tracking and strain) were decreased only for patients with right ventricular dysfunction, whereas myocardial performance index and systolic pulmonary artery pressure were increased. Mean brain natriuretic peptide value was 66+/-111pg/mL, also increased in patients with right ventricular dysfunction (136+/-146pg/mL). Mortality was 11% and related to age, malignancy and brain natriuretic peptide levels. The only echocardiographic variables capable of predicting events by univariate analysis were pulmonary pressure and right ventricular s' velocity. However, multivariate analysis showed only malignancy to predict mortality in this group. CONCLUSION: Lower tissue Doppler systolic velocities and elevated brain natriuretic peptide levels are associated with poorer prognosis in patients with pulmonary thromboembolism; but only malignancy emerged as an independent predictor of mortality.

3915588e: Einstein(Sao Paulo), 273-277(2013)
Link: [ PubMED ]
Title: Agreement of BNP and NT-proBNP and the influence of clinical and laboratory variables.
Author: Curiati,M.N., Silvestre,O.M., Pires,L.J., Mangini,S., Pires,P.V., Gaiotto,F.A., Laurino,A.M., Pego-Fernandes,P.M., Ferreira,C.E., Bacal,F.
Abstract:
OBJECTIVE: To correlate the serum levels of B type natriuretic peptide and the N-terminal fraction of the pro-B type natriuretic peptide, as well as to analyze the influence of age, obesity, renal failure, left ventricle ejection fraction, diastolic dysfunction, and anemia on serum levels of both markers. METHODS: An observational study in which the agreement was compared between these markers in consecutive samples of 138 patients. For the correlation, Pearson's test was used, and p<0.05 was considered statistically significant. RESULTS: A linear association was observed between the B type natriuretic peptide and N-terminal fraction of the pro-B type natriuretic peptide (r=0.907; p<0.001). When evaluating the categorized measurements as normal and altered, there was good agreement, with 90.6% of agreement classifications (p<0.001) in which altered values of the N-terminal fraction of the pro-B type natriuretic peptide and normal values of the B type natriuretic peptide represented 8.7% of the total; the opposite situation represented 1% of the total. Assessment of the influence of the clinical and laboratorial factors on the levels of natriuretic peptides showed that they rise according to age, but that they fall as the ejection fraction increases. Patients with anemia (p<0.001) or with renal failure (p=0.007) had higher values of both markers. There was no association between obesity and the B type natriuretic peptide. CONCLUSION: There was satisfactory agreement between the B type natriuretic peptide and the N-terminal fraction of the pro-B type natriuretic peptide. Age, creatinine levels, and hemoglobin, as well as ventricular function, influence the serum levels of both natriuretic peptides.

3915591e: PLoS One, e72301-e72301(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Efficacy and safety of a liposome-based vaccine against protein Tau, assessed in tau.P301L mice that model tauopathy.
Author: Theunis,C., Crespo-Biel,N., Gafner,V., Pihlgren,M., Lopez-Deber,M.P., Reis,P., Hickman,D.T., Adolfsson,O., Chuard,N., Ndao,D.M., Borghgraef,P., Devijver,H., Van Leuven,F., Pfeifer,A., Muhs,A.
Abstract:
Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimer's disease.

Address: Experimental Genetics Group, Department Human Genetics, KU Leuven, Leuven, Belgium

3915594e: PLoS One, e72067-e72067(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Uroplakin peptide-specific autoimmunity initiates interstitial cystitis/painful bladder syndrome in mice.
Author: Izgi,K., Altuntas,C.Z., Bicer,F., Ozer,A., Sakalar,C., Li,X., Tuohy,V.K., Daneshgari,F.
Abstract:
The pathophysiology of interstitial cystitis/painful bladder syndrome (IC/PBS) is enigmatic. Autoimmunity and impaired urothelium might lead the underlying pathology. A major shortcoming in IC/PBS research has been the lack of an appropriate animal model. In this study, we show that the bladder specific uroplakin 3A-derived immunogenic peptide UPK3A 65-84, which contains the binding motif for IA(d) MHC class II molecules expressed in BALB/c mice, is capable of inducing experimental autoimmune cystitis in female mice of that strain. A highly antigen-specific recall proliferative response of lymph node cells to UPK3A 65-84 was observed, characterized by selectively activated CD4+ T cells with a proinflammatory Th1-like phenotype, including enhanced production of interferon γ and interleukin-2. T cell infiltration of the bladder and bladder-specific increased gene expression of inflammatory cytokines were observed. Either active immunization with UPK3A 65-84 or adoptive transfer of peptide-activated CD4+ T cells induced all of the predominant IC/PBS phenotypic characteristics, including increased micturition frequency, decreased urine output per micturition, and increased pelvic pain responses to stimulation with von Frey filaments. Our study demonstrates the creation of a more specific experimental autoimmune cystitis model that is the first inducible model for IC/PBS that manifests all of the major symptoms of this debilitating condition.

Address: Department of Urology, Case Western Reserve University, Cleveland, Ohio, United States of America

3915598e: PLoS One, e71549-e71549(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Induction of encephalitis in rhesus monkeys infused with lymphocryptovirus-infected B-cells presenting MOG(34-56) peptide.
Author: Haanstra,K.G., Wubben,J.A., Jonker,M., 't Hart,B.A.
Abstract:
The overlapping epidemiology of multiple sclerosis (MS) and Epstein-Barr virus (EBV), the increased risk to develop MS after infectious mononucleosis (IM) and the localization of EBV-infected B-cells within the MS brain suggest a causal link between EBV and MS. However, the underlying mechanism is unknown. We hypothesize that EBV-infected B-cells are capable of eliciting a central nervous system (CNS) targeting autoimmune reaction. To test this hypothesis we have developed a novel experimental model in rhesus monkeys of IM-like disease induced by infusing autologous B-lymphoblastoid cells (B-LCL). Herpesvirus papio (HVP) is a lymphocryptovirus related to EBV and was used to generate rhesus monkey B-LCL. Three groups of five animals were included; each group received three intravenous infusions of B-LCL that were either pulsed with the encephalitogenic self peptide MOG(34-56) (group A), a mimicry peptide (981-1003) of the major capsid protein of cytomegalovirus (CMVmcp(981-1003); group B) or the citrullinated MOG(34-56) (cMOG(34-56); group C). Groups A and B received on day 98 a single immunization with MOG(34-56) in incomplete Freund's adjuvant (IFA). Group C monkeys were euthanized just prior to day 98 without booster immunization. We observed self-peptide-specific proliferation of T-cells, superimposed on similar strong proliferation of CD3+CD8+ T-cells against the B-LCL as observed in IM. The brains of several monkeys contained perivascular inflammatory lesions of variable size, comprising CD3+ and CD68+ cells. Moreover, clusters of CD3+ and CD20+ cells were detected in the meninges. The only evident clinical sign was substantial loss of bodyweight (>15%), a symptom observed both in early autoimmune encephalitis and IM. In conclusion, this model suggests that EBV-induced B-LCL can elicit a CNS targeting inflammatory (auto)immune reaction.

Address: Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands

3915601e: PLoS One, e71284-e71284(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Ghrelin receptor (GHS-R1A) antagonism suppresses both alcohol consumption and the alcohol deprivation effect in rats following long-term voluntary alcohol consumption.
Author: Suchankova,P., Steensland,P., Fredriksson,I., Engel,J.A., Jerlhag,E.
Abstract:
Alcohol dependence is a heterogeneous disorder where several signalling systems play important roles. Recent studies implicate that the gut-brain hormone ghrelin, an orexigenic peptide, is a potential mediator of alcohol related behaviours. Ghrelin increases whereas a ghrelin receptor (GHS-R1A) antagonist decreases alcohol consumption as well as operant self-administration of alcohol in rodents that have consumed alcohol for twelve weeks. In the present study we aimed at investigating the effect of acute and repeated treatment with the GHS-R1A antagonist JMV2959 on alcohol intake in a group of rats following voluntarily alcohol consumption for two, five and eight months. After approximately ten months of voluntary alcohol consumption the expression of the GHS-R1A gene (Ghsr) as well as the degree of methylation of a CpG island found in Ghsr was examined in reward related brain areas. In a separate group of rats, we examined the effect of the JMV2959 on alcohol relapse using the alcohol deprivation paradigm. Acute JMV2959 treatment was found to decrease alcohol intake and the effect was more pronounced after five, compared to two months of alcohol exposure. In addition, repeated JMV2959 treatment decreased alcohol intake without inducing tolerance or rebound increase in alcohol intake after the treatment. The GHS-R1A antagonist prevented the alcohol deprivation effect in rats. There was a significant down-regulation of the Ghsr expression in the ventral tegmental area (VTA) in high- compared to low-alcohol consuming rats after approximately ten months of voluntary alcohol consumption. Further analysis revealed a negative correlation between Ghsr expression in the VTA and alcohol intake. No differences in methylation degree were found between high- compared to low-alcohol consuming rats. These findings support previous studies showing that the ghrelin signalling system may constitute a potential target for development of novel treatment strategies for alcohol dependence.

Address: Institute of Neuroscience and Physiology, Department of Pharmacology, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden

3915606e: Diabetes Metab.Syndr.Obes., 293-301(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Plasminogen activator inhibitor-1, free fatty acids, and insulin resistance in patients with myocardial infarction.
Author: Gruzdeva,O., Uchasova,E., Dyleva,Y., Belik,E., Shurygina,E., Barbarash,O.
Abstract:
BACKGROUND: Insulin resistance is known to be a common feature of type 2 diabetes mellitus and is regarded as an important mechanism in the pathogenesis of this disease. The key pathogenetic mechanisms of insulin resistance progression are free fatty acids metabolism impairment and enhanced activity of plasminogen activator inhibitor 1. Both free fatty acids and plasminogen activator inhibitor 1 are recognized as risk factors for coronary heart disease. METHODS: THE PATIENTS WERE DIVIDED INTO TWO GROUPS: group 1 included 65 non-diabetic myocardial infarction patients and group 2 enrolled 60 diabetic myocardial infarction patients. The control group consisted of 30 sex- and age-matched volunteers. The concentration of serum free fatty acids, glucose, C-peptide, and insulin were measured on the 1st and 12th days of the study. All the patients had their postprandial glycemia, insulin, and C-peptide concentrations measured 2 hours after a standard carbohydrate breakfast containing 360 kcal (protein 20 g, carbohydrate 57 g, and fat 9 g). RESULTS: Free fatty acids levels in group 1 and in group 2 exceeded the control group values by 7-fold and 11-fold, respectively. Plasminogen activator inhibitor 1 concentration was 2.5-fold higher in group 1 and 4.6-fold higher in group 2 compared to the control group on the 1st day from the myocardial infarction onset. In addition, plasminogen activator inhibitor 1 concentration was significantly reduced in both groups on the 12th day from the myocardial infarction onset; however, it did not achieve the control group values. CONCLUSION: Increased postprandial glucose level, insulinemia, and elevated levels of free fatty acids and plasminogen activator inhibitor are associated with myocardial infarction-associated progression of insulin resistance. However, insulin resistance metabolic markers are of great predictive capacity in the assessment of risk of acute coronary events.

Address: Research Institute for Complex Issues of Cardiovascular Diseases under the Siberian Branch of the Russian Academy of Medical Sciences, Kemerovo, Russian Federation

3915607e: J.Pharmacol.Exp.Ther., 478-486(2013)
Link: [ PubMED ] [ DOI ]
Title: In Vivo Quantification of Calcitonin Gene-Related Peptide Receptor Occupancy by Telcagepant in Rhesus Monkey and Human Brain Using the Positron Emission Tomography Tracer [11C]MK-4232.
Author: Hostetler,E.D., Joshi,A.D., Sanabria-Bohorquez,S., Fan,H., Zeng,Z., Purcell,M., Gantert,L., Riffel,K., Williams,M., O'Malley,S., Miller,P., Selnick,H.G., Gallicchio,S.N., Bell,I.M., Salvatore,C.A., Kane,S.A., Li,C.C., Hargreaves,R.J., De Groot,T., Bormans,G., Van Hecken,A., Derdelinckx,I., De Hoon,J., Reynders,T., Declercq,R., De Lepeleire,I., Kennedy,W.P., Blanchard,R., Marcantonio,E.E., Sur,C., Cook,J.J., Van Laere,K., Evelhoch,J.L.
Abstract:
Calcitonin gene-related peptide (CGRP) is a potent neuropeptide whose agonist interaction with the CGRP receptor (CGRP-R) in the periphery promotes vasodilation, neurogenic inflammation and trigeminovascular sensory activation. This process is implicated in the cause of migraine headaches, and CGRP-R antagonists in clinical development have proven effective in treating migraine-related pain in humans. CGRP-R is expressed on blood vessel smooth muscle and sensory trigeminal neurons and fibers in the periphery as well as in the central nervous system. However, it is not clear what role the inhibition of central CGRP-R plays in migraine pain relief. To this end, the CGRP-R positron emission tomography (PET) tracer [(11)C]MK-4232 (2-[(8R)-8-(3,5-difluorophenyl)-6,8-[6-(11)C]dimethyl-10-oxo-6,9-diazaspiro[4.5]d ecan-9-yl]-N-[(2R)-2'-oxospiro[1,3-dihydroindene-2,3'-1H-pyrrolo[2,3-b]pyridine]- 5-yl]acetamide) was discovered and developed for use in clinical PET studies. In rhesus monkeys and humans, [(11)C]MK-4232 displayed rapid brain uptake and a regional brain distribution consistent with the known distribution of CGRP-R. Monkey PET studies with [(11)C]MK-4232 after intravenous dosing with CGRP-R antagonists validated the ability of [(11)C]MK-4232 to detect changes in CGRP-R occupancy in proportion to drug plasma concentration. Application of [(11)C]MK-4232 in human PET studies revealed that telcagepant achieved only low receptor occupancy at an efficacious dose (140 mg PO). Therefore, it is unlikely that antagonism of central CGRP-R is required for migraine efficacy. However, it is not known whether high central CGRP-R antagonism may provide additional therapeutic benefit.

Address: Merck Research Laboratories, West Point, Pennsylvania (E.D.H., A.D.J., S.S.-B., H.F., Z.Z., M.P., L.G., K.R., M.W., S.O., P.M. H.G.S., S.N.G., I.M.B., C.A.S., S.A.K., C.-C.L., R.J.H., C.S., J.J.C., J.L.E.)

3915608e: Neurology, 1191-1196(2013)
Link: [ PubMED ] [ DOI ]
Title: Interictal increase of CGRP levels in peripheral blood as a biomarker for chronic migraine.
Author: Cernuda-Morollon,E., Larrosa,D., Ramon,C., Vega,J., Martinez-Camblor,P., Pascual,J.
Abstract:
OBJECTIVE: To determine calcitonin gene-related peptide (CGRP) levels outside migraine attacks in peripheral blood as a potential biomarker for chronic migraine (CM). METHODS: Women older than 17 years and diagnosed with CM were recruited. Matched healthy women with no headache history and women with episodic migraine (EM) served as control groups, together with a series of patients with episodic cluster headache in a pain-free period. CGRP levels were determined in blood samples obtained from the right antecubital vein by ELISA outside a migraine attack and having taken no symptomatic medication the day before. For ethical reasons, preventatives were not stopped. RESULTS: We assessed plasma samples from 103 women with CM, 31 matched healthy women, 43 matched women with EM, and 14 patients with episodic cluster headache matched for age. CGRP levels were significantly increased in CM (74.90 pg/mL) as compared with control healthy women (33.74 pg/mL), women with EM (46.37 pg/mL), and patients with episodic cluster headache (45.87 pg/mL). Thresholds of 43.45 and 58.22 pg/mL optimize the sensitivity and specificity to differentiate CM from healthy controls and EM, respectively. In the CM group, CGRP levels were significantly increased in women with a history of migraine with aura vs those only experiencing migraine without aura. Variables such as age, analgesic overuse, depression, fibromyalgia, vascular risk factors, history of triptan consumption, or kind of preventative treatment did not significantly influence CGRP levels. CONCLUSION: Increased CGRP level measured in peripheral blood outside migraine attacks and in the absence of symptomatic medication could be a biomarker helping in the diagnosis of CM.

Address: From Neuroscience Area, Service of Neurology and Faculty of Medicine, University Hospital Central de Asturias and INEUROPA (E.C.-M, D.L., C.R., J.V., J.P.), and Oficina de Investigacion Biosanitaria OIB (P.M.-C), Oviedo, Asturias, Spain

3915618e: Nature(London), 416-420(2013)
Link: [ PubMED ] [ DOI ]
Title: DNA damage in germ cells induces an innate immune response that triggers systemic stress resistance.
Author: Ermolaeva,M.A., Segref,A., Dakhovnik,A., Ou,H.L., Schneider,J.I., Utermohlen,O., Hoppe,T., Schumacher,B.
Abstract:
DNA damage responses have been well characterized with regard to their cell-autonomous checkpoint functions leading to cell cycle arrest, senescence and apoptosis. In contrast, systemic responses to tissue-specific genome instability remain poorly understood. In adult Caenorhabditis elegans worms germ cells undergo mitotic and meiotic cell divisions, whereas somatic tissues are entirely post-mitotic. Consequently, DNA damage checkpoints function specifically in the germ line, whereas somatic tissues in adult C. elegans are highly radio-resistant. Some DNA repair systems such as global-genome nucleotide excision repair (GG-NER) remove lesions specifically in germ cells. Here we investigated how genome instability in germ cells affects somatic tissues in C. elegans. We show that exogenous and endogenous DNA damage in germ cells evokes elevated resistance to heat and oxidative stress. The somatic stress resistance is mediated by the ERK MAP kinase MPK-1 in germ cells that triggers the induction of putative secreted peptides associated with innate immunity. The innate immune response leads to activation of the ubiquitin-proteasome system (UPS) in somatic tissues, which confers enhanced proteostasis and systemic stress resistance. We propose that elevated systemic stress resistance promotes endurance of somatic tissues to allow delay of progeny production when germ cells are genomically compromised.

Address: Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases, Institute for Genetics, University of Cologne, Zulpicher Strasse 47a, 50674 Cologne, Germany

3915619e: Nat.Neurosci., 1436-1444(2013)
Link: [ PubMED ] [ DOI ]
Title: GluA1 phosphorylation at serine 831 in the lateral amygdala is required for fear renewal.
Author: Lee,S., Song,B., Kim,J., Park,K., Hong,I., An,B., Song,S., Lee,J., Park,S., Kim,J., Park,D., Lee,C.J., Kim,K., Shin,K.S., Tsien,R.W., Choi,S.
Abstract:
Fear renewal, a widely pursued model of post-traumatic stress disorder and phobias, refers to the context-specific relapse of conditioned fear after extinction. However, its molecular mechanisms are largely unknown. We found that renewal-inducing stimuli, generally believed to be insufficient to induce synaptic plasticity, enhanced excitatory synaptic strength, activity of synaptic GluA2-lacking AMPA receptors and Ser831 phosphorylation of synaptic surface GluA1 in the lateral nucleus of the amygdala (LAn) of fear-extinguished rats. Consistently, the induction threshold for LAn synaptic potentiation was considerably lowered after extinction, and renewal occluded this low-threshold potentiation. The low-threshold potentiation (a potential cellular substrate for renewal), but not long-term potentiation, was attenuated by dialysis into LAn neurons of a GluA1-derived peptide that competes with Ser831-phosphorylated GluA1. Microinjections of the same peptide into the LAn attenuated fear renewal, but not fear learning. Our findings suggest that GluA1 phosphorylation constitutes a promising target for clinical treatment of aberrant fear-related disorders.

Address: 1] School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea. [2]

3915625e: Inflamm.Res., 991-1001(2013)
Link: [ PubMED ] [ DOI ]
Title: Vimentin is a target of PKCβ phosphorylation in MCP-1-activated primary human monocytes.
Author: Thiagarajan,P.S., Akbasli,A.C., Kinter,M.T., Willard,B., Cathcart,M.K.
Abstract:
OBJECTIVE AND DESIGN: We designed a study to detect downstream phosphorylation targets of PKCβ in MCP-1-induced human monocytes. METHODS: Two-dimensional gel electrophoresis was performed for monocytes treated with MCP-1 in the presence or absence of PKCβ antisense oligodeoxyribonucleotides (AS-ODN) or a PKCβ inhibitor peptide, followed by phospho- and total protein staining. Proteins that stained less intensely with the phospho-stain, when normalized to the total protein stain, in the presence of PKCβ AS-ODN or the PKCβ inhibitor peptide, were sequenced. RESULTS: Of the proteins identified, vimentin was consistently identified using both experimental approaches. Upon 32P-labeling and vimentin immunoprecipitation, increased phosphorylation of vimentin was observed in MCP-1 treated monocytes as compared to the untreated monocytes. Both PKCβ AS-ODN and the PKCβ inhibitor reduced MCP-1-induced vimentin phosphorylation. The IP of monocytes with anti-vimentin antibody and immunoblotting with a PKCβ antibody revealed that increased PKCβ becomes associated with vimentin upon MCP-1 activation. Upon MCP-1 treatment, monocytes were shown to secrete vimentin and secretion depended on PKCβ expression and activity. CONCLUSIONS: We conclude that vimentin, a major intermediate filament protein, is a phosphorylation target of PKCβ in MCP-1-treated monocytes and that PKCβ phosphorylation is essential for vimentin secretion. Our recently published studies have implicated vimentin as a potent stimulator of the innate immune receptor Dectin-1 as reported by Thiagarajan et al. (Cardiovasc Res 99:494-504, 2013). Taken together our findings suggest that inhibition of PKCβ regulates vimentin secretion and, thereby, its interaction with Dectin-1 and downstream stimulation of superoxide anion production. Thus, PKCβ phosphorylation of vimentin likely plays an important role in propagating inflammatory responses.

Address: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH, 44195, USA

3915630e: Peptides, 1-8(2013)
Link: [ PubMED ] [ DOI ]
Title: Apelin and copeptin: Two opposite biomarkers associated with kidney function decline and cyst growth in autosomal dominant polycystic kidney disease.
Author: Lacquaniti,A., Chirico,V., Lupica,R., Buemi,A., Loddo,S., Caccamo,C., Salis,P., Bertani,T., Buemi,M.
Abstract:
Vasopressin (AVP) plays a detrimental role in autosomal dominant polycystic kidney disease (ADPKD). Copeptin represents a measurable substitute for circulating AVP whereas apelin counteracts AVP signaling. The aim of this study was to investigate the predictive value of apelin and copeptin for the progression of ADPKD disease. 52 ADPKD patients were enrolled and followed until the end of the observation period or the primary study endpoint was reached, defined by the combined outcome of decrease of glomerular filtration rate associated with a total renal volume increase. Receiver operating characteristics (ROC) analysis was employed for identifying the progression of renal disease and Kaplan-Meier curves assessed the renal survival. Adjusted risk estimates for progression endpoint and incident renal replacement therapy (RRT) were calculated using Cox proportional hazard regression analysis. ADPKD patients were characterized by lower apelin levels and higher copeptin levels when compared with healthy subjects. These biomarkers were strictly correlated with osmolality and markers of renal function. At ROC analysis, apelin and copeptin showed a very good diagnostic profile in identifying ADPKD progression. After the follow up of 24 months, 33 patients reached the endpoint. Cox proportional hazard regression analysis showed that apelin predicted renal disease progression and incident RRT independently of other potential confounders. Apelin is associated with kidney function decline in ADPKD, suggesting that it may be a new marker to predict kidney outcome.

Address: Department of Internal Medicine, University of Messina, Messina, Italy

3915634e: Biochem.Pharmacol., 1263-1271(2013)
Link: [ PubMED ] [ DOI ]
Title: Allosteric peptide regulators of chemokine receptors CXCR4 and CXCR7.
Author: Ehrlich,A., Ray,P., Luker,K.E., Lolis,E.J., Luker,G.D.
Abstract:
The chemokine CXCL12 and its shared seven-transmembrane receptors CXCR4 and CXCR7 regulate diseases including cancer, atherosclerosis, autoimmunity, and HIV infection, making these molecules promising drug targets. These molecules also control key processes in normal development and physiology, suggesting the need to selectively modulate CXCR4 and/or CXCR7 functions and signaling to reduce potential complications of long-term therapy. We previously identified two peptides that functioned as allosteric agonists driving CXCR4-dependent chemotaxis, providing key structural information to design a small number of additional peptides to investigate determinants of CXCL12 interactions and signaling through CXCR4 and CXCR7. In the current study, we show that the previously identified peptides only minimally activated CXCR4 signaling through the cytosolic adapter protein β-arrestin 2 and do not initiate signaling to ERK1/2. By comparison, peptides with diverse N-terminal amino acid sequences effectively activated CXCR7 signaling to β-arrestin 2. One peptide, designated as GSLW based on its N-terminal amino acids, activated CXCR7 signaling and potentiated CXCL12-CXCR7 signaling without blocking the scavenger function of CXCR7 to internalize CXCL12. These results advance our understanding of CXCR7 ligand recognition and signaling, and provide structural information to target allosteric binding sites on this receptor as chemical probes and potential therapeutic agents.

Address: Center for Molecular Imaging, Department of Radiology, University of Michigan Medical School, Ann Arbor, MI 48109-2200, USA

3915637e: Arch.Med.Res., 437-443(2013)
Link: [ PubMED ] [ DOI ]
Title: Short-term Glucose Metabolism and Gut Hormone Modulations after Billroth II Gastrojejunostomy in Nonobese Gastric Cancer Patients with Type 2 Diabetes Mellitus, Impaired Glucose Tolerance and Normal Glucose Tolerance.
Author: Zhang,X.J., Xiao,Z., Yu,H.L., Zhang,X.X., Cheng,Z., Tian,H.M.
Abstract:
BACKGROUND AND AIMS: Roux-en-Y gastric bypass (RYGB) is effective in controlling blood glucose in obese patients with type 2 diabetes (T2DM). The alterations of gut hormones involving in glucose metabolism may play an important role. Our aim was to explore the short-term effects of Billroth II gastrojejunostomy (a similar type of RYGB) on glucose metabolism and gut hormone modulations in nonobese patients with different levels of blood glucose tolerance. METHODS: Twenty one nonobese gastric cancer patients with different levels of blood glucose tolerance were submitted to Billroth II gastrojejunostomy. Among them, seven had T2DM, seven with impaired glucose tolerance (IGT) and the other seven had normal glucose tolerance (NGT). Body weight, glucose parameters, responses of plasma glucagon-like peptide-1 (GLP-1), peptide YY (PYY) and gastric inhibitory polypeptide (GIP) to 75 g glucose were measured at baseline and 3 months after surgery. RESULTS: Similar weight losses were observed in all groups. Blood glucose was reduced in T2DM and IGT patients. Fasting and 30-min plasma glucose were increased significantly in NGT. GLP-1 showed insignificant alterations in all groups. PYY was evaluated in T2DM and IGT but remained unchanged in the NGT group. Decreased fasting and AUC GIP were observed in patients with T2DM; however, fasting and 30-min GIP were increased in NGT patients. CONCLUSIONS: Billroth II gastrojejunostomy is effective in reducing blood glucose in nonobese patients with T2DM and IGT but could deteriorate early blood glucose in nonobese NGT in a 3-month time period. Variations of glucose and gut hormone changes in the three groups suggest a role of proximal intestine in the pathophysiology of T2DM.

Address: Laboratory of Endocrinology and Metabolism, West China Hospital, Sichuan University, Chengdu, Sichuan, PR China

3915638e: Osteoarthr.Cartil., 1383-1391(2013)
Link: [ PubMED ] [ DOI ]
Title: The innervation of synovium of human osteoarthritic joints in comparison with normal rat and sheep synovium.
Author: Eitner,A., Pester,J., Nietzsche,S., Hofmann,G.O., Schaible,H.G.
Abstract:
OBJECTIVE: To study whether osteoarthritis (OA) in the knee is associated with a change of the innervation pattern in the synovial layer. DESIGN: In synovial tissue from the normal knee joint of rat and sheep we studied the presence of vessels and of nerve fibres using transmission electron microscopy and immunohistochemistry. Synovial material was also obtained from patients who underwent total knee replacement surgery. This material was examined for inflammatory changes, and the presence of vessels and nerve fibres was assessed. RESULTS: The synovium in the parapatellar region of the normal knee joint of rat and sheep exhibited a dense capillary and neuronal network. It was entered by calcitonin gene-related peptide containing sensory fibres and tyrosine hydroxylase-positive sympathetic nerve fibres. Synovial material from patients with knee OA exhibited different degrees of inflammation. Synovial material without inflammation exhibited a similar vascular and neuronal network as the normal knee joint from rat and sheep. However, in synovium with inflammatory changes we found a significant decrease of nerve fibres in depth ranges close to the synovial lining layer depending on the degree of inflammation whereas deeper regions were less affected. CONCLUSIONS: Inflammatory changes in the synovium of OA joints are associated with a massive destruction of the capillary and neuronal network which is present in normal synovium. Due to the disappearance of the sensory fibres it is unlikely that OA pain is initiated directly in the synovium. The loss of normally innervated vascularisation may have multiple consequences for the physiological functions of the synovium.

E-Mail: Annett.Eitner@med.uni-jena.de
Address: Department of Physiology, University Hospital Jena, Friedrich Schiller University, D-07743 Jena, Germany

3915639e: Thromb.Res., 341-345(2013)
Link: [ PubMED ] [ DOI ]
Title: A pilot study of eptifibatide for treatment of acute pain episodes in sickle cell disease.
Author: Desai,P.C., Brittain,J.E., Jones,S.K., McDonald,A., Wilson,D.R., Dominik,R., Key,N.S., Parise,L.V., Ataga,K.I.
Abstract:
INTRODUCTION: The contribution of platelet activation to the pathogenesis of sickle cell disease (SCD) remains uncertain. We evaluated the safety and efficacy of eptifibatide, a synthetic peptide inhibitor of the αIIbβ3 receptor, in SCD patients during acute painful episodes. MATERIALS AND METHODS: In this single site, double-blind, placebo-controlled trial, eligible patients with SCD admitted for acute painful episodes were randomized to receive eptifibatide or placebo at a ratio of 2:1. RESULTS: Thirteen patients (SS - 10, Sβ(0) - 2, SC - 1) were randomized to receive either eptifibatide (N=9; 6 females; median age - 25years) or placebo (N=4; 3 females; median age - 31years). In the intent-to-treat analysis, there were no major bleeding episodes in either the eptifibatide or placebo arms (point estimate of difference: 0.00, 95% CI; -0.604, 0.372). There was one minor bleeding episode in the eptifibatide arm (point estimate of difference for any bleeding: 0.11, 95% CI: -0.502, 0.494). There was no significant difference in the proportion of patients with thrombocytopenia between the treatment groups (point estimate of difference: 0.11, 95% CI: -0.587, 0.495). There were no differences in the median times to discharge, median times to crisis resolution or the median total opioid use. CONCLUSIONS: In this small study, eptifibatide appeared to be safe, but did not improve the times to crisis resolution or hospital discharge. Adequately powered studies are required to evaluate the safety and efficacy of eptifibatide in SCD. Clinicaltrials.gov Identifier: NCT00834899.

Address: Division of Hematology/Oncology, University of North Carolina, Chapel Hill

3915640e: Int.J.Cardiol., 5167-5173(2013)
Link: [ PubMED ] [ DOI ]
Title: Eplerenone in systemic right ventricle: Double blind randomized clinical trial. The evedes study.
Author: Dos,L., Pujadas,S., Estruch,M., Mas,A., Ferreira-Gonzalez,I., Pijuan,A., Serra,R., Ordonez-Llanos,J., Subirana,M., Pons-Llado,G., Marsal,J.R., Garcia-Dorado,D., Casaldaliga,J.
Abstract:
BACKGROUND: There is no proven pharmacological strategy for the treatment of the failing systemic right ventricle (SRV) but myocardial fibrosis may play a role in its pathophysiology. METHODS: We designed a double-blind, placebo-controlled clinical trial to assess the effects of eplerenone 50mg during 12months on cardiac magnetic resonance parameters (SRV mass and ejection fraction) and neurohormonal and collagen turnover biomarker (CTB) levels. RESULTS: Twenty six patients with atrial switch repair for transposition of the great arteries were randomized to eplerenone (n=14) or placebo (n=12) and 14 healthy volunteers served as controls for comparison of baseline neurohormones and CTB levels. The study population showed a good baseline profile in terms of SRV mass (57.4+/-17g/m(2)) and ejection fraction (54.9+/-7.5%). However, levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), C terminal propeptide of type I procollagen (CICP) and C-terminal Telopeptide of type I Collagen (ICTP) were significantly elevated when compared to healthy controls. After one year of treatment, a trend toward reduction of CICP, N-terminal pro-Matrix Metalloproteinase 1 (NT-proMMP1), Tissue Inhibitor of Metalloproteinases 1 (TIMP1) and galectin 3 levels and a lower increase in ICTP in patients under eplerenone was observed. The reduction of SRV mass and the improvement of SRV function with eplerenone were not conclusive. CONCLUSIONS: Patients with SRV treated with eplerenone showed an improvement of an altered baseline CTB profile suggesting that reduction of myocardial fibrosis might be a therapeutic target in these patients.

Address: Integrated Adult Congenital Heart Disease Unit of Vall d'Hebron University Hospital and Santa Creu i Sant Pau University Hospital, Barcelona, Spain

3915644e: Semin.Oncol., 511-520(2013)
Link: [ PubMED ] [ DOI ]
Title: Non-clear cell renal cancer: disease-based management and opportunities for targeted therapeutic approaches.
Author: Linehan,W.M., Srinivasan,R., Garcia,J.A.
Abstract:
A better understanding of the biology of renal cell carcinoma (RCC) has significantly changed the treatment paradigm of the disease. Several novel vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) inhibitors have been approved recently by the US Food and Drug Administration. Unfortunately, the vast majority of clinical trials conducted today have been aimed to include patients with clear cell RCC, which remains the most common histologic subtype of the disease. Non-clear cell RCC represents approximately 20%-25% of all RCC patients. Non-clear cell RCC is made up of multiple histologic subtypes, each with a different molecular printing profile. Although VEGF and TORC inhibitors are commonly used in the management of this cohort of patients, non-clear cell histologies do not appear to be related to the von Hippel-Lindau gene (VHL). As such, the clinical efficacy of the existing agents is quite limited. There is a need to develop more rational therapeutic approaches that specifically target the biology of each of the different subtypes of non-clear cell RCC. In this review, we discuss molecular and clinical characteristics of each of the non-clear cell RCC subtypes and describe ongoing efforts to develop novel agents for this subset of patients.

E-Mail: WML@nih.gov
Address: Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

3915647e: Semin.Oncol., 472-481(2013)
Link: [ PubMED ] [ DOI ]
Title: Combination therapy in metastatic renal cell cancer.
Author: Ravaud,A., Gross-Goupil,M., Bellmunt,J.
Abstract:
The standard strategy in metastatic renal cell carcinoma (mRCC) is to use sequential treatment. Different pathways are known to be involved in the pathogenesis of mRCC and in the development of resistance to targeted drugs. Combinations of targeted drugs could theoretically achieve better inhibition of a given pathway, inhibition of different pathways, or inhibition of a pathway involved in the resistance to a given drug. However, there is as yet no clear evidence that combination therapy is of clinical benefit and excess toxicity has been observed. In mRCC the majority of studies have explored combinations of vascular endothelial growth factor (VEGF) inhibitors and mammalian target of rapamycin (mTOR) inhibitors. A new generation of VEGF tyrosine kinase inhibitors (TKIs) seems to be more tolerable, and therefore potentially easier to combine with other agents. Trials with PI3K/akt/mTORC1/2 inhibitor combinations and with other new targeted drugs are also ongoing.

E-Mail: alain.ravaud@chu-bordeaux.fr
Address: Department of Medical Oncology, Bordeaux University Hospital, Bordeaux, France

3915648e: Semin.Oncol., 465-471(2013)
Link: [ PubMED ] [ DOI ]
Title: Sequencing therapy in metastatic renal cell cancer.
Author: Escudier,B., Gore,M.
Abstract:
The sequencing of targeted therapy is well established in metastatic renal cancer. Switching between different vascular endothelial growth factor (VEGF)-targeted therapies or VEGF-targeted therapies and mammalian target of rapamycin (mTOR) inhibitors are both of proven benefit. The optimal sequence remains unclear. Unfortunately, there is a lack of biomarkers to drive decision making. In this article we review the current data and describe the treatment options for patients.

E-Mail: Bernard.Escudier@igr.fr
Address: Institut Gustave Roussy, 94805 Villejuif, France

3915649e: Surgery, 453-460(2013)
Link: [ PubMED ] [ DOI ]
Title: Efficacy of localization studies and intraoperative parathormone monitoring in the surgical management of hyperfunctioning ectopic parathyroid glands.
Author: Albuja-Cruz,M.B., Allan,B.J., Parikh,P.P., Lew,J.I.
Abstract:
BACKGROUND: Hyperfunctioning ectopic glands remain an operative challenge in patients with sporadic primary hyperparathyroidism. This study examines the incidence of ectopic glands and the utility of sestamibi scans (MIBI), surgeon-performed ultrasonography, and intraoperative parathormone monitoring in such patients undergoing parathyroidectomy. METHODS: We conducted a retrospective analysis of prospectively collected data from patients who underwent parathyroidectomy from 1980 to 2011 for sporadic primary hyperparathyroidism at a single institution. Demographics, localizing imaging studies, intraoperative parathyroid monitoring dynamics, and surgical outcome for patients with hyperfunctioning ectopic parathyroid glands were studied. RESULTS: Among 1,195 patients who underwent parathyroidectomy for sporadic primary hyperparathyroidism, 120 patients (10%) had hyperfunctioning ectopic glands, which were localized to the neck (n = 66) and mediastinum (n = 54). MIBI had a sensitivity of 85%, specificity of 97%, and positive predictive value (PPV) of 91% for ectopic glands in the neck, whereas in the mediastinum there was a sensitivity of 88%, specificity of 95%, and PPV of 86%. Surgeon-performed ultrasonography had a sensitivity of 81%, specificity of 98%, and PPV of 95% for neck ectopic glands. The overall accuracy of surgeon-performed ultrasonography, MIBI, and intraoperative parathyroid monitoring in the neck or mediastinum was 93%. Overall, operative success was 93% with a multiglandular disease rate of 5%. CONCLUSION: A high operative success rate for hyperfunctioning ectopic glands can be achieved using localization studies and intraoperative parathyroid monitoring. Nevertheless, surgeon judgment remains paramount in the operative direction of this patient population.

E-Mail: maria.albuja-cruz@ucdenver.edu
Address: Division of Surgical Endocrinology, DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, USA

3915650e: Micron, 33-38(2013)
Link: [ PubMED ] [ DOI ]
Title: Distribution of GnRH in the brain of the freshwater teleost Astyanax altiparanae ().
Author: Gomes,C.C., Costa,F.G., Borella,M.I.
Abstract:
GnRH is well known as a key decapeptide neurohormone involved in reproduction, stimulating the pituitary gland to release gonadotropins (LH and FSH), which, in turn, regulate steroidogenesis and gametogenesis. However, in addition to its reproductive functions, GnRH displays neuromodulatory roles with implications for sexual behavior. The pattern of distribution in the brain of GnRH may help reveal GnRH specific functions. Therefore, the main emphasis of this study is to detect the presence and distribution of GnRH in the brain of the freshwater teleost Astyanax altiparanae ("lambari"). The immunohistochemical method of peroxidase with an antibody raised against GnRH3 was used to detect the location of GnRHs in the brain and pituitary gland. Immunoreactivity to GnRH was found in the following encephalic areas: olfactory bulb, terminal nerve ganglion, preoptic area, nucleus of midbrain tegmentum, but also in torus longitudinalis, glomerular nucleus, and central and dorsal posterior nuclei of dorsal thalamus. In addition, cell bodies from neurons in the parvocellular and magnocellular periventricular nuclei and ventral tuberal nucleus along with many fibers including ones innervating the neurohypophysis were immunoreactive to a GnRH antiserum that detects all known eight GnRH peptides in teleosts. This is the first study describing the distribution of the complete GnRH system in the brain of A. altiparanae, which has great importance for aquaculture and ecology, and represents one of the major orders of South American teleosts - the Characiformes.

E-Mail: chayrra_bio@usp.br
Address: Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes 1524, Butanta, CEP 05508-900, Sao Paulo, SP, Brazil. Electronic address:

3915651e: Neuron, 712-724(2013)
Link: [ PubMED ] [ DOI ]
Title: Translational control of entrainment and synchrony of the suprachiasmatic circadian clock by mTOR/4E-BP1 signaling.
Author: Cao,R., Robinson,B., Xu,H., Gkogkas,C., Khoutorsky,A., Alain,T., Yanagiya,A., Nevarko,T., Liu,A.C., Amir,S., Sonenberg,N.
Abstract:
Protein synthesis is critical for circadian clock function, but little is known of how translational regulation controls the master pacemaker in mammals, the suprachiasmatic nucleus (SCN). Here we demonstrate that the pivotal translational repressor, the eukaryotic translational initiation factor 4E binding protein 1 (4E-BP1), is rhythmically regulated via the mechanistic target of rapamycin (mTOR) signaling in the SCN and preferentially represses vasoactive intestinal peptide (Vip) mRNA translation. Knockout (KO) of Eif4ebp1 (gene encoding 4E-BP1) leads to upregulation of VIP and higher amplitude of molecular rhythms in the SCN. Consequently, the 4E-BP1 null mice exhibit accelerated re-entrainment to a shifted light/dark cycle and are more resistant to the rhythm-disruptive effects of constant light. Conversely, in Mtor(+/-) mice VIP expression is decreased and susceptibility to the effects of constant light is increased. These results reveal a key role for mTOR/4E-BP1-mediated translational control in regulating entrainment and synchrony of the master clock.

Address: Department of Biochemistry and Goodman Cancer Research Center, McGill University, Montreal, QC H3A 1A3, Canada

3915656e: Clin.Exp.Obstet.Gynecol., 307-308(2013)
Link:
Title: Successful pregnancy after pulmonary embolism and heparin-induced thrombocytopenia--case report.
Author: Plesinac,S., Babovic,I., Karapandzic,V.P.
Abstract:
The authors present the case of a nulliparous 34-year-old patient. At the tenth week of gestation, she developed phlebothrombosis of veins of the right leg and massive pulmonary embolism. After thrombolytic and heparin therapy she developed rethrombosis and heparin-induced thrombocytopenia type II. Lepirudin was introduced in therapy and in the 12th week of gestation acenocumarol was added. After the 34th week, she received danaparoid sodium. After a week, by cesarean section, a healthy and mature female was delivered.

E-Mail: plesinac@hotmail.com
Address: Clinic for Gynecology and Obstetrics, Clinical Center, University of Belgrade, Belgrade, Serbia

3915657e: Clin.Exp.Obstet.Gynecol., 191-192(2013)
Link:
Title: A study to determine the efficacy of controlled ovarian hyperstimulation regimen using a gonadotropin releasing hormone agonist versus antagonist in women of advanced reproductive age with varying degrees of oocyte reserve on outcome following in vitro fertilization-embryo transfer.
Author: Check,J.H., Choe,J.K., Cohen,R., Wilson,C.
Abstract:
PURPOSE: To determine if the use of gonadotropin releasing hormone (GnRH) agonists (a) or antagonists (ant) allow better pregnancy rates when used in controlled ovarian hyperstimulation protocols in women of advanced reproductive age. Furthermore the study aimed to determine if the status of ovarian oocyte reserve has a confounding effect. MATERIALS AND METHODS: A 12-year retrospective review was performed on all in vitro fertilization-embryo transfer (IVF-ET) cycles in women aged 40-44. Pregnancy rates were determined according to whether a GnRH-a or GnRH-ant was used. The data were also stratified according to normal or low oocyte reserve. RESULTS: There was no significant difference in pregnancy rates according to whether a GnRH-a or GnRH-ant was used in women with normal oocyte reserve. Though a large majority of the women used a GnRH-ant, there was a 9% live pregnancy rate vs 0% in the women using a GnRH-a. CONCLUSION: Since it is unlikely that a larger study will ever be conducted, it is probably wise to use a GnRH-ant for the controlled ovarian hyperstimulation regimen in women aged 40-44 with diminished oocyte reserve.

E-Mail: laurie@ccivf.com
Address: Cooper Medical School of Rowan University, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology & Infertility, Camden, NJ, USA

3915659e: Pol.J.Vet.Sci., 359-368(2013)
Link: [ PubMED ]
Title: Morphology and immunohistochemical characteristics of the pterygopalatine ganglion in the chinchilla (Chinchilla laniger, Molina).
Author: Szczurkowski,A., Sienkiewicz,W., Kuchinka,J., Kaleczyc,J.
Abstract:
Histological and histochemical investigations revealed that the pterygopalatine ganglion (PPG) in the chinchilla is a structure closely connected with the maxillary nerve. Macro-morphological observations disclosed two different forms of the ganglion: an elongated stripe representing single agglomeration of nerve cells, and a ganglionated plexus comprising smaller aggregations of neurocytes connected with nerve fibres. Immunohistochemistry revealed that nearly 80% of neuronal cell bodies in PPG stained for acetylcholine transferase (CHAT) but only about 50% contained immunoreactivity to vesicular acetylcholine transporter (VACHT). Many neurons (40%) were vasoactive intestinal polypeptide (VIP)-positive. Double-staining demonstrated that approximately 20% of the VIP-immunoreactive neurons were VACHT-negative. Some neurons (10%) in PPG were simultaneously VACHT/nitric oxide synthase (NOS)- or Met-enkephaline (Met-ENK)/CHAT-positive, respectively. A small number of the perikarya stained for somatostatin (SOM) and solitary nerve cell bodies expressed Leu-ENK- and galanin-immunoreactivity. Interestingly about 5-8% of PPG neurons exhibited immunoreactivity to tyrosine hydroxylase (TH). Intraganglionic nerve fibres containing immunoreactivity to VACHT-, VIP- and Met-ENK- were numerous, those stained for calcitonin gene related peptide (CGRP)- and substance P (SP)- were scarce, and single nerve terminals were TH-, GAL-, VIP- and NOS-positive.

Address: Department of Comparative Anatomy, Institute of Biology, Jan Kochanowski University in Kielce, Swietokrzyska 15, 25-406 Kielce, Poland

3915660e: Pol.J.Vet.Sci., 231-239(2013)
Link: [ PubMED ]
Title: Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-α-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells.
Author: Ziolkowska,A., Mlynarczuk,J., Kotwica,J.
Abstract:
Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1 x 10(-5), 1 x 10(-7) M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-α-amidating mono-oxygenase (PGA). The influence of RU 486 (1 x 10(-5) M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary

Address: Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland

3915661e: J.Diabetes Res., 342479-342479(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: IL-10 induction from implants delivering pancreatic islets and hyaluronan.
Author: Bollyky,P.L., Vernon,R.B., Falk,B.A., Preisinger,A., Gooden,M.D., Nepom,G.T., Gebe,J.A.
Abstract:
Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes.

Address: Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Grant Building, 300 Pasteur Drive, Stanford, CA 94305-5107, USA

3915666e: Evid.-Based Complement.Altern.Med., 924541-924541(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Electroacupuncture Could Regulate the NF-κB Signaling Pathway to Ameliorate the Inflammatory Injury in Focal Cerebral Ischemia/Reperfusion Model Rats.
Author: Qin,W.Y., Luo,Y., Chen,L., Tao,T., Li,Y., Cai,Y.L., Li,Y.H.
Abstract:
The activated nuclear factor-KappaB signaling pathway plays a critical role in inducing inflammatory injury. It has been reported that electroacupuncture could be an effective anti-inflammatory treatment. We aimed to explore the complex mechanism by which EA inhibits the activation of the NF- κ B signal pathway and ameliorate inflammatory injury in the short term; the effects of NEMO Binding Domain peptide for this purpose were compared. Focal cerebral I/R was induced by middle cerebral artery occlusion for 2 hrs. Total 380 male Sprague-Dawley rats are in the study. The neurobehavioral scores, infarction volumes, and the levels of IL-1 β and IL-13 were detected. NF- κ B p65, I κ B α , IKK α , and IKK β were analyzed and the ability of NF- κ B binding DNA was investigated. The EA treatment and the NBD peptide treatment both reduced infarct size, improved neurological scores, and regulated the levels of IL-1 β and IL-13. The treatment reduced the expression of IKK α and IKK β and altered the expression of NF- κ B p65 and I κ B α in the cytoplasm and nucleus; the activity of NF- κ B was effectively reduced. We conclude that EA treatment might interfere with the process of NF- κ B nuclear translocation. And it also could suppress the activity of NF- κ B signaling pathway to ameliorate the inflammatory injury after focal cerebral ischemia/reperfusion.

Address: Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

3915670e: Endocrinology, 4352-4364(2013)
Link: [ PubMED ] [ DOI ]
Title: ERβ Selective Agonist Inhibits Angiotensin-Induced Cardiovascular Pathology in Female Mice.
Author: Pedram,A., Razandi,M., Korach,K.S., Narayanan,R., Dalton,J.T., Levin,E.R.
Abstract:
Cardiac hypertrophy in humans can progress to cardiac failure if the underlying impetus is poorly controlled. An important direct stimulator of hypertrophy and its progression is the angiotensin II (AngII) peptide. AngII also causes hypertension that indirectly contributes to cardiac hypertrophy. Others and we have shown that estrogens acting through the estrogen receptor (ER)-β can inhibit AngII-induced or other forms of cardiac hypertrophy in mice. However, the proliferative effects of estrogen in breast and uterus that promote the development of malignancy preclude using the steroid to prevent cardiac disease progression. We therefore tested whether an ERβ selective agonist, β-LGND2, can prevent hypertension and cardiac pathology in female mice. AngII infusion over 3 weeks significantly stimulated systolic and diastolic hypertension, cardiac hypertrophy, and cardiac fibrosis, all significantly prevented by β-LGND2 in wild-type but not in ERβ genetically deleted mice. AngII stimulated the Akt kinase to phosphorylate and inhibit the glycogen synthase kinase-3β kinase, leading to GATA4 transcription factor activation and hypertrophic mRNA expression. As a novel mechanism, all these actions were opposed by estradiol and β-LGND2. Our findings provide additional understanding of the antihypertrophic effects of ERβ and serve as an impetus to test specific receptor agonists in humans to prevent the worsening of cardiovascular disease.

E-Mail: ellis.levin@va.gov
Address: MD, Medical Service (111-I), Long Beach Veterans Affairs Medical Center, 5901 East Seventh Street, Long Beach, California 90822

3915673e: Investig.Ophthalmol.Vis.Sci., 6217-6223(2013)
Link: [ PubMED ] [ DOI ]
Title: Optimization of ceragenins for prevention of bacterial colonization of hydrogel contact lenses.
Author: Gu,X., Jennings,J.D., Snarr,J., Chaudhary,V., Pollard,J.E., Savage,P.B.
Abstract:
PURPOSE: We provided contact lens hydrogels with an antibacterial innate immune function using nonpeptide mimics of endogenous antimicrobial peptides. METHODS: Antimicrobial peptide mimics, ceragenins, were prepared for either covalent attachment to hydrogels or for controlled elution from lenses. The lipophilicity of the ceragenins was varied incrementally to provide differing levels of association with hydrophobic domains in lenses. Ceragenin-containing lenses were challenged repeatedly with Staphylococcus aureus or Pseudomonas aeruginosa in nutrient media. Bacterial growth and biofilm formation on lenses were quantified. RESULTS: A ceragenin covalently fixed in lenses effectively inhibited S. aureus biofilm formation on lenses in 10% tryptic soy broth (approximately 3-log reduction), but did not reduce biofilm formation in 100% tryptic soy broth. Ceragenins designed to elute from lenses were incorporated at 1% relative to the dry weight of the lenses. The ceragenin with the optimal lipid content, CSA-138, prevented bacterial colonization of lenses for 15 days with P. aeruginosa and for 30 days with S. aureus (daily exchange of growth media and reinoculation with 10(6) CFU). Measurement of CSA-138 elution showed that concentrations of the ceragenin never exceeded 5 μg/mL in a 24-hour period and that after 4 days of elution, concentrations dropped to <0.5 μg/mL, while maintaining antibacterial activity. CONCLUSIONS: Ceragenin CSA-138 appears well suited for providing an innate immune-like function to abiotic hydrogel contact lenses for extended periods of time. Elution of even low concentrations of CSA-138 (<0.5 μg) is sufficient to eliminate inocula of 10(6) CFU of S. aureus and P. aeruginosa.

Address: Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah

3915676e: Clin.Cancer Res., 5740-5748(2013)
Link: [ PubMED ] [ DOI ]
Title: The G-Protein-Coupled Receptor CLR Is Upregulated in an Autocrine Loop with Adrenomedullin in Clear Cell Renal Cell Carcinoma and Associated with Poor Prognosis.
Author: Nikitenko,L.L., Leek,R., Henderson,S., Pillay,N., Turley,H., Generali,D., Gunningham,S., Morrin,H.R., Pellagatti,A., Rees,M.C., Harris,A.L., Fox,S.B.
Abstract:
PURPOSE: The G-protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) and its ligand peptide adrenomedullin (encoded by ADM gene) are implicated in tumor angiogenesis in mouse models but poorly defined in human cancers. We therefore investigated the diagnostic/prognostic use for CLR in human tumor types that may rely on adrenomedullin signaling and in clear cell renal cell carcinoma (RCC), a highly vascular tumor, in particular. EXPERIMENTAL DESIGN: In silico gene expression mRNA profiling microarray study (n = 168 tumors) and cancer profiling cDNA array hybridization (n = 241 pairs of patient-matched tumor/normal tissue samples) were carried out to analyze ADM mRNA expression in 13 tumor types. Immunohistochemistry on tissue microarrays containing patient-matched renal tumor/normal tissues (n = 87 pairs) was conducted to study CLR expression and its association with clinicopathologic parameters and disease outcome. RESULTS: ADM expression was significantly upregulated only in RCC and endometrial adenocarcinoma compared with normal tissue counterparts (P < 0.01). CLR was localized in tumor cells and vessels in RCC and upregulated as compared with patient-matched normal control kidney (P < 0.001). Higher CLR expression was found in advanced stages (P < 0.05), correlated with high tumor grade (P < 0.01) and conferred shorter overall survival (P < 0.01). CONCLUSIONS: In human tissues ADM expression is upregulated in cancer type-specific manner, implicating potential role for adrenomedullin signaling in particular in RCC, where CLR localization suggests autocrine/paracrine mode for adrenomedullin action within the tumor microenvironment. Our findings reveal previously unrecognized CLR upregulation in an autocrine loop with adrenomedullin in RCC with potential application for this GPCR as a target for future functional studies and drug development.

Address: Cancer Research UK Viral Oncology Group and Sarcoma Biology Group, UCL Cancer Institute, University College London, London

3915677e: Braz.Dent.J., 204-212(2013)
Link: [ PubMED ] [ DOI ]
Title: Treatment of intrabony defects with anorganic bone matrix/p-15 or guided tissue regeneration in patients with aggressive periodontitis.
Author: Queiroz,A.C., Nobrega,P.B., Oliveira,F.S., Novaes,A.B.Jr., Taba,M.Jr., Palioto,D.B., Grisi,M.F., Souza,S.L.
Abstract:
Intrabony periodontal defects present a particular treatment problem, especially in patients with generalized aggressive periodontitis (G-AgP). Regenerative procedures have been indicated for this clinical situation. The aim of this study was to compare treatment outcomes of intrabony periodontal defects with either anorganic bone matrix/cell binding peptide (ABM/P-15) or guided tissue regeneration (GTR) in patients with G-AgP. Fifteen patients, with two intrabony defects >/=3 mm deep, were selected. Patients were randomly allocated to be treated with ABM/P-15 or GTR. At baseline and at 3 and 6 months after surgery, clinical and radiographic parameters and IL-1β and IL-6 gingival fluid concentrations were recorded. There was a significant probing pocket depth reduction (p<0.001) for both groups (2.27 +/- 0.96 mm for ABM/P-15 group and 2.57 +/- 1.06 mm for GTR group). Clinical attachment level gain (1.87 +/- 0.94 mm for ABM/P-15 group and 2.09 +/- 0.88 mm for GTR group) was also observed. There were no statistically significant differences in clinical parameters between the groups. The radiographic bone fill was more expressive in ABM/P-15 group (2.49 mm) than in GTR group (0.73 mm). In subtraction radiographs, the areas representing gain in density were 93.16% of the baseline defect for ABM/P-15 group versus 62.03% in GRT group. There were no statistically significant differences in inter-group and intra-group comparisons with regards to IL-1β and IL-6 quantification. Treatment of intrabony periodontal defects in patients with G-AgP with ABM/P-15 and GTR improved significantly the clinical outcomes. The use of ABM/P-15 promoted a better radiographic bone fill.

Address: Dental School, Federal University of Amazonas, Manaus, AM, Brazil

3915679e: Br.Dent.J., E6-E6(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Treatment of early caries lesions using biomimetic self-assembling peptides--a clinical safety trial.
Author: Brunton,P.A., Davies,R.P., Burke,J.L., Smith,A., Aggeli,A., Brookes,S.J., Kirkham,J.
Abstract:
OBJECTIVE: We previously reported that a rationally designed biomimetic self-assembling peptide, P11-4, nucleated hydroxyapatite de novo and was apparently capable of in situ enamel regeneration following infiltration into caries-like lesions. Our present aim was to determine the safety and potential clinical efficacy of a single application of P(1)(1)-4 on early enamel lesions. MATERIALS AND METHODS: Fifteen healthy adults with Class V 'white spot' lesions received a single application of P(1)(1)-4. Adverse events and lesion appearances were recorded over 180 days. RESULTS: Patients treated with P(1)(1)-4 experienced a total of 11 adverse events during the study, of which two were possibly related to the protocol. Efficacy evaluation suggested that treatment with P(1)(1)-4 significantly decreased lesion size (p = 0.02) after 30 days and shifted the apparent progression of the lesions from 'arrested/progressing' to 'remineralising' (p <0.001). A highly significant improvement in the global impression of change was recorded at day 30 compared with baseline (p <0.001). CONCLUSIONS: The results suggest that treatment of early caries lesions with P(1)(1)-4 is safe, and that a single application is associated with significant enamel regeneration, presumably by promoting mineral deposition within the subsurface tissue.

Address: Restorative Dentistry, Leeds Dental Institute, University of Leeds, Clarendon Way, Leeds, LS2 9LU, UK

3915681e: Neurobiol.Aging, 159-168(2014)
Link: [ PubMED ] [ DOI ]
Title: SAMP8 mice have altered hippocampal gene expression in long term potentiation, phosphatidylinositol signaling, and endocytosis pathways.
Author: Armbrecht,H.J., Siddiqui,A.M., Green,M., Farr,S.A., Kumar,V.B., Banks,W.A., Patrick,P., Shah,G.N., Morley,J.E.
Abstract:
The senescence-accelerated mouse (SAMP8) strain exhibits decreased learning and memory and increased amyloid β (Aβ) peptide accumulation at 12 months. To detect differences in gene expression in SAMP8 mice, we used a control mouse that was a 50% cross between SAMP8 and CD-1 mice and which showed no memory deficits (50% SAMs). We then compared gene expression in the hippocampus of 4- and 12-month-old SAMP8 and control mice using Affymetrix gene arrays. At 12 months, but not at 4 months, pathway analysis revealed significant differences in the long term potentiation (6 genes), phosphatidylinositol signaling (6 genes), and endocytosis (10 genes) pathways. The changes in long term potentiation included mitogen-activated protein kinase (MAPK) signaling (N-ras, cAMP responsive element binding protein [CREB], protein phosphatase inhibitor 1) and Ca-dependent signaling (inositol triphosphate [ITP] receptors 1 and 2 and phospholipase C). Changes in phosphatidylinositol signaling genes suggested altered signaling through phosphatidylinositol-3-kinase, and Western blotting revealed phosphorylation changes in serine/threonine protein kinase AKT and 70S6K. Changes in the endocytosis pathway involved genes related to clathrin-mediated endocytosis (dynamin and clathrin). Endocytosis is required for receptor recycling, is involved in Aβ metabolism, and is regulated by phosphatidylinositol signaling. In summary, these studies demonstrate altered gene expression in 3 SAMP8 hippocampal pathways associated with memory formation and consolidation. These pathways might provide new therapeutic targets in addition to targeting Aβ metabolism itself.

Address: Geriatric Research, Education and Clinical Center (GRECC), St Louis Veterans Affairs Medical Center, St Louis, MO, USA

3915685e: Can.J.Neurol.Sci., 722-725(2013)
Link: [ PubMED ]
Title: Calcitonin gene related peptide gene polymorphism in migraine patients.
Author: Guldiken,B., Sipahi,T., Tekinarslan,R., Kabayel,L., Ozkan,H., Unlu,A., Yamasan,B.E., Okman-Kilic,T., Turgut,N.
Abstract:
OBJECTIVE: Calcitonin gene related peptide (CGRP), which has a vasodilator effect, is held responsible for neurogenic inflammation and vasodilatation of the cranial vessels in migraine pathophysiology. In this study, we investigated the association between α CGRP gene polymorphism (CALCA T-692C) and migraine. MATERIAL AND METHODS: One hundred and thirty-four female migraineurs and 96 healthy female cases were enrolled in the study. The patient group was further subdivided into migraine with and without aura groups. The CALCA T-692C gene polymorphism was identified using polymerase chain reaction (PCR) technique and restriction fragment length polymorphism (RFLP). RESULTS: The genotype and allele frequencies of CALCA T-692C gene polymorphism did not differ between the migraine and control groups. Between the migraine with and without aura subgroups, there was no difference. No association was seen between the CALCA T-692C gene polymorphisms and migraine attack severity and frequency. CONCLUSION: Our study did not show any association between CALCA T-692C gene polymorphism and migraine.

Address: Department of Neurology, Trakya University Medical Faculty

3915688e: Exp.Parasitol., 414-420(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).
Author: Mousa,A.A., Cao,S., Aboge,G.O., Terkawi,M.A., Kirdasy,A.E., Salama,A., Attia,M., Aboulaila,M., Zhou,M., Kamyingkird,K., Moumouni,P.F., Masatani,T., Aziz,S.A., Moussa,W.M., Chahan,B., Fukumoto,S., Nishikawa,Y., Ballal,S.S., Xuan,X.
Abstract:
Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.

Address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan

3915692e: Curr.Protein Pept.Sci., 472-480(2013)
Link:
Title: Emerging roles for vasoactive peptides in diagnostic and therapeutic strategies against atherosclerotic cardiovascular diseases.
Author: Watanabe,T., Sato,K., Itoh,F., Noguchi,Y., Fujimoto,K., Koyama,T., Shichiri,M.
Abstract:
Coronary artery disease (CAD) arising from atherosclerosis remains the most common cause of death and morbidity worldwide, although its risk factors, such as hypertension, dyslipidemia, and diabetes, have been individually treated with increasingly improved outcomes. Therefore, it is important to develop diagnostic and therapeutic windows for CAD. Many classical vasoactive hormones, inflammatory cytokines, and oxidative products have been implicated as potential biomarkers. Our recent studies have shown that high levels of the pro-atherogenic vasoactive agents, serotonin and urotensin II, which are potent vasoconstrictors, can be used as biomarkers for CAD. In subsequent trials, we unraveled anti- and pro-atherogenic roles for more recently identified peptides. Anti-atherogenic peptides include the adipocytokine adiponectin, the neuronal growth factor heregulin-β(1) (neuregulin-1 type I), the incretin hormone, glucagon-like peptide-1 (GLP-1), and a peptide recently identified by an in silico approach, salusin-α. Atherogenic roles have been demonstrated by cellular, animal, and clinical experiments, which indicate that human adiponectin, heregulin-β(1), GLP-1, and salusin-α attenuate the development of atherosclerotic lesions by suppressing macrophage foam cell formation via down-regulation of acyl-CoA:cholesterol acyltransferase-1. Circulating levels of these peptides in the blood are markedly decreased in CAD patients compared with those in non-CAD patients. Receiver operating characteristic curve analyses have shown that salusin-α is the most useful biomarker for detecting CAD among the four peptides examined. Therefore, salusin-α, alone or in various combinations with heregulin-β(1), adiponectin, and/or GLP-1, is a candidate biomarker for predicting CAD. Further, anti-atherogenic peptides could potentially serve as useful therapeutic targets for atherosclerotic cardiovascular diseases.

E-Mail: watanabe@toyaku.ac.jp
Address: Laboratory of Cardiovascular Medicine, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-City, Tokyo 192-0392, Japan

3915702e: Eur.J.Gynaecol.Oncol., 278-279(2013)
Link:
Title: A rare case of lymphangiomyomatosis treated with leuprolide acetate: five-years follow-up.
Author: Bastu,E., Akhan,S.E., Karamustafaoglu,B., Gungor-Ugurlucan,F., Sozen,H., Iyibozkurt,A.C.
Abstract:
Lymphangiomyomatosis (LAM) is a rare and systemic disease that is characterized by the abnormal proliferation of smooth muscle-like cells in the lungs and along the axial lymphatic system. The authors herein present a rare case of LAM that was treated with long-term use of leuprolide acetate, a gonadotropin-releasing hormone analogue (GnRHa).

E-Mail: ercan.bastu@istanbul.edu.tr
Address: Department of Obstetrics and Gynecology, Istanbul University School of Medicine, Istanbul, Turkey

3915706e: PLoS One, e72347-e72347(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Pituitary adenylate cyclase-activating polypeptide 6-38 blocks cocaine- and amphetamine-regulated transcript peptide-induced hypophagia in rats.
Author: Burgos,J.R., Iresjo,B.M., Smedh,U.
Abstract:
Cocaine- and amphetamine-regulated transcript peptides (CARTp) suppress nutritional intake after administration into the fourth intracerebral ventricle. Recent in vitro studies have shown that PACAP 6-38, a pituitary adenylate cyclase-activating polypeptide (PACAP) fragment, could act as a competitive antagonist against CARTp 55-102 on a common CARTp-sensitive receptor structure. Here, we show for the first time in vivo that the reduction in solid food intake induced by exogenous CARTp 55-102 (0.3 nmol: 1.5 μg) administered fourth i.c.v. is blocked by pretreatment with PACAP 6-38 (3 nmol). The PACAP 6-38 fragment had no effect by itself either when given into the fourth ventricle or subcutaneously. Although effective to block the CARTp-effect on feeding and short-term body weight, PACAP 6-38 failed to attenuate CARTp-associated gross motor behavioral changes suggesting at least two CARTp-sensitive receptor subtypes. In conclusion, PACAP 6-38 acts as a functional CARTp antagonist in vivo and blocks its effects on feeding and short term weight gain.

Address: Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, Sahlgrenska University Hospital, Gothenburg, Sweden

3915707e: PLoS One, e71513-e71513(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Peripheral effects of nesfatin-1 on glucose homeostasis.
Author: Li,Z., Gao,L., Tang,H., Yin,Y., Xiang,X., Li,Y., Zhao,J., Mulholland,M., Zhang,W.
Abstract:
AIMS/HYPOTHESIS: The actions of peripherally administered nesfatin-1 on glucose homeostasis remain controversial. The aim of this study was to characterize the mechanisms by which peripheral nesfatin-1 regulates glucose metabolism. METHODS: The effects of nesfatin-1 on glucose metabolism were examined in mice by continuous infusion of the peptide via osmotic pumps. Changes in AKT phosphorylation and Glut4 were investigated by Western blotting and immnuofluorescent staining. Primary myocytes, adipocytes and hepatocytes were isolated from male mice. RESULTS: Continuous peripheral infusion of nesfatin-1 altered glucose tolerance and insulin sensitivity in mice fed either normal or high fat diet, while central administration of nesfatin-1 demonstrated no effect. Nesfatin-1 increases insulin secretion in vivo, and in vitro in cultured min6 cells. In addition, nesfatin-1 up-regulates the phosphorylation of AKT in pancreas and min6 islet cells. In mice fed normal diet, peripheral nesfatin-1 significantly increased insulin-stimulated phosphorylation of AKT in skeletal muscle, adipose tissue and liver; similar effects were observed in skeletal muscle and adipose tissue in mice fed high fat diet. At basal conditions and after insulin stimulation, peripheral nesfatin-1 markedly increased GLUT4 membrane translocation in skeletal muscle and adipose tissue in mice fed either diet. In vitro studies showed that nesfatin-1 increased both basal and insulin-stimulated levels of AKT phosphorylation in cells derived from skeletal muscle, adipose tissue and liver. CONCLUSIONS: Our studies demonstrate that nesfatin-1 alters glucose metabolism by mechanisms which increase insulin secretion and insulin sensitivity via altering AKT phosphorylation and GLUT 4 membrane translocation in the skeletal muscle, adipose tissue and liver.

Address: Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China

3915710e: PLoS One, e70842-e70842(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: In vivo CD8+ T cell dynamics in the liver of Plasmodium yoelii immunized and infected mice.
Author: Cabrera,M., Pewe,L.L., Harty,J.T., Frevert,U.
Abstract:
Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naive, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69+/-0.34 mum/min in naive mice to 5.74+/-0.66 mum/min, 9.26+/-0.92 mum/min, and 7.11+/-0.73 mum/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naive controls. To examine cellular mechanisms of immunity in situ, naive mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68+/-0.10 mum/min, 1.53+/-0.22 mum/min, and 1.06+/-0.26 mum/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS280-288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.

Address: Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine, New York, United States of America

3915712e: PLoS One, e70731-e70731(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: IVIg treatment reduces catalytic antibody titers of renal transplanted patients.
Author: Mahendra,A., Peyron,I., Dollinger,C., Gilardin,L., Sharma,M., Wootla,B., Padiolleau-Lefevre,S., Friboulet,A., Boquet,D., Legendre,C., Kaveri,S.V., Thaunat,O., Lacroix-Desmazes,S.
Abstract:
Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is a treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, to desensitize the patients and prevent the development of acute graft rejection. Here, we followed for a period of 24 months the levels of catalytic IgG towards the synthetic peptide Pro-Phe-Arg-methylcoumarinimide in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.

Address: Centre de Recherche des Cordeliers, Universite Pierre et Marie Curie, Unite Mixte de Recherche S 872, Paris, France

3915713e: PLoS One, e70644-e70644(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: A panel of 4 microRNAs facilitates the prediction of left ventricular contractility after acute myocardial infarction.
Author: Devaux,Y., Vausort,M., McCann,G.P., Kelly,D., Collignon,O., Ng,L.L., Wagner,D.R., Squire,I.B.
Abstract:
BACKGROUND: Prediction of clinical outcome after acute myocardial infarction (AMI) is challenging and would benefit from new biomarkers. We investigated the prognostic value of 4 circulating microRNAs (miRNAs) after AMI. METHODS: We enrolled 150 patients after AMI. Blood samples were obtained at discharge for determination of N-terminal pro-brain natriuretic peptide (Nt-proBNP) and levels of miR-16, miR-27a, miR-101 and miR-150. Patients were assessed by echocardiography at 6 months follow-up and the wall motion index score (WMIS) was used as an indicator of left ventricular (LV) contractility. We assessed the added predictive value of miRNAs against a multi-parameter clinical model including Nt-proBNP. RESULTS: Patients with anterior AMI and elevated Nt-proBNP levels at discharge from the hospital were at high risk of subsequent impaired LV contractility (follow-up WMIS>1.2, n = 71). A combination of the 4 miRNAs (miR-16/27a/101/150) improved the prediction of LV contractility based on clinical variables (P = 0.005). Patients with low levels of miR-150 (odds ratio [95% confidence interval] 0.08 [0.01-0.48]) or miR-101 (0.19 [0.04-0.97]) and elevated levels of miR-16 (15.9 [2.63-95.91]) or miR-27a (4.18 [1.36-12.83]) were at high risk of impaired LV contractility. The 4 miRNA panel reclassified a significant proportion of patients with a net reclassification improvement of 66% (P = 0.00005) and an integrated discrimination improvement of 0.08 (P = 0.001). CONCLUSION: Our results indicate that panels of miRNAs may aid in prognostication of outcome after AMI.

E-Mail: yvan.devaux@crp-sante.lu
Address: Laboratory of Cardiovascular Research, Centre de Recherche Public de la Sante, Luxembourg, Luxembourg

3915715e: PLoS One, e67999-e67999(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Cellular trafficking of thymosin β-4 in HEPG2 cells following serum starvation.
Author: Pichiri,G., Coni,P., Nemolato,S., Cabras,T., Fanari,M.U., Sanna,A., Di Felice,E., Messana,I., Castagnola,M., Faa,G.
Abstract:
Thymosin β-4 (Tβ4) is an ubiquitous multi-functional regenerative peptide, related to many critical biological processes, with a dynamic and flexible conformation which may influence its functions and its subcellular distribution. For these reasons, the intracellular localization and trafficking of Tβ4 is still not completely defined and is still under investigation in in vivo as well as in vitro studies. In the current study we used HepG2 cells, a human hepatoma cell line; cells growing in normal conditions with fetal bovine serum expressed high levels of Tβ4, restricted to the cytoplasm until 72 h. At 84 h, a diffuse Tβ4 cytoplasmic immunostaining shifted to a focal perinuclear and nuclear reactivity. In the absence of serum, nuclear reactivity was localized in small granules, evenly dispersed throughout the entire nuclear envelop, and was observed as earlier as at 48 h. Cytoplasmic immunostaining for Tβ4 in HepG2 cells under starvation appeared significantly lower at 48 h and decreased progressively at 72 and at 84 h. At these time points, the decrease in cytoplasmic staining was associated with a progressive increase in nuclear reactivity, suggesting a possible translocation of the peptide from the cytoplasm to the nuclear membrane. The normal immunocytochemical pattern was restored when culture cells submitted to starvation for 84 h received a new complete medium for 48 h. Mass spectrometry analysis, performed on the nuclear and cytosolic fractions of HepG2 growing with and without serum, showed that Tβ4 was detectable only in the cytosolic and not in the intranuclear fraction. These data suggest that Tβ4 is able to translocate from different cytoplasmic domains to the nuclear membrane and back, based on different stress conditions within the cell. The punctuate pattern of nuclear Tβ4 immunostaining associated with Tβ4 absence in the nucleoplasm suggest that this peptide might be localized in the nuclear pores, where it could regulate the pore permeability.

E-Mail: pichiri@unica.it
Address: Divisione di Anatomia Patologica, Dipartimento di Citomorfologia, University of Cagliari, Cagliari, Italy

3915720e: PLoS Pathog., 003547-003547(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Local CD4 and CD8 T-cell reactivity to HSV-1 antigens documents broad viral protein expression and immune competence in latently infected human trigeminal ganglia.
Author: Van Velzen,M., Jing,L., Osterhaus,A.D., Sette,A., Koelle,D.M., Verjans,G.M.
Abstract:
Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

Address: Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands

3915721e: J.Neurosci., 13600-13611(2013)
Link: [ PubMED ] [ DOI ]
Title: Prenatal exposure to nicotine stimulates neurogenesis of orexigenic peptide-expressing neurons in hypothalamus and amygdala.
Author: Chang,G.Q., Karatayev,O., Leibowitz,S.F.
Abstract:
Animal and clinical studies show that gestational exposure to nicotine increases the propensity of offspring to consume nicotine, but the precise mechanism mediating this behavioral phenomenon is unclear. The present study in Sprague Dawley rats examined the possibility that the orexigenic peptide systems, enkephalin (ENK) and orexin (OX), which are stimulated by nicotine in adult animals and promote consummatory behavior, may be similarly responsive to nicotine's stimulatory effect in utero while having long-term behavioral consequences. The results demonstrated that nicotine exposure during gestation at low doses (0.75 or 1.5 mg/kg/d) significantly increased mRNA levels and density of neurons that express ENK in the hypothalamic paraventricular nucleus and central nucleus of the amygdala, OX, and another orexigenic peptide, melanin-concentrating hormone, in the perifornical lateral hypothalamus in preweanling offspring. These effects persisted in the absence of nicotine, at least until puberty. Colabeling of the cell proliferation marker BrdU with the neuronal marker NeuN and peptides revealed a marked stimulatory effect of prenatal nicotine on neurogenesis, but not gliogenesis, and also on the number of newly generated neurons expressing ENK, OX, or melanin-concentrating hormone. During adolescence, offspring also exhibited significant behavioral changes, increased consumption of nicotine and other substances of abuse, ethanol and a fat-rich diet, with no changes in chow and water intake or body weight. These findings reveal a marked sensitivity during gestation of the orexigenic peptide neurons to low nicotine doses that may increase the offspring's propensity to overconsume substances of abuse during adolescence.

Address: The Rockefeller University, New York, NY, USA

3915722e: Am.J.Clin.Nutr., 1123-1135(2013)
Link: [ PubMED ] [ DOI ]
Title: An undigested gliadin peptide activates innate immunity and proliferative signaling in enterocytes: the role in celiac disease.
Author: Nanayakkara,M., Lania,G., Maglio,M., Discepolo,V., Sarno,M., Gaito,A., Troncone,R., Auricchio,S., Auricchio,R., Barone,M.V.
Abstract:
BACKGROUND: On ingestion of gliadin, the major protein component of wheat and other cereals, the celiac intestine is characterized by the proliferation of crypt enterocytes with an inversion of the differentiation/proliferation program. Gliadins and A-gliadin peptide P31-43, in particular, act as growth factors for crypt enterocytes in patients with celiac disease (CD). The effects of gliadin on crypt enterocyte proliferation and activation of innate immunity are mediated by epidermal growth factors (EGFs) and innate immunity mediators [interleukin 15 (IL15)]. OBJECTIVE: The aim of this study was to determine the molecular basis of proliferation and innate immune response to gliadin peptides in enterocytes. DESIGN: The CaCo-2 cell line was used to study EGF-, IL15-, and P31-43-induced proliferation. Silencing messenger RNAs and blocking EGF receptor and IL15 antibodies have been used to study proliferation in CaCo-2 cells and intestinal biopsy samples from patients with CD and control subjects. RESULTS: In the CaCo-2 cell model, IL15 and EGF cooperated to induce proliferation in intestinal epithelial cells at both the transcriptional and posttranscriptional levels, and the respective receptors interacted to activate each other's signaling. In addition, the effects of the P31-43 peptide on CaCo-2 cell proliferation and downstream signaling were mediated by cooperation between EGF and IL15. The increased crypt enterocyte proliferation in intestinal biopsy samples from patients with CD was reduced by EGF receptor and IL15 blocking antibodies only when used in combination. CONCLUSIONS: EGF receptor/IL15R-α cooperation regulates intestinal epithelial cell proliferation induced by EGF, IL15, and the gliadin peptide P31-43. Increased proliferation of crypt enterocytes in the intestine of CD patients is mediated by EGF/IL15 cooperation.

Address: Department of Pediatrics and European Laboratory for the Investigation of Food Induced Disease, University of Naples, Federico II, Naples, Italy, and the Department of Medicine, University of Chicago, Chicago, IL

3915725e: Sci.Transl.Med., 9ra111-9ra111(2013)
Link: [ PubMED ] [ DOI ]
Title: Epidermal EGFR controls cutaneous host defense and prevents inflammation.
Author: Lichtenberger,B.M., Gerber,P.A., Holcmann,M., Buhren,B.A., Amberg,N., Smolle,V., Schrumpf,H., Boelke,E., Ansari,P., Mackenzie,C., Wollenberg,A., Kislat,A., Fischer,J.W., Rock,K., Harder,J., Schroder,J.M., Homey,B., Sibilia,M.
Abstract:
The epidermal growth factor receptor (EGFR) plays an important role in tissue homeostasis and tumor progression. However, cancer patients treated with EGFR inhibitors (EGFRIs) frequently develop acneiform skin toxicities, which are a strong predictor of a patient's treatment response. We show that the early inflammatory infiltrate of the skin rash induced by EGFRI is dominated by dendritic cells, macrophages, granulocytes, mast cells, and T cells. EGFRIs induce the expression of chemokines (CCL2, CCL5, CCL27, and CXCL14) in epidermal keratinocytes and impair the production of antimicrobial peptides and skin barrier proteins. Correspondingly, EGFRI-treated keratinocytes facilitate lymphocyte recruitment but show a considerably reduced cytotoxic activity against Staphylococcus aureus. Mice lacking epidermal EGFR (EGFR(Δep)) show a similar phenotype, which is accompanied by chemokine-driven skin inflammation, hair follicle degeneration, decreased host defense, and deficient skin barrier function, as well as early lethality. Skin toxicities were not ameliorated in a Rag2-, MyD88-, and CCL2-deficient background or in mice lacking epidermal Langerhans cells. The skin phenotype was also not rescued in a hairless (hr/hr) background, demonstrating that skin inflammation is not induced by hair follicle degeneration. Treatment with mast cell inhibitors reduced the immigration of T cells, suggesting that mast cells play a role in the EGFRI-mediated skin pathology. Our findings demonstrate that EGFR signaling in keratinocytes regulates key factors involved in skin inflammation, barrier function, and innate host defense, providing insights into the mechanisms underlying EGFRI-induced skin pathologies.

Address: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Austria

3915728e: Molecules, 9949-9965(2013)
Link: [ PubMED ] [ DOI ]
Title: Neuroprotective, anti-amyloidogenic and neurotrophic effects of apigenin in an Alzheimer's disease mouse model.
Author: Zhao,L., Wang,J.L., Liu,R., Li,X.X., Li,J.F., Zhang,L.
Abstract:
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles in the brain. Amyloid-β peptides (Aβ) are considered to play a critical role in the onset and progression of AD. Apigenin (4',5,7-trihydroxyflavone) is a pharmacologically active agent. Even though some evidence suggests that it has potential neuroprotective effects, no preexisting study has reported any therapeutic effects of apigenin in AD models. In the present study, we examined the effects of apigenin on cognitive function in APP/PS1 double transgenic AD mice and explored its mechanism(s) of action. Three-month oral treatment with apigenin rescued learning deficits and relieved memory retention in APP/PS1 mice. Apigenin also showed effects affecting APP processing and preventing Aβ burden due to the down-regulation of BACE1 and β-CTF levels, the relief of Aβ deposition, and the decrease of insoluble Aβ levels. Moreover, apigenin exhibited superoxide anion scavenging effects and improved antioxidative enzyme activity of superoxide dismutase and glutathione peroxidase. In addition, apigenin restored neurotrophic ERK/CREB/BDNF pathway in the cerebral cortex. In conclusion, apigenin may ameliorate AD-associated learning and memory impairment through relieving Aβ burden, suppressing amyloidogenic process, inhibiting oxidative stress, and restoring ERK/CREB/BDNF pathway. Therefore, apigenin appears to represent an alternative medication for the prevention and/or therapy of AD.

E-Mail: zhaole@bjciq.gov.cn
Address: College of Life and Environmental Sciences, Minzu University of China, 27 South Street, Zhongguancun, Beijing 100081, China

3915734e: J.Neuropathol.Exp.Neurol., 846-860(2013)
Link: [ PubMED ] [ DOI ]
Title: Disruption of CDH2/N-cadherin-based adherens junctions leads to apoptosis of ependymal cells and denudation of brain ventricular walls.
Author: Oliver,C., Gonzalez,C.A., Alvial,G., Flores,C.A., Rodriguez,E.M., Batiz,L.F.
Abstract:
Disruption/denudation of the ependymal lining has been associated with the pathogenesis of various human CNS disorders, including hydrocephalus, spina bifida aperta, and periventricular heterotopia. It has been traditionally considered that ependymal denudation is a consequence of mechanical forces such as ventricular enlargement. New evidence indicates that ependymal disruption can precede ventricular dilation, but the cellular and molecular mechanisms involved in the onset of ependymal denudation are unknown. Here, we present a novel model to study ependymal cell pathophysiology and demonstrate that selective disruption of N-cadherin-based adherens junctions is sufficient to provoke progressive ependymal denudation. Blocking N-cadherin function using specific peptides that interfere with the histidine-alanine-valine extracellular homophilic interaction domain caused early pathologic changes characterized by disruption of zonula adherens and abnormal intracellular accumulation of N-cadherin. These changes then triggered massive apoptosis of ependymal cells and denudation of brain ventricular walls. Because no typical extrinsic mechanical factors such as elevated pressure or stretching forces are involved in this model, the critical role of N-cadherin-based adherens junctions in ependymal survival/physiology is highlighted. Furthermore, the results suggest that abnormal adherens junctions between ependymal cells should be considered as key components of the pathogenesis of CNS disorders associated with ependymal denudation.

Address: Instituto de Anatomia, Histologia y Patologia, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile

3915735e: J.Neuropathol.Exp.Neurol., 833-845(2013)
Link: [ PubMED ] [ DOI ]
Title: Skeletal muscle biopsy analysis in reducing body myopathy and other FHL1-related disorders.
Author: Malfatti,E., Olive,M., Taratuto,A.L., Richard,P., Brochier,G., Bitoun,M., Gueneau,L., Laforet,P., Stojkovic,T., Maisonobe,T., Monges,S., Lubieniecki,F., Vasquez,G., Streichenberger,N., Lacene,E., Saccoliti,M., Prudhon,B., Alexianu,M., Figarella-Branger,D., Schessl,J., Bonnemann,C., Eymard,B., Fardeau,M., Bonne,G., Romero,N.B.
Abstract:
FHL1 mutations have been associated with various disorders that include reducing body myopathy (RBM), Emery-Dreifuss-like muscular dystrophy, isolated hypertrophic cardiomyopathy, and some overlapping conditions. We report a detailed histochemical, immunohistochemical, electron microscopic, and immunoelectron microscopic analyses of muscle biopsies from 18 patients carrying mutations in FHL1: 14 RBM patients (Group 1), 3 Emery-Dreifuss muscular dystrophy patients (Group 2), and 1 patient with hypertrophic cardiomyopathy and muscular hypertrophy (Group 2). Group 1 muscle biopsies consistently showed RBs associated with cytoplasmic bodies. The RBs showed prominent FHL1 immunoreactivity whereas desmin, αB-crystallin, and myotilin immunoreactivity surrounded RBs. By electron microscopy, RBs were composed of electron-dense tubulofilamentous material that seemed to spread progressively between the myofibrils and around myonuclei. By immunoelectron microscopy, FHL1 protein was found exclusively inside RBs. Group 2 biopsies showed mild dystrophic abnormalities without RBs; only minor nonspecific myofibrillar abnormalities were observed under electron microscopy. Molecular analysis revealed missense mutations in the second FHL1 LIM domain in Group 1 patients and ins/del or missense mutations within the fourth FHL1 LIM domain in Group 2 patients. Our findings expand the morphologic features of RBM, clearly demonstrate the localization of FHL1 in RBs, and further illustrate major morphologic differences among different FHL1-related myopathies.

Address: Unite de Morphologie Neuromusculaire, Institut de Myologie, Groupe Hospitalier Universitaire La Pitie-Salpetriere, Paris, France

3915738e: J.Anim.Sci., 4750-4756(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of composite antimicrobial peptides in weanling piglets challenged with deoxynivalenol: II. Intestinal morphology and function.
Author: Xiao,H., Tan,B.E., Wu,M.M., Yin,Y.L., Li,T.J., Yuan,D.X., Li,L.
Abstract:
Deoxynivalenol (DON) affects animal and human health and targets the gastrointestinal tract. The objective of this study was to evaluate the ability of composite antimicrobial peptides (CAP) to repair intestinal injury in piglets challenged with DON. A total of 28 piglets (Duroc x Landrace x Large Yorkshire) weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (7 pigs/treatment): negative control, basal diet (NC), basal diet + 0.4% composite antimicrobial peptide (CAP), basal diet + 4 mg/kg DON (DON), and basal diet + 4 mg/kg DON + 0.4% CAP (DON + CAP). After an adaptation period of 7 d, blood samples were collected on d 15 and 30 after the initiation of treatment for determinations of the concentrations of D-lactate and diamine oxidase. At the end of the study, all piglets were slaughtered to obtain small intestines for the determination of intestinal morphology, epithelial cell proliferation, and protein expression in the mammalian target of rapamycin (mTOR) signaling pathway. The results showed that DON increased serum concentrations of D-lactate and diamine oxidase, and these values in the CAP and DON + CAP treatments were less than those in the NC and DON treatments, respectively (P < 0.05). The villous height/crypt depth in the jejunum and ileum and the goblet cell number in the ileum in the CAP and DON + CAP treatments were greater than those in the NC and DON treatments (P < 0.05). The proliferating cell nuclear antigen (PCNA) labeling indexes for the jejunum and ileum in the DON + CAP treatment were greater than those in the DON treatment (P < 0.05). The DON decreased (P < 0.05) the relative protein expression of phosphorylated Akt (Protein Kinase B) and mTOR in the jejunal and ileal mucosa and of phosphorylated 4E-binding protein 1 (p-4EBP1) in the jejunal mucosa, whereas CAP increased (P < 0.05) the protein expression of p-4EBP1 in the jejunum. These findings showed that DON could enhance intestinal permeability, damage villi, cause epithelial cell apoptosis, and inhibit protein synthesis, whereas CAP improved intestinal morphology and promoted intestinal epithelial cell proliferation and protein synthesis, indicating that CAP may repair the intestinal injury induced by DON.

Address: Observation and Experiment Station of Animal Nutrition and Feed Science in South-Central China, Ministry of Agriculture

3915739e: J.Anim.Sci., 4772-4780(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of composite antimicrobial peptides in weanling piglets challenged with deoxynivalenol: I. Growth performance, immune function, and antioxidation capacity.
Author: Xiao,H., Wu,M.M., Tan,B.E., Yin,Y.L., Li,T.J., Xiao,D.F., Li,L.
Abstract:
The mycotoxin deoxynivalenol (DON) is a food contaminant that leads to reduced feed intake and reduced BW gain, as well as organ impairment. On the other hand, antimicrobial peptides have been shown to have positive effects on growth performance, nutrient digestibility, and immune function. The purpose of this study was to investigate the protective effects of composite antimicrobial peptides (CAP) on piglets challenged with DON. After a 7-d adaptation period, 28 individually housed piglets (Duroc x Landrace x Large Yorkshire) weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (7 pigs/treatment): negative control, basal diet (NC), basal diet + 0.4% CAP (CAP), basal diet + 4 mg/kg DON (DON), and basal diet + 4 ppm DON + 0.4% CAP (DON + CAP). On d 15 and 30 after the initiation of treatment, blood samples were collected for the determination of blood profile. Piglets were monitored for 30 d to assess performance and then were slaughtered to obtain organs for the determination of the relative weight of organs. The results showed that dietary supplementation with DON decreased (P < 0.05) ADFI, ADG, and G:F, whereas dietary supplementation with CAP improved ADG and G:F (P < 0.05). The relative weight of the kidney and pancreas was greater and the relative weight of the spleen was lighter in the DON treatment than in the other 3 treatments (P < 0.05). There were no effects (P > 0.05) on other relative weights of viscera, except the relative weight of the gallbladder, but the diamine oxidase activity in the liver decreased in DON-treated piglets (P < 0.05). Piglets in the DON treatment had increased serum concentrations of alkaline phosphatase, alanine transaminase, and aspartate aminotransferase and a dramatic decrease in total protein (P < 0.05), whereas there were no differences (P > 0.05) between the DON + CAP treatment and the other treatments. The DON treatment decreased the numbers of red blood cells and platelets, as well as the serum catalase concentrations, and decreased the serum concentrations of H2O2, maleic dialdehyde, and nitric oxide (P < 0.05). The numbers of platelets and thrombocytocrit, as well as the serum concentrations of catalase, were greater, whereas the maleic dialdehyde concentrations were decreased, in both the CAP and DON + CAP treatments compared with the other treatments (P < 0.05). Compared with the control treatment, DON decreased peripheral lymphocyte proliferation on d 15, whereas supplementation with CAP increased it on d 15 and 30 (P < 0.05). These findings indicate that CAP could improve feed efficiency, immune function, and antioxidation capacity and alleviate organ damage, and thus, it has a protective effect in piglets challenged with DON.

Address: Hunan Provincial Engineering Research Center for Healthy Livestock and Poultry Production, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan 410125, China

3915741e: Arthritis Res.Ther., R91-R91(2013)
Link: [ PubMED ] [ DOI ]
Title: SCORE and REGICOR function charts underestimate the cardiovascular risk in Spanish patients with rheumatoid arthritis.
Author: Gomez-Vaquero,C., Corrales,A., Zacarias,A., Rueda-Gotor,J., Blanco,R., Gonzalez-Juanatey,C., Llorca,J., Gonzalez-Gay,M.A.
Abstract:
INTRODUCTION: Our objective was to determine which one of the two function charts available in Spain to calculate the cardiovascular (CV) risk, SCORE or REGICOR, should be used in patients with rheumatoid arthritis (RA). METHODS: A series of RA patients seen over a 1-year period without history of CV events were assessed. SCORE, REGICOR, modified (m)SCORE and mREGICOR according to the European League Against Rheumatism (EULAR) recommendations were applied. Carotid ultrasonography (US) was performed. Carotid intima-media thickness (cIMT) > 0.90 mm and/or carotid plaques was used as the gold standard test for severe subclinical atherosclerosis and high CV risk (US+). The area under the receiver operating curves (AUC) for the predicted risk for mSCORE and mREGICOR were calculated according to the presence of severe carotid US findings (US+). RESULTS: We included 370 patients (80% women; mean age 58.9+/-13.7 years); 36% had disease duration [greater than or equal to] 10 years; rheumatoid factor (RF) and/or anticyclic citrullinated peptide (anti-CCP) were positive in 68%; and 17% had extra-articular manifestations. The EULAR multiplier factor was used in 122 (33%) of the patients. The mSCORE was 2.16 +/- 2.49% and the mREGICOR 4.36 +/- 3.46%. Regarding US results, 196 (53%) patients were US+. The AUC mSCORE was 0.798 (CI 95%: 0.752-0.844) and AUC mREGICOR 0.741 (95% CI; 0.691-0.792). However, mSCORE and mREGICOR failed to identify 88% and 91% of US+ patients. More than 50% of patients with mSCORE [greater than or equal to] 1% or mREGICOR > 1% were US+. CONCLUSIONS: Neither of these two functions charts was useful to estimate CV risk in Spanish RA patients.

3915744e: BMC Endocr.Disord., 32-32(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Effect of glycemic control on soluble RAGE and oxidative stress in type 2 diabetic patients.
Author: Motawi,T.M., Abou-Seif,M.A., Bader,A.M., Mahmoud,M.O.
Abstract:
BACKGROUND: The interaction of advanced glycation end products (AGEs) and its receptor (RAGE) has played an important role in the pathogenesis of diabetes and its complications. A soluble form of RAGE (sRAGE) has been reported as a decoy receptor for AGEs. Oxidative stress is demonstrated in pathological condition such as atherosclerosis and diabetes mellitus. It has been suggested to be involved in the pathogenesis of both macro- and microvascular complications. This study was designed to evaluate the effect of glycemic control on sRAGE and oxidative stress markers in type 2 diabetic patients. METHODS: Seventy patients with type 2 diabetes and 20 healthy subjects were recruited into the study. Blood glutathione (GSH) and plasma total nitric oxide (NOx) levels were measured using commercially available colorimetric kits, blood superoxide dismutase (SOD) activity was measured by the method of Marklund and Marklund, and plasma C-peptide, oxidized LDL (ox-LDL), sRAGE, and VCAM-1 levels were measured using competitive ELISA kits. RESULTS: Plasma sRAGE levels were significantly lower (p < 0.05) while VCAM-1 levels were significantly higher (p < 0.05) in poorly controlled diabetic patients compared with healthy control. Blood GSH levels were significantly lower in diabetic patients compared with healthy control (p < 0.05). Plasma C-peptide, NOx, ox-LDL levels, and SOD activity were not significantly different in diabetic patients compared with healthy control. Plasma levels of sRAGE were negatively associated with circulating VCAM-1 levels in diabetic patients. CONCLUSION: Poor glycemic control decreases plasma sRAGE and increases VCAM-1 levels while good glycemic control improves these abnormalities which provides benefit to diabetic patients.

E-Mail: mohamed_omar_bio@yahoo.com
Address: Department of Biochemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt

3915749e: Ren.Fail., 1273-1277(2013)
Link: [ PubMED ] [ DOI ]
Title: NGAL and NT-proBNP levels in diabetic patients with macroproteinuria.
Author: Taskapan,H., Taskapan,M.C., Orman,I., Ulutas,O., Yigit,A., Ozyalin,F., Yologlu,S.
Abstract:
BACKGROUND: In patients with heart failure plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) levels are correlated to urine neutrophil gelatinase-associated lipocalin (NGAL) levels. We prospectively evaluated the relationship among glomerular filtration rate (eGFR), urine albumin-to-creatinine ratio (ACR), urine and serum NGAL and NT-proBNP levels in 20 type II diabetic patients with macroalbuminuria at 4-month intervals. RESULTS: Compared with 20 age, gender-matched healthy controls, diabetic patients had higher urine and serum NGAL, serum NT-proBNP and lower eGFR. The eGFR of the patients at the baseline, the 4th and the 8th month were 29.6 +/- 12.0, 27.8 +/- 13.7 and 22.9 +/- 10.4 mL/min/1.73 m(2), respectively. No significant change in urine NGAL levels was detected (p > 0.05), whereas there were significant increases in NT-proBNP, serum NGAL and urine ACR and significant decrease in eGFR as the study progressed (p < 0.05). Both the baseline and the 4th month urine ACR were positively correlated to NT-proBNP levels measured at the same periods (r: 0.451; p: 0.046; r: 0.489; p: 0.029 respectively). In all measurements, urine ACR was negatively correlated to serum albumin levels measured at the same periods (r: -0.792; p: 0.000; r: -0.716; p: 0.000; r: -0.531; p: 0.016 respectively). None of eGFR measurements was correlated with NT-proBNP (p > 0.05). Neither serum NGAL nor urinary NGAL levels are associated with NT-proBNP (p > 0.05). CONCLUSION: Our findings show an association between NT-proBNP and proteinuria in type II diabetic patients with macroalbuminuria but not with serum and urine NGAL.

Address: Department of Nephrology

3915751e: Reprod.Domest.Anim.Suppl., 44-52(2013)
Link: [ PubMED ] [ DOI ]
Title: Hypothalamic integration of nutrient status and reproduction in the sheep.
Author: Daniel,J.A., Foradori,C.D., Whitlock,B.K., Sartin,J.L.
Abstract:
Nutrient availability is a determinant of reproductive success. It is well known that inadequate nutrition results in reproductive failure due to a number of factors including delay of puberty or anoestrous in post-pubertal animals. The lack of nutrients is detected primarily by changes in circulating nutrient molecules and hormones and communicated directly or indirectly to the hypothalamus and brain stem for integration. The general effect is that low nutrition leads to increased appetite stimulation and reduced reproductive performance. When nutrition is adequate, the reverse is true. Both aspects will be the focus of this review. One result of the lack of nutrients is a reduction in luteinizing hormone (LH) concentrations and pulse frequency. Nutrient signals, such as glucose availability, hormonal signals, such as insulin and leptin, and neuroendocrine signals, such as neuropeptide Y and corticotropin-releasing hormone, have been clearly demonstrated to interact to produce changes in LH and reproductive success. Other signals, such as fatty acids, ghrelin, agouti-related peptide, melanin-concentrating hormone, orexin, melanocyte-stimulating hormone, kisspeptin, neurokinin, dynorphin and gonadotropin inhibitory hormone may also play a role in integrating nutrition and reproduction. This review will focus on the major features of the reciprocal control of appetite and reproduction in sheep.

Address: Berry College, Mt Berry, GA, USA

3915753e: Korean Circ.J., 481-487(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Cardiac rehabilitation increases exercise capacity with a reduction of oxidative stress.
Author: Fukuda,T., Kurano,M., Fukumura,K., Yasuda,T., Iida,H., Morita,T., Yamamoto,Y., Takano,N., Komuro,I., Nakajima,T.
Abstract:
BACKGROUND AND OBJECTIVES: Reactive oxygen species (ROS) mediate various signaling pathways that underlie vascular inflammation in atherogenesis and cardiovascular diseases. Cardiac rehabilitation (CR) has a variety of multiple beneficial effects, including anti-inflammatory effects. The purpose of the present study was to investigate the effects of CR on ROS in patients with cardiovascular diseases. SUBJECTS AND METHODS: The serum level of derivatives of reactive oxidative metabolites, an index of oxidative stress, was measured in 100 patients with cardiovascular diseases before, and, subsequently, 3 and 6 months after, CR. A biological antioxidant potential (BAP) test was applied to assess the antioxidant power of the serum. RESULTS: The resting reactive oxidative metabolite levels decreased 3-6 months after CR {pre: 351+/-97 Carratelli unit (CARR U), 3 months: 329+/-77 CARR U, 6 months: 325+/-63 CARR U, all p<0.01} with the increase of the percentage of the predicted values of VO2 peak and the percentage of the predicted values of VO2 at the anaerobic threshold (VO2 AT) and the decrease of the B-type natriuretic peptide (BNP). The BAP test and antioxidative/oxidative stress ratio increased 6 months after CR. The % changes of the antioxidative/oxidative stress ratio was positively correlated with the % changes of VO2 AT, and negatively correlated with the % changes of the BNP. CONCLUSION: These results suggest that intensive supervised CR significantly improved exercise capacity, which may be attributable to an adaptive response involving more efficient oxidative metabolites or the increased capacity of endogenous anti-oxidative systems in patients with cardiovascular diseases.

Address: Health Center, Hitotsubashi University, Tokyo, Japan.

3915754e: Korean Circ.J., 462-467(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The Impact of Trimetazidine Treatment on Left Ventricular Functions and Plasma Brain Natriuretic Peptide Levels in Patients with Non-ST Segment Elevation Myocardial Infarction Undergoing Percutaneous Coronary Intervention.
Author: Demirelli,S., Karakelleoglu,S., Gundogdu,F., Tas,M.H., Kaya,A., Duman,H., Degirmenci,H., Hamur,H., Simsek,Z.
Abstract:
BACKGROUND AND OBJECTIVES: The aim of this study was to investigate the impact of treatment with oral trimetazidine (TMZ) applied before and after percutaneous coronary interventions (PCI) on short-term left ventricular functions and plasma brain natriuretic peptide (BNP) levels in patients with non-ST segment elevation myocardial infarction (NSTEMI) undergoing PCI. SUBJECTS AND METHODS: The study included 45 patients who were undergoing PCI with the diagnosis of NSTEMI. The patients were randomized into two groups. The first group (n=22) of the patients hospitalized with the diagnosis of NSTEMI was given conventional therapy plus 60 mg TMZ just prior to PCI. Treatment with TMZ was continued for one month after the procedure. TMZ treatment was not given to the second group (n=23). Echocardiography images were recorded and plasma BNP levels were measured just prior to the PCI and on the 1st and 30th days after PCI. RESULTS: The myocardial performance index (MPI) was greater in the second group (p=0.02). In the comparison of BNP levels, they significantly decreased in both of the groups during the 30-day follow-up period (29.0+/-8 and 50.6+/-33, p<0.01 respectively). However, decreasing of BNP levels was higher in the group administered with TMZ. The decrease of left ventriclular end-diastolic volume was observed in all groups at 30 days after intervention, but was higher in the group administered with TMZ (p=0.01). CONCLUSION: Trimetazidine treatment commencing prior to PCI and continued after PCI in patients with NSTEMI provides improvements in MPI, left ventricular end diastolic volume and a decrease in BNP levels.

Address: Department of Cardiology, Erzurum Regional Training and Research Hospital, Erzurum, Turkey

3915759e: World J.Gastroenterol., 5111-5117(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Diagnostic accuracy of a new point-of-care screening assay for celiac disease.
Author: Benkebil,F., Combescure,C., Anghel,S.I., Besson Duvanel,C., Schappi,M.G.
Abstract:
AIM: To determine the diagnostic accuracy of a new point-of-care assay detecting anti-deamidated gliadin peptides in celiac disease (CD) patients. METHODS: One-hundred-and-twelve patients (age range: 1.8-79.2 years old) with clinical symptoms suggestive of CD and/or first-degree relatives (FDR) of CD patients (n = 66), and confirmed CD on a gluten-free diet (GFD) (n = 46), were prospectively enrolled in the study at Gastroenterology outpatient clinics for adult patients and from the Gastroenterology Consultation Ward at the Pediatric Department of the University Hospital of Geneva. Written informed consent was obtained from all subjects enrolled. The study received approval from the local ethics committee. The original CD diagnosis had been based on serum-positive IgA anti-tissue transglutaminase enzyme-linked immunosorbent assay (ELISA) (QuantaLite, Inova Diagnostics, San Diego, CA, United States) and on biopsy results. Serum samples from all study participants were tested by the new CD lateral flow immunochromatographic assay (CD-LFIA) device, Simtomax(R) Blood Drop (Augurix SA, BioArk, Monthey, Switzerland) to detect immunoglobulin (Ig)A and IgG antibodies against deamidated gliadin peptides. The diagnostic performance was evaluated using receiver operating characteristic curves with 95%CIs. A cut-off of 2 on the Rann colorimetric scale was used to calculate the device's sensitivity and specificity. RESULTS: CD-LFIA was highly accurate in detecting untreated celiac patients. In the group of patients with CD symptoms and/or FDR, eight new cases of CD were detected by ELISA and biopsy. All of these new cases were also correctly identified by CD-LFIA. The test yielded four false positive and four false negative results. The false positive results were all within the groups with clinical symptoms suggestive of CD and/or FDR, whereas the false negative results were all within the GFD group. The test yeld a sensitivity of 78.9% (95%CI: 54.4-93.9) and specificity of 95.7% (95%CI: 89.4-98.8), and the area under the curve reached 0.893 (95%CI: 0.798-0.988). The Kappa coefficient, calculated according to the values obtained by two readers from the same device, was of 0.96 (SE: 0.06). When the GFD patients were excluded from the analysis, the area under the curve reached 0.989 (95%CI: 0.971-1.000) and the Kappa coefficient, calculated according to the values obtained by two readers from the same device, became 0.96 (SE: 0.07). Furthermore, using the Rann scale cut-off of 2 without the GFD patients, sensitivity was 100% and specificity was 93.1% (95%CI: 83.3-98.1). CONCLUSION: The new CD-LFIA rapid screening test shows good diagnostic accuracy, sensitivity and specificity, and may rule out CD in patients with CD-related symptoms.

Address: Vidymed, 1007 Lausanne, Switzerland

3915771e: J.Am.Heart Assoc., 000284-000284(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Long-term miR-669a therapy alleviates chronic dilated cardiomyopathy in dystrophic mice.
Author: Quattrocelli,M., Crippa,S., Montecchiani,C., Camps,J., Cornaglia,A.I., Boldrin,L., Morgan,J., Calligaro,A., Casasco,A., Orlacchio,A., Gijsbers,R., D'Hooge,J., Toelen,J., Janssens,S., Sampaolesi,M.
Abstract:
BACKGROUND: Dilated cardiomyopathy (DCM) is a leading cause of chronic morbidity and mortality in muscular dystrophy (MD) patients. Current pharmacological treatments are not yet able to counteract chronic myocardial wastage, thus novel therapies are being intensely explored. MicroRNAs have been implicated as fine regulators of cardiomyopathic progression. Previously, miR-669a downregulation has been linked to the severe DCM progression displayed by Sgcb-null dystrophic mice. However, the impact of long-term overexpression of miR-669a on muscle structure and functionality of the dystrophic heart is yet unknown. METHODS AND RESULTS: Here, we demonstrate that intraventricular delivery of adeno-associated viral (AAV) vectors induces long-term (18 months) miR-669a overexpression and improves survival of Sgcb-null mice. Treated hearts display significant decrease in hypertrophic remodeling, fibrosis, and cardiomyocyte apoptosis. Moreover, miR-669a treatment increases sarcomere organization, reduces ventricular atrial natriuretic peptide (ANP) levels, and ameliorates gene/miRNA profile of DCM markers. Furthermore, long-term miR-669a overexpression significantly reduces adverse remodeling and enhances systolic fractional shortening of the left ventricle in treated dystrophic mice, without significant detrimental consequences on skeletal muscle wastage. CONCLUSIONS: Our findings provide the first evidence of long-term beneficial impact of AAV-mediated miRNA therapy in a transgenic model of severe, chronic MD-associated DCM.

Address: Translational Cardiomyology Lab, Stem Cell Biology and Embryology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium

3915772e: J.Pharm.Sci., 3998-4008(2013)
Link: [ PubMED ] [ DOI ]
Title: Mechanistic study of the uptake/permeation of cell-penetrating peptides across a caco-2 monolayer and their stimulatory effect on epithelial insulin transport.
Author: Kamei,N., Onuki,Y., Takayama,K., Takeda-Morishita,M.
Abstract:
Our recent studies have demonstrated the potential of cell-penetrating peptides (CPPs) to significantly stimulate the intestinal absorption of therapeutic peptides and proteins. This study examined the mechanisms underlying the intestinal epithelial uptake and permeation of CPPs and their contribution to the enhanced absorption of insulin. Fluorescein-tagged octaarginine (R8) and penetratin were used as the promising CPPs, and in vitro uptake and permeation assays were conducted using Caco-2 cell monolayer. The assay conducted under low temperature conditions revealed that energy-dependent pathways are not involved in d-form arginines (d-R8) uptake or its stimulatory effect on insulin uptake. The Km value (3.82 μM), calculated from the dose dependence of d-R8 uptake, suggested that a part of the d-R8 uptake was saturated at the functional concentration (60 μM d-R8). An analysis based on the binding parameters of insulin and d-R8 also showed an increase in the uptake clearance of the insulin/d-R8 complex, even at a saturated concentration of d-R8, implying that this complex is taken up by Caco-2 cells via pathways that differ from those that take up unbound d-R8. Thus, this study suggests that CPPs such as oligoarginines stimulate the intestinal epithelial transport of peptide and protein drugs via energy-independent unsaturable internalization.

Address: Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan

3915777e: Acta Physiol.Sin., 417-432(2013)
Link: [ PubMED ]
Title: An engineered multidomain fungicidal peptide against plant fungal pathogens.
Author: Qiu,X.Q., Tong,C.Y., Zhong,Z.Q., Wang,W.Q., Zuo,Y.W., Huang,Y., Zhang,L., Zhang,X.L., Zhang,H.X., Li,R.Q., Wang,J., Chen,H., Li,Y.Y., Wu,G., Wang,H.
Abstract:
Fungal pathogens represent major problems for human health and agriculture. As eukaryotic organisms, fungi share some important features with mammalian cells. Therefore, current anti-fungal antibiotics often can not distinguish between fungi and mammalian cells, resulting in serious side effects in mammalian cells. Accordingly, there is strong impetus to develop antifungal alternatives that are both safe and effective. The E1 family of colicin are channel-forming bacteriocins produced by Escherichia coli, which are bactericidal only to E. coli and related species. To target the channel-forming domain of colicin to fungal cell membrane, we engineered a sexual mating pheromone of Candida albicans, α-factor pheromone to colicin Ia. A peptide was constructed consisting of an α mating pheromone of C. albicans fused to the channel-forming domain of colicin Ia to create a new fusion protein, pheromonicin-CA (PMC-CA). Indirect immunolabeling showed that the PMC-CA bound to fungal cells and inhibited growth in the laboratory and field. In the field, the protective activity of pheromonicin against rice blast disease was significantly greater, on a molar basis, than that of triazoles, tricyclazole or isoprothiolane. These results suggest that fusion peptides may be of value as fungicidal agents under agricultural conditions.

Address: Laboratory of Biomembrane and Membrane Protein, West China Hospital

3915778e: Nuklearmedizin, 170-177(2013)
Link: [ PubMED ] [ DOI ]
Title: Early prediction of tumour response to PRRT. The sequential change of tumour-absorbed doses during treatment with 177Lu-octreotate.
Author: Ezziddin,S., Reichmann,K., Yong-Hing,C., Damm,M., Risse,J., Ahmadzadehfar,H., Logvinski,T., Guhlke,S., Biersack,H.J., Sabet,A.
Abstract:
[177Lu-DOTA0,Tyr3]-octreotate (177Lu-octreotate) in peptide receptor radionuclide therapy (PRRT) offers direct intra-therapeutic dosimetry. The aim of this study was to compare tumour and non-tumour parameters and assess intra-individual variations. Patients, methods: Retrospective analysis of 53 consecutive PRRT treatment cycles (mean activity of 7.53 +/- 0.46 GBq 177Lu-octreotate, intended four cycles at intervals of 10-14 weeks, standard nephroprotection) in 27 GEP NET patients. Extended planar dosimetry with serial whole-body imaging on selected, non-superimposed tumour and non-tumour regions; liver (LM), bone (BM), and other (OM) metastases. The per-cycle variation was compared with post-treatment response (CT/MRI three months post-treatment, modified SWOG criteria). Results: Residence time in tumor lesions (133-147 h) exceeded that in kidneys (93 h). Tumour-to-kidney absorbed dose ratios ranged from 14 to 28 (LM, BM, OM). Intra-individual per-cycle dose variation was insignificant for kidneys, but significant for metastases (LM, BM, and OM; p < 0.05). The mean per-cycle decrease of tumour absorbed dose (ΔD/A0[%]) was linked to morphologic response after PRRT. A mean decrease of >20% was predictive of a partial or minor remission in all 11 evaluable patients, while absent significant dose reduction indicated stable or progressive disease in 4/5 patients. The dose decrease was unrelated to volume effects and also observed for BM. Conclusion: Besides confirmation of a favourable tumour-to-kidney parameter relation for 177Lu-octreotate, stepwise intra-lesional comparison seems to imply a prognostic impact of tumor dosimetry: The early per-cycle change ΔD/A0 between treatment cycles may predict the outcome after PRRT. Larger studies are needed to confirm this finding.

E-Mail: samer.ezziddin@ukb.uni-bonn.de
Address: Samer Ezziddin, MD, Department of Nuclear Medicine, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, Tel. +49/(0)228/28 71 91 74, Fax +49/(0)228/28 79 01 91 74, E-mail:

3915779e: Transl.Psychiatry, e293-e293(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: CSF protein biomarkers predicting longitudinal reduction of CSF β-amyloid42 in cognitively healthy elders.
Author: Mattsson,N., Insel,P., Nosheny,R., Zetterberg,H., Trojanowski,J.Q., Shaw,L.M., Tosun,D., Weiner,M.
Abstract:
β-amyloid (Aβ) plaque accumulation is a hallmark of Alzheimer's disease (AD). It is believed to start many years prior to symptoms and is reflected by reduced cerebrospinal fluid (CSF) levels of the peptide Aβ1-42 (Aβ42). Here we tested the hypothesis that baseline levels of CSF proteins involved in microglia activity, synaptic function and Aβ metabolism predict the development of Aβ plaques, assessed by longitudinal CSF Aβ42 decrease in cognitively healthy people. Forty-six healthy people with three to four serial CSF samples were included (mean follow-up 3 years, range 2-4 years). There was an overall reduction in Aβ42 from a mean concentration of 211-195 pg ml(-1) after 4 years. Linear mixed-effects models using longitudinal Aβ42 as the response variable, and baseline proteins as explanatory variables (n=69 proteins potentially relevant for Aβ metabolism, microglia or synaptic/neuronal function), identified 10 proteins with significant effects on longitudinal Aβ42. The most significant proteins were angiotensin-converting enzyme (ACE, P=0.009), Chromogranin A (CgA, P=0.009) and Axl receptor tyrosine kinase (AXL, P=0.009). Receiver-operating characteristic analysis identified 11 proteins with significant effects on longitudinal Aβ42 (largely overlapping with the proteins identified by linear mixed-effects models). Several proteins (including ACE, CgA and AXL) were associated with Aβ42 reduction only in subjects with normal baseline Aβ42, and not in subjects with reduced baseline Aβ42. We conclude that baseline CSF proteins related to Aβ metabolism, microglia activity or synapses predict longitudinal Aβ42 reduction in cognitively healthy elders. The finding that some proteins only predict Aβ42 reduction in subjects with normal baseline Aβ42 suggest that they predict future development of the brain Aβ pathology at the earliest stages of AD, prior to widespread development of Aβ plaques.

Address: Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Molndal, Sweden [3] Department of Radiology and Biomedical Imaging, University of California, San Francisco, CA, USA

3915787e: Br.J.Pharmacol., 1041-1052(2013)
Link: [ PubMED ] [ DOI ]
Title: Sertraline inhibits the transport of PAT1 substrates in vivo and in vitro.
Author: Nielsen,C.U., Frolund,S., Abdulhadi,S., Sari,H., Langthaler,L., Nohr,M.K., Kall,M.A., Brodin,B., Holm,R.
Abstract:
BACKGROUND AND PURPOSE: Intestinal nutrient transporters may mediate the uptake of drugs. The aim of this study was to investigate whether sertraline interacts with the intestinal proton-coupled amino acid transporter 1 PAT1 (SLC36A1). EXPERIMENTAL APPROACH: In vitro investigations of interactions between sertraline and human (h)PAT1, hSGLT1 (sodium-glucose linked transporter 1) and hPepT1 (proton-coupled di-/tri-peptide transporter 1) were conducted in Caco-2 cells using radiolabelled substrates. In vivo pharmacokinetic investigations were conducted in male Sprague-Dawley rats using gaboxadol (10 mg.kg(-1) , p.o.) as a PAT1 substrate and sertraline (0-30.6 mg.kg(-1) ). Gaboxadol was quantified by hydrophilic interaction chromatography followed by MS/MS detection. KEY RESULTS: Sertraline inhibited hPAT1-mediated L-[(3) H]-Pro uptake in Caco-2 cells. This interaction between sertraline and PAT1 appeared to be non-competitive. The uptake of the hSGLT1 substrate [(14) C]-α-methyl-D-glycopyranoside and the hPepT1 substrate [(14) C]-Gly-Sar in Caco-2 cells was also decreased in the presence of 0.3 mM sertraline. In rats, the administration of sertraline (0.1-10 mM, corresponding to 0.3-30.6 mg.kg(-1) , p.o.) significantly reduced the maximal gaboxadol plasma concentration and AUC after its administration p.o. CONCLUSIONS AND IMPLICATIONS: Sertraline is an apparent non-competitive inhibitor of hPAT1-mediated transport in vitro. This inhibitory effect of sertraline is not specific to hPAT1 as substrate transport via hPepT1 and hSGLT1 was also reduced in the presence of sertraline. In vivo, sertraline reduced the amount of gaboxadol absorbed, suggesting that the inhibitory effect of sertraline on PAT1 occurs both in vitro and in vivo. Hence, sertraline could alter the bioavailability of drugs absorbed via PAT1.

Address: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

3915789e: Mol.Pharm., 3603-3611(2013)
Link: [ PubMED ] [ DOI ]
Title: Multivalency of Non-Peptide Integrin αVβ3 Antagonist Slows Tumor Growth.
Author: Kim,Y.S., Li,F., Kong,R., Bai,Y., Li,K.C., Fan,Y., O'Neill,B.E., Li,Z.
Abstract:
Multivalency is a powerful strategy for achieving high-affinity molecular binding of compounds to increase their therapeutic potency or imaging potential. In our study, multivalent non-peptide integrin αvβ3 antagonists (IA) were designed for antitumor therapy. Docking and molecular dynamics were employed to explore the binding modes of IA monomer, dimer, and trimer. In silico, one IA unit binds tightly in the active site with similar pose to native ligand RGD and other parts of dimer and trimer contribute extra binding affinities by interacting with residues in vicinity of the original site. In vitro studies demonstrated that increasing valency results in increasing antiproliferative and antiorganizational effects against endothelial cells (HUVECs), and a much weaker effect on melanoma B16F10 cells. The antitumor efficacies of the IA multivalent compounds were evaluated in subcutaneous B16F10 melanoma tumor-bearing mice. At 30 mg/kg dose, the mean masses of tumors harvested 18 days after inoculation were significantly reduced (p < 10(-7)) by 36 +/- 9%, 49 +/- 8%, and 71 +/- 7% for the IA monomer, dimer, and trimer groups, relative to control. The importance of multivalency was demonstrated to be highly significant beyond the additive effect of the extra pharmacological sites (p = 0.00011). These results suggest that the major target of these anti-αvβ3 compounds is the neovasculature rather than the cancer cells, and the success of a multivalent strategy depends on the details of the components and linker. This is the first integrin αvβ3 multivalent ligand showing clear enhancement in antitumor effectiveness.

Address: Department of Translational Imaging, section signDepartment of Systems Medicine & Bioengineering, The Methodist Hospital Research Institute, Weill Medical College of Cornell University , 6670 Bertner Avenue, Houston, Texas 77030, United States

3915791e: Indian J.Endocrinol.Metab., 611-619(2013)
Link: [ PubMED ] [ DOI ]
Title: Glycemic variability: Clinical implications.
Author: Satya Krishna,S.V., Kota,S.K., Modi,K.D.
Abstract:
Glycemic control and its benefits in preventing microvascular diabetic complications are convincingly proved by various prospective trials. Diabetes control and complications trial (DCCT) had reported variable glycated hemoglobin (HbA1C) as a cause of increased microvascular complications in conventional glycemic control group versus intensive one. However, in spite of several indirect evidences, its link with cardiovascular events or macrovascular complications is still not proved. Glycemic variability (GV) is one more tool to explain relation between hyperglycemia and increased cardiovascular risk in diabetic patients. In fact GV along with fasting blood sugar, postprandial blood sugar, HbA1C, and quality of life has been proposed to form glycemic pentad, which needs to be considered in diabetes management. Postprandial spikes in blood glucose as well as hypoglycemic events, both are blamed for increased cardiovascular events in Type 2 diabetics. GV includes both these events and hence minimizing GV can prevent future cardiovascular events. Modern diabetes management modalities including improved sulfonylureas, glucagon like peptide-1 (GLP-1)-based therapy, newer basal insulins, and modern insulin pumps address the issue of GV effectively. This article highlights mechanism, clinical implications, and measures to control GV in clinical practice.

Address: Department of Endocrinology, Medwin Hospital, Hyderabad, Andhra Pradesh, India

3915794e: Saudi Dent.J., 75-82(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Expression of human β defensins (HBDs) 1, 2 and 3 in gingival crevicular fluid of patients affected by localized aggressive periodontitis.
Author: Ebrahem,M.A.
Abstract:
AIM: To study the effect of nonsurgical periodontal therapy on the expression frequencies of human β-defensin (HBD)-1, -2, and -3 in the gingival crevicular fluid (GCF) of patients affected by localized aggressive periodontitis. MATERIALS AND METHODS: Twenty patients affected by localized aggressive periodontitis (age range, 20-35 years) and 20 healthy subjects (age range, 21-37 years) were examined with clinical periodontal parameters and radiographic examination with the long-cone parallel technique. All periodontitis patients underwent nonsurgical periodontal therapy combined with doxycycline treatment and a maintenance program (including brushing with regular toothpaste). GCF samples were collected from patients and healthy control subjects at baseline as well as 3 months after periodontal therapy for the patient group. RESULTS: In the patient group, the expression frequencies of HBD-1, -2, and -3 mRNA at baseline were 30%, 85%, and 35%, respectively, which changed after periodontal therapy to 80%, 45%, and 85%, respectively (all P < 0.001). In the healthy control subjects, the expression frequencies were 95%, 40%, and 95% for HBD-1, -2, and -3, respectively, which were different from those of diseased patients at baseline (all P < 0.001). CONCLUSIONS: The appropriate expression of HBD peptides in health and disease may contribute to the maintenance of periodontal homeostasis, possibly through its antimicrobial effects and the promotion of adaptive immune responses.

Address: Department of Oral Medicine, Periodontology and Oral Diagnosis, Faculty of Dental Medicine, Minia University, Egypt

3915795e: J.Pharmacol.Pharmacother., 198-203(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of structural analogues of apelin-12 in acute myocardial infarction in rats.
Author: Pisarenko,O.I., Serebryakova,L.I., Studneva,I.M., Pelogeykina,Y.A., Tskitishvili,O.V., Bespalova,Z.D., Sidorova,M.V., Az'muko,A.A., Khatri,D.N., Pal'keeva,M.E., Molokoedov,A.S.
Abstract:
OBJECTIVE: To examine cardioprotective effects of Rho-terminal fragment of adipokine apelin-12 (A12), its novel structural analogue [MeArg(1), NLe(10)]-A12 (I), and [d-Ala(12)]-A12 (II), a putative antagonist of APJ receptor, employing in vivo model of ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: Peptides were synthesized by the automatic solid phase method using Fmoc technology. Anesthetized open-chest male Wistar rats were subjected to left anterior descending (LAD) coronary artery occlusion and coronary reperfusion. Hemodynamic variables and electrocardiogram (ECG) were monitored throughout the experiment. Myocardial injury was assessed by infarct size (IS), activity of necrosis markers in plasma, and metabolic state of the area at risk (AAR). RESULTS: Intravenous injection of A12, I, or II at the onset of reperfusion led to a transient reduction of the mean arterial pressure. A12 or I administration decreased the percent ratio of IS/AAR by 40% and 30%, respectively, compared with control animals which received saline. Both peptides improved preservation of high-energy phosphates, reduced lactate accumulation in the AAR, and lowered CK-MB and LDH activities in plasma at the end of reperfusion compared with these indices in control. Treatment with II did not significantly affect either the IS/AAR, % ratio, or activities of both markers of necrosis compared with control. The overall metabolic protection of the AAR in the treated groups increased in the following rank: II < A12 < I. CONCLUSIONS: The structural analogue of apelin-12 [MeArg(1), NLe(10)]-A12 may be a promising basis to create a new drug for the treatment of acute coronary syndrome.

Address: Russian Cardiology Research-and-Production Complex, Institute of Experimental Cardiology, Moscow, Russian Federation

3915797e: Endocrinology, 3978-3983(2013)
Link: [ PubMED ] [ DOI ]
Title: Oral L-Arginine Stimulates GLP-1 Secretion to Improve Glucose Tolerance in Male Mice.
Author: Clemmensen,C., Smajilovic,S., Smith,E.P., Woods,S.C., Brauner-Osborne,H., Seeley,R.J., D'Alessio,D.A., Ryan,K.K.
Abstract:
Pharmacological and surgical interventions that increase glucagon-like peptide 1 (GLP-1) action are effective to improve glucose homeostasis in type 2 diabetes mellitus. In light of this, nutritional strategies to enhance postprandial GLP-1 secretion, particularly in the context of diet-induced obesity, may provide an alternative therapeutic approach. Importantly, recent evidence suggests the amino acid l-arginine, a well-known insulin secretagogue, can also stimulate release of GLP-1 from isolated rat intestine. Here we tested the hypothesis that oral l-arginine acts as a GLP-1 secretagogue in vivo, to augment postprandial insulin secretion and improve glucose tolerance. To test this, we administered l-arginine or vehicle by oral gavage, immediately prior to an oral glucose tolerance test in lean and diet-induced obese mice. In both lean and obese mice oral l-arginine increased plasma GLP-1 and insulin and substantially improved glucose clearance. To directly assess the contribution of GLP-1 receptor (GLP-1R)-signaling to these improvements, l-arginine was given to Glp1r knockout mice and their wild-type littermates. In this experiment oral l-arginine significantly augmented insulin secretion and improved glucose clearance in WT mice, but not in Glp1r knockout littermates. Taken together these findings identify l-arginine as a GLP-1 secretagogue in vivo and demonstrate that improvement of glucose tolerance by oral l-arginine depends on GLP-1R-signaling. These findings raise the intriguing possibility that l-arginine-based nutritional and/or pharmaceutical therapies may benefit glucose tolerance by improving the postprandial GLP-1 response in obese individuals.

E-Mail: karen.ryan@uc.edu
Address: 2170 East Galbraith Road, A125, Cincinnati, Ohio 45237

3915798e: Endocrinology, 4270-4280(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular evolution of kiss2 genes and peptides in vertebrates.
Author: Osugi,T., Ohtaki,N., Sunakawa,Y., Son,Y.L., Ohkubo,M., Iigo,M., Amano,M., Tsutsui,K.
Abstract:
The kiss1 peptide (kisspeptin), a product of the kiss1 gene, is one of the key neuropeptides regulating vertebrate reproduction. In 2009, we identified a paralogous gene of kiss1 in the brain of amphibians and named it kiss2. Currently, the presence of the kiss2 gene and the kiss2 peptide is still obscure in amniotes compared with that in other vertebrates. Therefore, we performed genome database analyses in primates and reptiles to investigate the molecular evolution of the kiss2 gene in vertebrates. Because the mature kiss2 peptide has been identified only in amphibians, we further performed immunoaffinity purification and mass spectrometry to identify the mature endogenous kiss2 peptide in the brains of salmon and turtle that possessed the kiss2 gene. Here we provide the first evidence for the presence of a kiss2-like gene in the genome database of primates including humans. Synthetic amidated human KISS2 peptide activated human GPR54 expressed in COS7 cells, but nonamidated KISS2 peptide was inactive. The endogenous amidated kiss2 peptide may not be produced in primates because of the lack of an amidation signal in the precursor polypeptide. The kiss2-like gene may be nonfunctional in crocodilians because of premature stop codons. We identified the mature amidated kiss2 peptide in turtles and fish and analyzed the localization of kiss2 peptide mRNA expression in fish. The present study suggests that the kiss2 gene may have mutated in primates and crocodilians and been lost in birds during the course of evolution. In contrast, the kiss2 gene and mature kiss2 peptide are present in turtles and fish.

E-Mail: k-tsutsui@waseda.jp
Address: PhD, Professor, Laboratory of Integrative Brain Sciences, Department of Biology, Waseda University, Center for Medical Life Science of Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan

3915799e: Hypertension, 759-766(2013)
Link: [ PubMED ] [ DOI ]
Title: Circulating endothelin-1 alters critical mechanisms regulating cerebral microcirculation.
Author: Faraco,G., Moraga,A., Moore,J., Anrather,J., Pickel,V.M., Iadecola,C.
Abstract:
Endothelin-1 (ET1) is a potent vasoconstrictor peptide implicated in the cerebrovascular alterations occurring in stroke, subarachnoid hemorrhage, and brain trauma. Brain or circulating levels of ET1 are elevated in these conditions and in risk factors for cerebrovascular diseases. Most studies on the cerebrovascular effects of ET1 have focused on vascular smooth muscle constriction, and little is known about the effect of the peptide on cerebrovascular regulation. We tested the hypothesis that ET1 increases cerebrovascular risk by disrupting critical mechanisms regulating cerebral blood flow. Male C57Bl6/J mice equipped with a cranial window were infused intravenously with vehicle or ET1, and somatosensory cortex blood flow was assessed by laser Doppler flowmetry. ET1 infusion increased mean arterial pressure and attenuated the blood flow increase produced by neural activity (whisker stimulation) or neocortical application of the endothelium-dependent vasodilator acetylcholine but not A23187. The cerebrovascular effects of ET1 were abrogated by the ET(A) receptor antagonist BQ123 and were not related to vascular oxidative stress. Rather, the dysfunction was dependent on Rho-associated protein kinase activity. Furthermore, in vitro studies demonstrated that ET1 suppresses endothelial nitric oxide (NO) production, assessed by its metabolite nitrite, an effect associated with Rho-associated protein kinase-dependent changes in the phosphorylation state of endothelial NO synthase. Collectively, these novel observations demonstrate that increased ET1 plasma levels alter key regulatory mechanisms of the cerebral circulation by modulating endothelial NO synthase phosphorylation and NO production through Rho-associated protein kinase. The ET1-induced cerebrovascular dysfunction may increase cerebrovascular risk by lowering cerebrovascular reserves and increasing the vulnerability of the brain to cerebral ischemia.

E-Mail: coi2001@med.cornell.edu
Address: Brain and Mind Research Institute, 407 E 61st St, Room 303, New York, NY 10065

3915802e: Peptides, 21-26(2013)
Link: [ PubMED ] [ DOI ]
Title: The satiety signal oleoylethanolamide stimulates oxytocin neurosecretion from rat hypothalamic neurons.
Author: Romano,A., Cassano,T., Tempesta,B., Cianci,S., Dipasquale,P., Coccurello,R., Cuomo,V., Gaetani,S.
Abstract:
The anandamide monounsaturated analogue oleoylethanolamide (OEA) acts as satiety signal released from enterocytes upon the ingestion of dietary fats to prolong the interval to the next meal. This effect, which requires intact vagal fibers and intestinal PPAR-α receptors, is coupled to the increase of c-fos and oxytocin mRNA expression in neurons of the paraventricular nucleus (PVN) and is prevented by the intracerebroventricular administration of a selective oxytocin antagonist, thus suggesting a necessary role of oxytocinergic neurotransmission in the pro-satiety effect of OEA. By brain microdialysis and immunohistochemistry, in this study we demonstrate that OEA treatment can stimulate oxytocin neurosecretion from the PVN and enhance oxytocin expression at both axonal and somatodendritic levels of hypothalamic neurons. Such effects, which are maxiμm 2h after OEA administration, support the hypothesis that the satiety-inducing action of OEA is mediated by the activation of oxytocin hypothalamic neurons.

Address: Department of Physiology and Pharmacology, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy

3915808e: Eur.J.Pharm.Biopharm., 12-19(2013)
Link: [ PubMED ] [ DOI ]
Title: Cell penetrating peptides fail to induce an innate immune response in epithelial cells in vitro: implications for continued therapeutic use.
Author: Carter,E., Lau,C.Y., Tosh,D., Ward,S.G., Mrsny,R.J.
Abstract:
Cell penetrating peptides (CPPs) offer the exciting potential of effectively delivering macromolecules to the cytoplasm of a cell that are otherwise impermeable to the plasma membrane. Although the use of these peptides has so far been well tolerated in clinical trials, it is important to remember that some of these CPPs were originally derived from pathogenic material. We therefore sought to determine if three of the most widely studied CPPs; HIV-TAT, Antennapedia and Transportan, initiated an immune response in epithelial cells. Using conditions where these peptides efficiently delivered a rhodamine tagged BSA cargo to the interior of epithelial cells, we failed to observe an effect on cell viability as determined by MTT assay (P>0.05). Further, CPP-mediated delivery of this protein cargo failed to activate NFκB, which would be indicative of toll-like receptor signalling. Finally, no significant increase in the release of the inflammatory cytokines interleukin (IL)-8 and IL-6 was detected in epithelial cells exposed to CPP complexes for 72 h (P>0.05). Together, these results indicate that these commonly used CPPs are passive carriers that do not initiate epithelial cell-associated 'danger signals' during the process of cytoplasmic delivery of a model protein cargo.

Address: Department of Pharmacy and Pharmacology, University of Bath, Bath, UK

3915809e: Eur.J.Pharm.Biopharm., 5-11(2013)
Link: [ PubMED ] [ DOI ]
Title: Is there a future for cell-penetrating peptides in oligonucleotide delivery?
Author: Lee,S.H., Castagner,B., Leroux,J.C.
Abstract:
Cell-penetrating peptides have been widely investigated as delivery vehicles for oligonucleotides (e.g., siRNA and antisense oligonucleotides). Different delivery strategies can be used, such as co-incubation, direct conjugation, non-covalent complex, and modification on the surface of liposome or polymer complexes. However, several challenges remain for their preclinical and clinical development. Endosomal escape, lack of cell/tissue specificity, and toxicity are major concerns in the design of cell-penetrating peptide-mediated delivery systems. In this commentary, we highlight recent reports of cell-penetrating peptide incorporation into oligonucleotide delivery systems and underline the remaining challenges, particularly for preclinical and clinical applications.

Address: Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Zurich, Switzerland

3915810e: Atherosclerosis, 86-91(2013)
Link: [ PubMED ] [ DOI ]
Title: Ankle-brachial index in relation to the natriuretic peptide system polymorphisms and urinary sodium excretion in Chinese.
Author: Hu,B.C., Li,Y., Liu,M., Sheng,C.S., Wang,J.G.
Abstract:
OBJECTIVE: Recent studies have demonstrated that the natriuretic pepetides induce endothelial regeneration and angiogenesis after vascular injury through the autocrine or paracrine action, and might have an inhibitory effect on atherosclerosis. We therefore systematically investigated single nucleotide polymorphisms (SNPs) in the natriuretic peptide system in relation to ankle-brachial index (ABI) in a Chinese population. METHODS: The study population was recruited from a mountainous area 500 km south of Shanghai from 2003 to 2009. Using the SNapShot method, we first genotyped 951 subjects enrolled in 2005 for 16 SNPs and then the remaining 1355 subjects as validation for 5 SNPs selected from the primary study. ABI and plasma proBNP were measured using the Omron VP-2000/1000 device and the Elecsys proBNP immunoassay, respectively. RESULTS: Overall, the genetic associations were not significant (P >/= 0.07). However, in the primary study, there was significant (Pint </= 0.045) interaction between 3 SNPs (rs6668352, rs198388, and rs198389) at the NPPA-NPPB locus and urinary sodium excretion in relation to ABI, and the rs6668352 polymorphism had the strongest association (Pint = 0.018). In the primary combined with the validation study populations, the interaction between the rs6668352 polymorphism and urinary sodium excretion in relation to ABI remained statistically significant (Pint = 0.0036). After adjustment for covariates, the rs6668352 A allele carriers, compared with GG homozygotes, had a higher ABI (mean +/- standard error, 1.103 +/- 0.006 vs. 1.084 +/- 0.004, P = 0.009) and lower risk of peripheral arterial disease (PAD, defined as an ABI < 0.90, odds ratio 0.37, 95% confidence interval: 0.14-0.98, P = 0.04) in the subjects of high sodium intake. CONCLUSION: The minor alleles of 3 SNPs at the NPPA-NPPB locus are associated with a lower risk of PAD, especially in the subjects of high sodium intake.

Address: Centre for Epidemiological Studies and Clinical Trials, The Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, China

3915811e: J.Pharm.Pharm.Sci., 279-288(2013)
Link: [ PubMED ]
Title: The antiinflammatory action and pharmacokinetics of a novel glucosamine-based di-peptide aminosugar.
Author: Gilzad-Kohan,M.H., Kaur,K., Jamali,F.
Abstract:
PURPOSE: We have previously shown favorable in vitro gut permeability for three novel di-peptide esters of glucosamine (GlcN) likely facilitated by the peptide transporter 1 (PepT1). Herein, we report the development of a novel assay for the determination of bioavailability of the peptide ester of interest, the anti-inflammatory properties of a glycine-valine ester derivative of GlcN (GVG) as well as its pharmacokinetics under healthy and inflammatory conditions. METHODS: A pre-column derivatization (with 9-fluorenylmethoxycarbonyl) HPLC assay was developed to study bioavailability of GVG, GlcN or cleaved GlcN in the rats that were cannulated in their right jugular vein for blood collection. The compounds of interest were orally administered to both healthy and arthritic rats. Serial blood samples and urine were collected and assayed for the compounds. The stability of the GVG was also tested after incubation with the rat feces. Efficacy of GVG was tested in inflamed rats (injection of 0.2 mL of Mycobacterium butyricum in squalene) following GVG (20 and 30 mg/kg/day GlcN equivalent) or GlcN (20 and 90 mg/kg/day) administration. Arthritis index was calculated at the end of the experiment. RESULTS: The assay was linear (ranged between 0.05-20 μg/mL) and reproducible (intra- and inter-day<10%). Among the tested compounds, only GVG showed a significantly higher plasma concentrations and urinary excretion than GlcN ( approximately 3-fold increase). GVG showed a favorable stability in the rat feces. Adjuvant arthritis was completely prevented with doses greater than 20 mg/kg/day with GVG being 3-fold more potent than GlcN. CONCLUSION: The examined glycine-valine-GlcN di-peptide aminosugar is a potent anti-inflammatory compound due to its favorable properties to deliver GlcN into the systemic circulation.

Address: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

3915812e: Arthritis Res.Ther., R89-R89(2013)
Link: [ PubMED ] [ DOI ]
Title: The phenotype and genotype of rheumatoid arthritis in the Democratic Republic of Congo.
Author: Malemba,J.J., Mbuyi-Muamba,J.M., Mukaya,J., Bossuyt,X., Emonds,M.P., Deiteren,K., Westhovens,R., Verschueren,P.
Abstract:
INTRODUCTION: The aim of this study was to describe the phenotype and genotype of rheumatoid arthritis (RA) in Congolese patients. METHODS: All consecutive RA patients attending Kinshasa University Hospital in a three year time period were included. Demographics, clinical features and tobacco consumption were noted. Disease activity score 28 based on the erythrocyte sedimentation rate (DAS-28-ESR), health sssessment questionnaire (HAQ), anti-cyclic citrullinated peptide (anti-CCP) antibodies and rheumatoid factor (RF) were determined. Radiographs were scored according to Sharp-van der Heijde. On a subset of patients and controls human leukocyte antigen (HLA)-DRB1 typing was performed. RESULTS: 114 females and 14 males aged 51.2 +/- 14.9 were included. Mean duration of symptoms was 4 years. Moderate tobacco consumption was reported in a minority of patients. DAS-28 at first visit was > 5.1 and HAQ[greater than or equal to] 0.5 in all patients. X-rays showed joint erosion and/or joint space narrowing, mostly of a moderate grade in 55.8%. Anti-CCP and/or RF were present in 48.6% of patients with available data (n=72) and in 3.0% of controls (n=67). Radiographic changes and nodules were more frequent in RF or anti-CCP positive patients. One copy of the shared epitope was found in 13 patients (35.1%) and 3 controls (12.5%). Two copies were found in one patient (2.7%) and in one control (4.2%). CONCLUSIONS: Congolese patients with RA consult long after disease onset. Despite this delay the majority presents without major damage and are RF, anti-CCP and SE negative. We put forward the hypothesis that besides different environmental factors there is probably also a particular genetic risk profile in Congolese patients, different from the HLA-DRB1 shared epitope.

3915813e: BMC Res.Notes, 331-331(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Hypercortisolaemia due to ectopic adrenocorticotropic hormone secretion by a nasal paraganglioma: a case report and review of the literature.
Author: Thomas,T., Zender,S., Terkamp,C., Jaeckel,E., Manns,M.P.
Abstract:
BACKGROUND: Adrenocorticotropic hormone-producing extraadrenal paragangliomas are extremely rare. We present a case of severe hypercortisolemia due to ectopic adrenocorticotropic hormone secretion by a nasal paraganglioma. CASE PRESENTATION: A 70-year-old Caucasian woman, was emergently admitted to our department with supraventricular tachycardia, oedema of face and extremities and hypertensive crisis. Initial laboratory evaluation revealed severe hypokalemia and hyperglycemia without ketoacidosis, although no diabetes mellitus was previously known. Computed tomography revealed a large tumor obliterating the left paranasal sinus and a left-sided adrenal mass. After cardiovascular stabilisation, a thorough hormonal assessment was performed revealing marked adrenocorticotropic hormone-dependent hypercortisolism. Due to the presence of a cardiac pacemaker magnetic resonance imaging of the hypophysis was not possible. [68Ga-DOTA]-TATE-Positron-Emission-Tomography was performed, showing somatostatin-receptor expression of the paranasal lesion but not of the adrenal lesion or the hypophysis. The paranasal tumor was resected and found to be an adrenocorticotropic hormone-producing paraganglioma of low-proliferative rate. Postoperatively the patient became normokaliaemic, normoglycemic and normotensive without further need for medication. Genetic testing showed no mutation of the succinatdehydrogenase subunit B- and D genes, thus excluding hereditary paragangliosis. CONCLUSION: Detection of the adrenocorticotropic hormone source in Cushing's syndrome can prove extremely challenging, especially when commonly used imaging modalities are unavailable or inconclusive. The present case was further complicated by the simultaneous detection of two tumorous lesions of initially unclear biochemical behaviour. In such cases, novel diagnostic tools - such as somatostatin-receptor imaging - can prove useful in localising hormonally active neuroendocrine tissue. The clinical aspects of the case are discussed and relevant literature is reviewed.

E-Mail: thomas.theodoros@mh-hannover.de
Address: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany

3915814e: J.Dairy Sci., 6440-6448(2013)
Link: [ PubMED ] [ DOI ]
Title: Alterations in milk metabolome and coagulation ability during the lactation of dairy cows.
Author: Harzia,H., Ilves,A., Ots,M., Henno,M., Joudu,I., Kaart,T., Ling,K., Kart,O., Kilk,K., Soomets,U.
Abstract:
Milk composition has been known to change during lactation. To help understand the changes in metabolic profile throughout the whole lactation, liquid chromatography mass-spectrometry was used to analyze 306 milk samples from 82 primi- and multiparous dairy cows. Changes in metabolic profile common to all cows throughout lactation were ascertained based on principal component and general linear model analysis. Sets of specific markers; for instance, 225, 397, and 641-642 m/z (positive mode), and 186, 241, and 601-604 (negative mode), with at least a 1.5-fold higher intensity during the first 60d compared with the last 60d of lactation were observed. The metabolome was affected by parity and milking time. Markers, identified as peptides differentiating parity, were observed. A significant increase for citrate was observed in evening milk. Milk coagulation traits were strongly animal specific. The curd firmness values were influenced by milking time. Sets of markers were associated with curd firmness in positive (197 m/z) and negative (612, 737, 835, 836, 902, 1000, 1038, and 1079 m/z) ion mode.

Address: Institute of Veterinary Medicine and Animal Science, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia

3915815e: Pathol.Int., 398-407(2013)
Link: [ PubMED ] [ DOI ]
Title: An immunohistochemical study of human platelets using a rabbit antibody against H18-K24 of apolipoprotein CIII (HATKTAK).
Author: Yamanaka,T., Sakamoto,H., Nakagawa,T., Tanaka,S., Matsumoto,K., Ueno,M.
Abstract:
H18-K24 of human apolipopotein CIII (Apo CIII) (HATKTAK) is an activator of the macromolecular activators of phagocytosis from platelets (MAPPs). Using a rabbit antibody against HATKTAK, we performed an immunohistochemical study of human platelets. Indirect ELISA showed that this antibody reacts with Apo CIII-derived peptides with a C-terminal of HATKTAK, but not with Apo CIII. Immunoelectron microscopy revealed that reaction of anti-HATKTAK antibody occurred in the pseudopods of activated platelets. In blood coagula produced from the peripheral blood and formalin-fixed after various incubation periods, reaction of this antibody with platelets appeared rapidly with a peak at 3 to 6 h of incubation, and then diminished gradually. Leukocytes in the blood coagula were stained strongly positive. In tissue sections, fresh thrombi and hemorrhages with slight fibrin formation revealed a positive response of platelets to anti-HATKTAK antibody, whereas older ones with leukocytic infiltration, fibrin formation and organization did not. In addition to platelets, endothelial cells and leukocytes were stained positive by anti-HATKTAK antibody. All of the positive reactions by anti-HATKTAK antibody disappeared or diminished by co-incubation with HATKTAK. In conclusion, the anti-HATKTAK antibody reveals platelets during the early phase of activation.

Address: Inflammation Pathology, Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa, Japan

3915816e: J.Neuroendocrinol., 929-938(2013)
Link: [ PubMED ] [ DOI ]
Title: Glucagon-like peptide-1 receptor agonist administration suppresses both water and saline intake in rats.
Author: McKay,N.J., Daniels,D.
Abstract:
Glucagon-like peptide-1 (GLP-1) plays an important role in energy homeostasis. Injections of GLP-1 receptor (GLP-1R) agonists suppress food intake, and endogenous GLP-1 is released when nutrients enter the gut. There is also growing evidence that the GLP-1 system is involved in the regulation of body fluid homeostasis. GLP-1R agonists suppress water intake independent of their effects on food intake. It is unknown, however, whether this suppressive effect of GLP-1R agonists extends to saline intake. Accordingly, we tested the effect of the GLP-1R agonists liraglutide (0.05 μg) and exendin-4 (0.05 μg) on water and saline intake, as stimulated either by angiotensin II (AngII) or by water deprivation with partial rehydration (WD-PR). Each agonist suppressed AngII-induced water intake; however, only exendin-4 suppressed saline intake. WD-PR-induced water and saline intakes were both attenuated by each agonist. Analysis of drinking microstructure after WD-PR found a reliable effect of the agonists on burst number. Furthermore, exendin-4 conditioned a robust taste avoidance to saccharine; however, there was no similar effect of liraglutide. To evaluate the relevance of the conditioned taste avoidance, we tested whether inducing visceral malaise by injection of lithium chloride (LiCl) suppressed fluid intake. Injection of LiCl did not suppress water or saline intakes. Overall, these results indicate that the fluid intake suppression by GLP-1R activation is not selective to water intake, is a function of post-ingestive feedback, and is not secondary to visceral malaise.

Address: Behavioral Neuroscience Program, Department of Psychology, The State University of New York at Buffalo, Buffalo, NY, USA

3915819e: J.Food Sci., C1152-C1158(2013)
Link: [ PubMED ] [ DOI ]
Title: Partial characterization of ultrafiltrated soy protein hydrolysates with antioxidant and free radical scavenging activities.
Author: Jimenez-Ruiz,E.I., Calderon de la Barca,A.M., Sotelo-Mundo,R.R., Arteaga-Mackinney,G.E., Valenzuela-Melendez,M., Pena-Ramos,E.A.
Abstract:
Soy protein isolate (SPI) was hydrolyzed with Flavourzyme(R) (SHF) or chymotrypsin (SHC). Hydrolysates were sequencially fractionated by ultrafiltration using different membrane pore sizes (50, 10, and 3 kDa). The antioxidant ability of each hydrolysate protein fraction was tested in a liposome oxidizing system and their free radical scavenging activity (FRSA) was evaluated with the DPPH method (diphenylpicrilhydrazine radical). Molecular weight (MW) distribution, solubility, surface hydrophobicity, and amino acid composition of each SPI hydrolysate fraction were measured and their effect on antioxidant and scavenging activities was established by multivariate correlation. The most active ultrafiltrated peptide fractions (P < 0.05), from SHF and SHC, had of MW of <3 kDa (F3 and C3, respectively). These fractions decreased liposome oxidation by 83.2% and 84.5%, respectively, and also showed the highest FRSA (F3: 21.3% and C3: 24.4%). In addition to molecular size, the antioxidant activity and FRSA of soy protein fractions were related to their amino acid composition, especially to an increased content of Phe and a lowered content of Lys. Also, hydrophobicity of ultrafiltrated peptide fractions was an important characteristic (P < 0.001) associated with their ability to trap free radicals. Ultrafiltered peptide fractions with low MW have a high potential to be used as natural alternatives to prevent lipid oxidation in foods.

Address: Centro de Investigacion en Alimentacion y Desarrollo, A.C. Carr. a la Victoria km 0.6, Hermosillo Sonora, C.P. 83304, Mexico

3915821e: BioMed Res.Int., 397391-397391(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Concerted action of ANP and dopamine D1-receptor to regulate sodium homeostasis in nephrotic syndrome.
Author: Fernandes-Cerqueira,C., Sampaio-Maia,B., Quelhas-Santos,J., Moreira-Rodrigues,M., Simoes-Silva,L., Blazquez-Medela,A.M., Martinez-Salgado,C., Lopez-Novoa,J.M., Pestana,M.
Abstract:
The edema formation in nephrotic syndrome (NS) is associated with a blunted response to atrial natriuretic peptide (ANP). The natriuretic effects of ANP have been related to renal dopamine D1-receptors (D1R). We examined the interaction between ANP and renal D1R in rats with puromycin aminonucleoside-induced NS (PAN-NS). Urinary sodium, cyclic guanosine monophosphate (cGMP) excretion, and D1R protein expression and localization in renal tubules were evaluated in PAN-NS and control rats before and during volume expansion (VE). The effects of zaprinast (phosphodiesterase type 5 inhibitor), alone or in combination with Sch-23390 (D1R antagonist), were examined in both groups. The increased natriuresis and urinary cGMP excretion evoked by acute VE were blunted in PAN-NS despite increased levels of circulating ANP. This was accompanied in PAN-NS by a marked decrease of D1R expression in the renal tubules. Infusion of zaprinast in PAN-NS resulted in increased urinary excretion of cGMP and sodium to similar levels of control rats and increased expression of D1R in the plasma membrane of renal tubular cells. Combined administration of Sch-23390 and zaprinast prevented natriuresis and increased cGMP excretion induced by zaprinast alone. We conclude that D1R may play a major role in the ANP resistance observed in PAN-NS.

Address: Nephrology Research & Development Unit, Faculty of Medicine, University of Porto & Hospital S. Joao EPE, Alameda Prof. Hernani Monteiro, 4200-319 Porto, Portugal

3915822e: Open J.Clin.Diagn., 37-51(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: SCNH2 is a novel apelinergic family member acting as a potent mitogenic and chemotactic factor for both endothelial and epithelial cells.
Author: Fang,C., Avis,I., Bianco,C., Held,N., Morris,J., Ylaya,K., Hewitt,S.M., Aplin,A.C., Nicosia,R.F., Fung,L.A., Lewis,J.D., Stetler-Stevenson,W.G., Salomon,D.S., Cuttitta,F.
Abstract:
The gut hormone apelin is a major therapeutic focus for several diseases involving inflammation and aberrant cell growth. We investigated whether apelin-36 contained alternative bioactive peptides associated with normal physiology or disease. Amino acid sequence analysis of apelin-36 identified an amidation motif consistent with the formation of a secondary bioactive peptide (SCNH2). SCNH2 is proven to be mitogenic and chemotactic in normal/malignant cells and augments angiogenesis via a PTX-resistant/CT-X-sensitive G protein-coupled receptor (GPCR). Notably, SCNH2 is substantially more potent and sensitive than apelin-13 and vascular endothelial growth factor-A. Endogenous SCNH2 is highly expressed in human tumors and placenta and in mouse embryonic tissues. Our findings demonstrate that SCNH2 is a new apelinergic member with critical pluripotent roles in angiogenesis related diseases and embryogenesis via a non-APJ GPCR.

Address: Angiogenesis Core Facility, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, USA

3915825e: Quat.Geochronol., 158-172(2013)
Link: [ PubMED ] [ DOI ]
Title: Intra-crystalline protein diagenesis (IcPD) in . Part II: Breakdown and temperature sensitivity.
Author: Demarchi,B., Collins,M.J., Tomiak,P.J., Davies,B.J., Penkman,K.E.
Abstract:
Artificial diagenesis of the intra-crystalline proteins isolated from Patella vulgata was induced by isothermal heating at 140 degrees C, 110 degrees C and 80 degrees C. Protein breakdown was quantified for multiple amino acids, measuring the extent of peptide bond hydrolysis, amino acid racemisation and decomposition. The patterns of diagenesis are complex; therefore the kinetic parameters of the main reactions were estimated by two different methods: 1) a well-established approach based on fitting mathematical expressions to the experimental data, e.g. first-order rate equations for hydrolysis and power-transformed first-order rate equations for racemisation; and 2) an alternative model-free approach, which was developed by estimating a "scaling" factor for the independent variable (time) which produces the best alignment of the experimental data. This method allows the calculation of the relative reaction rates for the different temperatures of isothermal heating. High-temperature data were compared with the extent of degradation detected in sub-fossil Patella specimens of known age, and we evaluated the ability of kinetic experiments to mimic diagenesis at burial temperature. The results highlighted a difference between patterns of degradation at low and high temperature and therefore we recommend caution for the extrapolation of protein breakdown rates to low burial temperatures for geochronological purposes when relying solely on kinetic data.

Address: BioArCh, Departments of Biology, Archaeology and Chemistry, University of York, York YO10 5DD, UK

3915833e: Cell Biol.Int., 1300-1307(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of PTHrP on expression of MMP9 and MMP13 in sika deer antler chondrocytes.
Author: Wang,S.T., Gao,Y.J., Duan,C.C., Li,D.D., Tian,X.C., Zhang,Q.L., Guo,B., Yue,Z.P.
Abstract:
Deer antlers are the only mammalian appendages to display an annual cycle of full regeneration. However, little is known about the molecular mechanisms of antler regeneration. Our previous study has demonstrated that parathyroid hormone-related peptide (PTHrP) can promote proliferation of antler chondrocytes and inhibit its differentiation, but the mechanism underlying such regulation is not fully understood. We have determined the role of PTHrP on the mRNA expression of matrix metalloproteinase-9 (MMP9) and MMP13 in the antler chondrocytes. The possible pathways that transduce PTHrP effects were examined. In situ hybridization showed that MMP9 and MMP13 were mainly localized in the dermal fibroblasts, perichondrium, and cartilage in the sika deer antler, of which MMP9 and MMP13 were highly expressed in the chondrocytes. Exogenous PTHrP could inhibit the expression of MMP9 and MMP13 in the antler chondrocytes. The inhibitory effect of PTHrP on MMP9 was abolished by JNK inhibitor, SP600125, while P38MAPK inhibitor SB203850 and PKC inhibitor GF109203X could rescue the inhibitory effect of PTHrP on MMP13. The results suggest that PTHrP can inhibit MMP9 expression by JNK signaling pathway and MMP13 expression by p38MAPK and PKC signaling pathways in the antler chondrocytes. Thus PTHrP is involved in the control of antler chondrocytes maturation and cartilage matrix degradation.

Address: Department of Histology and Embryology, College of Veterinary Medicine, Jilin University, Changchun, 130062, P. R. China

3915836e: Cell Tissue Res., 431-440(2013)
Link: [ PubMED ] [ DOI ]
Title: Distribution of neuropeptides in the antennal lobes of male Spodoptera littoralis.
Author: Kromann,S.H., Hansson,B.S., Ignell,R.
Abstract:
Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.

E-Mail: skromann@ice.mpg.de
Address: Department of Plant Protection Biology, Unit of Chemical Ecology, Swedish University of Agricultural Sciences, Box 102, 230 53, Alnarp, Sweden,

3915840e: Am.J.Clin.Pathol., 387-394(2013)
Link: [ PubMED ] [ DOI ]
Title: MYD88 L265P somatic mutation: its usefulness in the differential diagnosis of bone marrow involvement by B-cell lymphoproliferative disorders.
Author: Ondrejka,S.L., Lin,J.J., Warden,D.W., Durkin,L., Cook,J.R., Hsi,E.D.
Abstract:
OBJECTIVES: To examine the usefulness of the MYD88 L265P somatic mutation in identifying cases of lymphoplasmacytic lymphoma (LPL) from other lymphoplasmacytic neoplasms in bone marrow biopsy specimens. METHODS: We studied 64 bone marrow biopsy specimens with involvement by various small B-cell lymphomas or plasma cell myeloma. RESULTS: The MYD88 L265P somatic mutation was present in 13/13 cases of LPL, 1/13 cases of hairy cell leukemia, and absent in the other mature B-cell neoplasms tested. A test set of diagnostically challenging bone marrow cases with lymphoplasmacytoid morphology (B-cell lymphoma, not otherwise specified) was selected for additional review and reclassified, without knowledge of the MYD88 L265P status. Of those 16 cases, 7 were positive for MYD88, including 4/4 cases that were reclassified as LPL during the review. CONCLUSIONS: Although not entirely specific, MYD88 L265P is a useful adjunct for bone marrow diagnosis in separating LPL from other small B-cell lymphomas and plasma cell myeloma.

Address: Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH, USA

3915841e: Am.J.Clin.Pathol., 314-323(2013)
Link: [ PubMED ] [ DOI ]
Title: MS-based monitoring of proteolytic decay of synthetic reporter peptides for quality control of plasma and serum specimens.
Author: Findeisen,P., Thumfart,J.O., Costina,V., Hofheinz,R., Neumaier,M.
Abstract:
OBJECTIVES: To determine the preanalytical quality of serum and plasma by monitoring the time-dependent ex vivo decay of a synthetic reporter peptide (RP) with liquid chromatography/mass spectrometry (LC/MS). METHODS: Serum and plasma specimens were spiked with the RP and proteolytic fragments were monitored with LC/MS at different preanalytical time points ranging from 2 to 24 hours after blood withdrawal. RESULTS: The concentration of fragments changed in a time-dependent manner, and respective peptide profiles were used to classify specimens according to their preanalytical time span. Classification accuracy was high, with values always above 0.89 for areas under receiver operating characteristic curves. CONCLUSIONS: This "proteomics degradation clock" can be used to estimate the preanalytical quality of serum and plasma and might have impact on quality control procedures of biobanking repositories.

E-Mail: peter.findeisen@umm.de
Address: Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, Theodor-Kutzer-Ufer 1, 68167 Mannheim, Germany

3915842e: J.Pharm.Sci., 3539-3544(2013)
Link: [ PubMED ] [ DOI ]
Title: Development of a nose cream containing the synthetic antimicrobial peptide P60.4Ac for eradication of methicillin-resistant Staphylococcus aureus carriage.
Author: Goblyos,A., Schimmel,K.J., Valentijn,A.R., Fathers,L.M., Cordfunke,R.A., Chan,H.L., Oostendorp,J., Nibbering,P.H., Drijfhout,J.W., Hiemstra,P.S., Hartigh,J.D.
Abstract:
Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing problem, and current treatment options are suboptimal. Nasal carriage of MRSA is a major risk factor for infection, but nasal eradication strategies are increasingly considered to be insufficiently effective. In this study, a water-in-oil cream formulation was developed for nasal application with an antimicrobial peptide, P60.4Ac, aimed at the eradication of MRSA carriage. Quality control of the cream included the measurement of the content and release of the peptide by a validated high-performance liquid chromatography method. Stability of the peptide in the formulation was investigated including the evaluation of the effect of stress conditions. Preliminary shelf-life study of the drug formulation demonstrated that the peptide is stable in the formulation at least for 5 months. Microbial-killing assays with MRSA LUH14616 as a target demonstrated the dose-dependent antimicrobial activity of the peptide formulation.

Address: Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands

3915851e: Physiol.Behav., 173-181(2013)
Link: [ PubMED ] [ DOI ]
Title: Oral processing effort, appetite and acute energy intake in lean and obese adults.
Author: Mattes,R.D., Considine,R.V.
Abstract:
Chewing reportedly contributes to satiation and satiety signals. Attempts to document and quantify this have led to small and inconsistent effects. The present trial manipulated oral processing effort though required chewing of gums of different hardness and measured appetitive sensations, energy intake, gastric emptying, GI transit time, and concentrations of glucose, insulin, GLP-1, ghrelin and pancreatic polypeptide. Sixty adults classified by sex and BMI (15 each of lean females, obese females, lean males and obese males) were tested in a randomized, controlled, cross-over trial with three arms. They chewed nothing, soft gum or hard gum for 15min while sipping grape juice (10% of individual energy needs) containing acetaminophen and lactulose on one day each separated by 7days. Electromyographic recordings and self-reports were obtained during and after chewing to quantify oral processing effort. Blood was sampled through an indwelling catheter and appetite ratings were obtained at baseline and at 0, 15, 30, 45, 60, 90, 120, 180 and 240min after chewing initiation. Breath samples were collected at 10min intervals for the first 2h and at 30min intervals for the next 2h. No effects of chewing were observed for appetitive sensations or gut peptide concentrations. Energy intake tended to decline in lean and increase in obese participants so that daily energy intake differed significantly between the two groups when chewing either gum, while no difference was observed on the non-chewing day. Serum glucose and insulin were significantly lower at selected time points 90-240min after chewing compared to baseline and the non-chewing day. These data indicate chewing effort does not affect appetitive sensations or gut peptide secretion, but may exert a small differential effect on acute energy intake in lean and obese individuals and lead to greater post-prandial declines of serum glucose and insulin. The efficacy of gum chewing as a substitute for eating for weight management remains uncertain.

E-Mail: mattes@purdue.edu
Address: Purdue University, Department of Nutrition Science, West Lafayette, IN, USA. Electronic address:

3915853e: Mol.Biochem.Parasitol., 24-27(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of a family of four UDP-polypeptide N-acetylgalactosaminyl transferases in Cryptosporidium species.
Author: Bhat,N., Wojczyk,B.S., Decicco,M., Castrodad,C., Spitalnik,S.L., Ward,H.D.
Abstract:
Although mucin-type O-glycans are critical for Cryptosporidium infection, the enzymes catalyzing their synthesis have not been studied. Here, we report four UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl transferases (ppGalNAc-Ts) from the genomes of C. parvum, C. hominis and C. muris. All are Type II membrane proteins which include a cytoplasmic tail, a transmembrane domain, a stem region, a glycosyltransferase family 2 domain and a C-terminal ricin B lectin domain. All are expressed during C. parvum infection in vitro, with Cp-ppGalNAc-T1 and -T4 expressed at 24h and Cp-ppGalNAc-T2 and -T3 at 48 and 72h post-infection, suggesting that their expression may be developmentally regulated. C. parvum sporozoite lysates display ppGalNAc-T enzymatic activity against non-glycosylated and pre-glycosylated peptides suggesting that they contain enzymes capable of glycosylating both types of substrates. The importance of mucin-type O-glycans in Cryptosporidium-host cell interactions raises the possibility that Cp-ppGalNAc-Ts may serve as targets for intervention in cryptosporidiosis.

Address: Tufts Medical Center, Boston, MA, United States

3915855e: J.Ethnopharmacol., 389-392(2013)
Link: [ PubMED ] [ DOI ]
Title: Modulation effect of Smilax glabra flavonoids on ryanodine receptor mediated intracellular Ca2+ release in cardiomyoblast cells.
Author: Shou,Q., Pan,S., Tu,J., Jiang,J., Ling,Y., Cai,Y., Chen,M., Wang,D.
Abstract:
ETHNOPHARMACOLOGICAL RELEVANCE: Smilax glabra rhizome, a plant material from Liliaceae family, is a widely used traditional Chinese medicine for anti-cardiac hypertrophy treatment. We have previously found that Smilax glabra flavonoids (SGF) exerted such anti-cardiac hypertrophy activity. However, the mechanism of this activity of SGF has not been clarified yet. MATERIALS AND METHODS: This study was aimed to investigate the inhibitory role of SGF on intracellular Ca2+ release in rat cardiomyoblast cells (H9C2). Intracellular Ca2+ release was determined by Ca2+ indicator fluorescence (fluo 4-AM) in H9C2 cell line. RESULTS: SGF at concentrations of 0.25, 0.5, 1.0mg/ml significantly inhibited the phenylephrine or angiotensin II induced intracellular Ca2+ release in a dose-dependent manner. Furthermore, SGF could also inhibit ryanodine receptor (RyR) agonist caffeine induced Ca2+ release and phenylephrine (PE)-induced Ca2+ release under the condition in which inositol trisphosphate (IP3) receptors were blocked with 2-Aminoethoxydiphenyl borate (2-APB). Nevertheless, SGF had no impact on PE-induced Ca2+ release under the condition in which RyRs were blocked with tetracaine. CONCLUSIONS: Our results suggest that the protective effects of SGF are mediated via targeting inhibition of RyR mediated intracellular Ca2+ release.

Address: Experimental Animal Research Center, Zhejiang Chinese Medical University, Hangzhou 310053, China

3915857e: Neurobiol.Aging, 203-212(2014)
Link: [ PubMED ] [ DOI ]
Title: Selective benefits of simvastatin in bitransgenic APPSwe,Ind/TGF-β1 mice.
Author: Papadopoulos,P., Tong,X.K., Hamel,E.
Abstract:
Cognitive and cerebrovascular deficits are 2 landmarks of Alzheimer's disease (AD) to target for effective therapy. Here, we evaluated the efficacy of simvastatin in bitransgenic A/T mice overexpressing a mutated form of the human amyloid precursor protein (APPSwe,Ind) and a constitutively active form of transforming growth factor-β1. These mice feature the AD amyloid β (Aβ) and cerebrovascular pathology. Simvastatin significantly decreased insoluble Aβ peptide levels and Aβ plaque load despite no effect on β-site amyloid precursor protein-cleaving enzyme and Aβ-degrading enzyme neprilysin protein levels. However, simvastatin failed to improve spatial learning and memory deficits and the decreased baseline levels of the memory-related protein early growth response-1 (Egr-1) in the hippocampus CA1 area. The impaired hyperemic response to whisker stimulation in A/T mice was not improved with treatment, but simvastatin fully restored constitutive nitric oxide synthesis in vessel walls and exacerbated agonist-mediated dilatory deficits. These findings point to the efficacy of simvastatin on selective AD features in a complex model of the disease, likely reflecting the challenges faced by recent clinical trials in assessing statin efficacy.

Address: Laboratory of Cerebrovascular Research, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada

3915858e: Neurobiol.Aging, 213-222(2014)
Link: [ PubMED ] [ DOI ]
Title: Nardilysin prevents amyloid plaque formation by enhancing α-secretase activity in an Alzheimer's disease mouse model.
Author: Ohno,M., Hiraoka,Y., Lichtenthaler,S.F., Nishi,K., Saijo,S., Matsuoka,T., Tomimoto,H., Araki,W., Takahashi,R., Kita,T., Kimura,T., Nishi,E.
Abstract:
Amyloid β (Aβ) peptide, the main component of senile plaques in patients with Alzheimer's disease (AD), is derived from proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretases. Alpha-cleavage of APP by α-secretase has a potential to preclude the generation of Aβ because it occurs within the Aβ domain. We previously reported that a metalloendopeptidase, nardilysin (N-arginine dibasic convertase; NRDc) enhances α-cleavage of APP, which results in the decreased generation of Aβ in vitro. To clarify the in vivo role of NRDc in AD, we intercrossed transgenic mice expressing NRDc in the forebrain with an AD mouse model. Here we demonstrate that the neuron-specific overexpression of NRDc prevents Aβ deposition in the AD mouse model. The activity of α-secretase in the mouse brain was enhanced by the overexpression of NRDc, and was reduced by the deletion of NRDc. However, reactive gliosis adjacent to the Aβ plaques, one of the pathological features of AD, was not affected by the overexpression of NRDc. Taken together, our results indicate that NRDc controls Aβ formation through the regulation of α-secretase.

Address: Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan

3915861e: Cytokine, 208-212(2013)
Link: [ PubMED ] [ DOI ]
Title: The association between TNF-α and insulin resistance in euglycemic women.
Author: Walsh,J.M., McGowan,C.A., Byrne,J.A., Rath,A., McAuliffe,F.M.
Abstract:
Chronic low levels of inflammation have links to obesity, diabetes and insulin resistance. We sought to assess the relationship between cytokine tumor necrosis factor (TNF-α) and insulin resistance in a healthy, euglycemic population. This is a prospective study of 574 non-diabetic mother and infant pairs. Maternal body mass index (BMI), TNF-α, glucose and insulin were measured in early pregnancy and at 28 weeks. Insulin resistance was calculated by HOMA index. At delivery birthweight was recorded and cord blood analysed for fetal C-peptide and TNF-α. In a multivariate model, maternal TNF-α in early pregnancy was predicted by maternal insulin resistance at the same time-point, (β=0.54, p<0.01), and maternal TNF-α at 28 weeks was predicted by maternal insulin resistance in early pregnancy (β=0.24, p<0.01) and at 28 weeks (β=0.39, p<0.01). These results, in a large cohort of healthy, non-diabetic women have shown that insulin resistance, even at levels below those diagnostic of gestational diabetes, is associated with maternal and fetal inflammatory response. These findings have important implications for defining the pathways of fetal programming of later metabolic syndrome and childhood obesity.

Address: UCD Obstetrics and Gynaecology, School of Medicine and Medical Science, University College Dublin, Ireland

3915864e: Anim.Reprod.Sci., 62-67(2013)
Link: [ PubMED ] [ DOI ]
Title: Different types of opioid receptors involved in the suppression of LH secretion in lactating sheep.
Author: Dobek,E., Gorski,K., Romanowicz,K., Misztal,T.
Abstract:
To learn the involvement of endogenous opioid peptides (EOP) in the regulation of reproductive activity in ruminants, the effects of different opioid antagonists on luteinizing hormone (LH) secretion were determined in sheep during the early stage of lactation. The opioid receptor antagonists: naloxone (all types of receptors, n=5), naloxonazine (μ receptor, n=5), GNTI- (κ receptor, n=5), naltrindole (δ receptor, n=5) or the vehicle (control, n=5) were infused intracerebroventricularly in a series of five 30-min infusions (60μg/60μl) at 30-min intervals. The period of the experiment included the non-suckling (10:00-12.30) and suckling (12.30-15.00) periods. Blood samples were collected from 10.00 to 15.00 at 10-min intervals, and plasma LH concentration was assayed by the radioimmunoassay method. The obtained results showed that blocking of the EOP action within the central nervous system in lactating sheep caused a significant (p<0.001) increase in LH concentration in all treated groups, in comparison to the control. In the naloxone-treated group, a significant (p<0.05) increase in LH secretion also occurred during suckling. The amplitude of LH pulses increased significantly in the naloxonazine- (p<0.01) and naltrindole- (p<0.05) treated ewes compared to the control; there were no significant differences in the frequency of LH pulses among the groups. In conclusion, our study indicates that EOP play a crucial role in the mechanism inhibiting GnRH/LH axis activity in lactating sheep and that the ligands for μ opioid receptor may have the highest inhibitory effect.

Address: Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jablonna n/Warsaw, Poland

3915867e: Transplant.Proc., 2539-2542(2013)
Link: [ PubMED ] [ DOI ]
Title: Cell stress response in rat chronic small bowel allograft rejection.
Author: Wang,J., Li,Y., Li,J.
Abstract:
BACKGROUND: We sought to assess the expression of five cell stress molecules: Heat shock protein (HSP)-60, HSP-70, interferon (IFN)-α, rat retinoic acid early induible-1 like transcript (RRLT), and vasoactive intestinal peptide (VIP) during rat chronic small bowel allograft rejection. METHODS: A rat model of small bowel allograft rejection was induced on postoperative day 90 using F344 rat as donor and Lewis rat as recipient. RESULTS: We demonstrated increased expression of lamina propria HSP-60 (mean rank grade 8.3 and 3.2, respectively), graft RRLT (relative mRNA amount 5.1 +/- 1.8 and 3.0 +/- 1.3, respectively) and IFN-α (relative mRNA amount 108.3 +/- 49.2 and 59.2 +/- 22.0, respectively) in allograft group compared with the isograft group. CONCLUSION: These data suggest the presence of cell stress during chronic small bowel allograft rejection.

Address: Department of Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China

3915868e: Transplant.Proc., 2506-2512(2013)
Link: [ PubMED ] [ DOI ]
Title: Oxytocin ameliorates the immediate myocardial injury in rat heart transplant through downregulation of neutrophil-dependent myocardial apoptosis.
Author: Al-Amran,F., Shahkolahi,M.
Abstract:
BACKGROUND: Cardiac oxytocin (OT) is structurally identical to that found in the hypothalamus, indicating that this active form of OT is derived from the same gene. The abundance of OT and its receptors in atrial myocytes suggests that, directly and/or via the release of the cardiac hormone atrial natriuretic peptide, this hormone regulates the force of cardiac contractions. Previous studies have demonstrated a role of OT in myocardial inflammatory responses. We sought to study protective myocardial and anti-inflammatory effects of OT in the immediate post-transplant period. METHODS: We grouped adult male Albino rats into sham, control, and OT-treated groups. Control and treated groups underwent heterotopic cervical heart transplantation. Myocardial injury was assessed by measuring plasma cardiac troponin I, and myocardial proinflammatory cytokines as well as by performing histopathologic assessments injury score, and of apoptotic degree. Myocardial myeloperoxidase, neutrophil infiltration, neutrophil chemotactic mediators as well as formation of reactive oxygen and reactive nitrogen species were measured in the myocardium at 3 hours after reperfusion to assess neutrophil-dependent myocardial injury. RESULTS: OT downregulates neutrophil chemotactic molecules--KC/CXCL1 and MIP-2/CXCL2. The decrement in myocardial PMN infiltration was associated with reduced reactive oxygen and reactive nitrogen species formation in the myocardium at 3 hours after reperfusion following global ischemia. OT reduced postmyocardial ischemia/reperfusion apoptotic processed. CONCLUSION: OT ameliorated immediate myocardial injury in heart grafts, through downregulation of the inflammatory response, of reactive oxygen species, and of neutrophil dependent apoptosis.

E-Mail: fadhil.al-amran@ucdenver.edu
Address: Surgical Department, Medical College, Kufa University, Najaf, Iraq

3915869e: Ann.Med., 455-464(2013)
Link: [ PubMED ] [ DOI ]
Title: Fatty acids as determinants of in-vivo lipid peroxidation: the EFFGE study in Eastern Finnish hypertensive and non-hypertensive subjects.
Author: Kaikkonen,J.E., Vilppo,T., Asikainen,J., Voutilainen,S., Kurl,S., Salonen,J.T.
Abstract:
BACKGROUND: The degree of fatty acid (FA) unsaturation as a determinant of lipid peroxidation has been inadequately studied. METHODS: We examined associations of plasma free F2α-isoprostanes (F2-IsoPs), an indicator of in-vivo lipid peroxidation, with the levels/intake of FAs, adjusted for the risk factors of cardiovascular disease (CVD) in 1211 Finnish men and women, of whom 50% were hypertensive, aged 59.3 +/- 8.3 years, mean +/- SD. RESULTS: Elevated age- and sex-adjusted plasma free levels of ω-6 and ω-3 polyunsaturated Fas (PUFAs), saturated FAs (SFAs), and the PUFA/SFA and the ω-6/ω-3 PUFA ratios were all associated with decreased F2-IsoPs. High dietary SFA intake was associated with elevated F2-IsoP concentrations. In a multivariable regression (with clinical, nutritional, and behavioral CVD risk factors), female gender, body mass index (BMI), serum apolipoprotein A1, and NT-proBNP (natriuretic peptide) were positively associated with the F2-IsoPs, whereas the dietary PUFA/SFA ratio, plasma β-carotene, the ω-6/ω-3 PUFA ratio, and protein intake showed inverse associations. CONCLUSIONS: We propose that elevated lipid peroxidation is associated with several risk factors of CVD, such as a low PUFA/SFA ratio, whereas the FA precursors of lipid peroxidation, i.e. ω-3 and ω-6 PUFAs are associated with attenuated F2-IsoP levels. These findings provide mechanistic support for earlier observations linking PUFA to improved cardiovascular health.

E-Mail: jari.kaikkonen@utu.fi
Address: The Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku, Finland

3915874e: J.Vet.Intern.Med., 1097-1104(2013)
Link: [ PubMED ] [ DOI ]
Title: Comparative effect of carperitide and furosemide on left atrial pressure in dogs with experimentally induced mitral valve regurgitation.
Author: Suzuki,S., Fukushima,R., Yamamoto,Y., Ishikawa,T., Hamabe,L., Kim,S., Yoshiyuki,R., Fukayama,T., Machida,N., Tanaka,R.
Abstract:
BACKGROUND: The effects of carperitide on left atrial pressure (LAP) in dogs with mitral valve disease (mitral regurgitation, MR) have not been documented. OBJECTIVE: The objective was to compare the short-term effects of carperitide versus furosemide on LAP and neurohumoral factors in MR dogs. ANIMALS: Six healthy Beagle dogs weighing 9.8-12.6 kg (2 males and 4 females; aged 3 years) were used. METHODS: Experimental, randomized, cross-over, and interventional study. Carperitide 0.1 μg/kg/min or furosemide 0.17 mg/kg/h (1 mg/kg/6 h) was administered to dogs with surgically induced MR for 6 hours, and after a 14 day wash-out period, the other drug was administered. LAP, plasma renin activity, plasma aldosterone, and echocardiographic variables were measured. RESULTS: Left atrial pressure was decreased similarly after the administration of carperitide 0.1 μg/kg/min and furosemide 0.17 mg/kg/h (1 mg/kg/6 h) compared with baseline in dogs with MR (Baseline 14.75 +/- 3.74 mmHg, carperitide 10.24 +/- 4.97 mmHg, P < .01, furosemide 10.77 +/- 5.06 mmHg, P < .05). Plasma renin activity and plasma aldosterone were significantly lower after the administration of carperitide than after the administration of furosemide (P < .05, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Carperitide significantly decreased LAP in dogs with acute MR caused by experimental chordal rupture. Carperitide can have additional benefits from the viewpoint of minimal activation of neurohumoral factors in the treatment of dogs with MR. Additional studies in dogs with spontaneous disease are warranted.

Address: Department of Veterinary Surgery, Faculty of Veterinary Medicine, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo, Japan

3915875e: J.Med.Primatol., 318-324(2013)
Link: [ PubMED ] [ DOI ]
Title: Somatometric measurements, and clinical chemistry and hematology parameters in Tiβn macaque (Macaca thiβna).
Author: Wu,D., Jiang,W.Y., Yang,F., Wei,S.Y., Zhou,L., Yi,Y., Wang,H.X., Zhang,Y.A., Yue,F.
Abstract:
BACKGROUND: Limited physiological data for Tiβn macaques are available at present. This study will provide more rationale for evaluating this species. METHODS: Thirty-seven Tiβn macaques (15 males and 22 females) were used in this study. Somatometric measurements, clinical chemistry and hematology parameters, insulin, and C-peptide were analyzed. RESULTS: Females had higher values of waist and waist hip ratio (WHR) than males in somatometric measurements. There were no significant differences between the two genders in hematology. Significant differences between males and females were only found for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in biochemistry testing. In addition, females had higher fasting insulin and C-peptide than males. There was a strongly positive correlation between age and some somatometric parameters. CONCLUSIONS: These physiological data will provide veterinarians and researchers with baseline values to evaluate experimental results using Tiβn macaques.

Address: Cell Therapy Center, Beijing Institute of Geriatrics, Xuanwu Hospital, Capital Medical University, Beijing, China

3915876e: J.Clin.Periodontol., 933-941(2013)
Link: [ PubMED ] [ DOI ]
Title: LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid.
Author: McCrudden,M.T., Orr,D.F., Yu,Y., Coulter,W.A., Manning,G., Irwin,C.R., Lundy,F.T.
Abstract:
AIM: To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease. MATERIALS AND METHODS: Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS: The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites. CONCLUSIONS: LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis.

Address: Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, UK

3915877e: Ann.Otol.Rhinol.Laryngol., 440-444(2013)
Link: [ PubMED ]
Title: Salivary concentration of epidermal growth factor in adults with reflux laryngitis before and after treatment.
Author: Eckley,C.A., Sardinha,L.R., Rizzo,L.V.
Abstract:
OBJECTIVES: The diagnosis of laryngopharyngeal reflux (LPR) is controversial. There is no correlation between the number of reflux episodes and the severity of the inflammatory response at the esophagus or the laryngopharyngeal segment. Some authors have suggested that decreased salivary epidermal growth factor (EGF) concentrations in patients with gastroesophageal reflux disease and LPR point to a breakdown in the local defenses. Our objective was to establish whether treatment of the disease influences low salivary EGF concentrations. METHODS: The spontaneous whole saliva of 20 adults with LPR was sampled at a tertiary teaching hospital before and after a 16-week course of full-dose proton pump inhibitor and compared to that of 12 healthy controls. Salivary EGF concentrations were established with a commercially available enzyme-linked immunosorbent assay kit. RESULTS: Although the mean salivary EGF concentrations were higher before treatment than after treatment and control of the disease (25,083 versus 19,359 pg/mL), this difference was not statistically significant (p = 0.065). The mean salivary EGF concentration of healthy control subjects was significantly higher (54,509 pg/mL; p < 0.0001). CONCLUSIONS: Both before and after treatment, patients with reflux laryngitis present lower salivary EGF concentrations than healthy control subjects, suggesting a primary deficit in their protective mechanisms.

Address: Department of Otolaryngology, Santa Casa School of Medicine and Hospitals of Sao Paulo, Sao Paulo, Brazil

3915883e: PLoS One, e71992-e71992(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Sex differences in the beneficial cardiac effects of chronic treatment with atrial natriuretic peptide in spontaneously hypertensive rats.
Author: Romero,M., Caniffi,C., Bouchet,G., Elesgaray,R., Laughlin,M.M., Tomat,A., Arranz,C., Costa,M.A.
Abstract:
INTRODUCTION: The aim of this study was to investigate both the effects of chronic treatment with atrial natriuretic peptide (ANP) on systolic blood pressure (SBP), cardiac nitric oxide (NO) system, oxidative stress, hypertrophy, fibrosis and apoptosis in spontaneously hypertensive rats (SHR), and sex-related differences in the response to the treatment. METHODS: 10 week-old male and female SHR were infused with ANP (100 ng/hr/rat) or saline (NaCl 0.9%) for 14 days (subcutaneous osmotic pumps). SBP was recorded and nitrites and nitrates excretion (NOx) were determined. After treatment, NO synthase (NOS) activity, eNOS expression, thiobarbituric acid-reactive substances (TBARS) and glutathione concentration were determined in left ventricle, as well as the activity of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). Morphological studies in left ventricle were performed in slices stained with hematoxylin-eosin or Sirius red to identify collagen as a fibrosis indicator; immunohistochemistry was employed for identification of transforming growth factor β; and apoptosis was evaluated by Tunel assay. RESULTS: Female SHR showed lower SBP, higher NO-system activity and less oxidative stress, fibrosis and hypertrophy in left ventricle, as well as higher cardiac NOS activity, eNOS protein content and NOx excretion than male SHR. Although ANP treatment lowered blood pressure and increased NOS activity and eNOS expression in both sexes, cardiac NOS response to ANP was more marked in females. In left ventricle, ANP reduced TBARS and increased glutathione concentration and activity of CAT and SOD enzymes in both sexes, as well as GPx activity in males. ANP decreased fibrosis and apoptosis in hearts from male and female SHR but females showed less end-organ damage in heart. Chronic ANP treatment would ameliorate hypertension and end-organ damage in heart by reducing oxidative stress, increasing NO-system activity, and diminishing fibrosis and hypertrophy.

Address: Catedra De Fisiologia, Facultad De Farmacia Y Bioquimica, Universidad De Buenos Aires, Instituto De Quimica Y Metabolismo Del Farmaco, Consejo Nacional De Investigaciones Cientificas Y Tecnicas, Buenos Aires, Argentina

3915886e: PLoS One, e71650-e71650(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Inhibitory peptide of mitochondrial μ-calpain protects against photoreceptor degeneration in rhodopsin transgenic S334ter and P23H rats.
Author: Ozaki,T., Ishiguro,S., Hirano,S., Baba,A., Yamashita,T., Tomita,H., Nakazawa,M.
Abstract:
Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-microCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated Royal College of Surgeons rats, one of the animal models of RP. Because of the high rate of rhodopsin mutations in RP patients, the present study was performed to confirm the protective effects of Tat-microCL against retinal degeneration in rhodopsin transgenic S334ter and P23H rats. We examined the effects of intravitreal injection or topical application of the peptide on retinal degeneration in S334ter and P23H rats by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electroretinogram (ERG), immunohistochemistry for AIF, and histological staining. In S334ter rats, we found that intravitreal injection or topical application of the peptide prevented photoreceptor cell death from postnatal (PN) 15 to 18 days, the time of early-stage retinal degeneration. Topical application of the peptide also delayed attenuation of ERG responses from PN 28 to 56 days. In P23H rats, topical application of the peptide protected against photoreceptor cell death and nuclear translocation of AIF on PN 30, 40, and 50 days, as the primary stages of degeneration. We observed that topical application of the peptide inhibited the thinning of the outer nuclear layer and delayed ERG attenuations from PN 30 to 90 days. Our results demonstrate that the mitochondrial μ-calpain and AIF pathway is involved in early-stage retinal degeneration in rhodopsin transgenic S334ter and P23H rats, and inhibition of this pathway shows curative potential for rhodopsin mutation-caused RP.

Address: Department of Ophthalmology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

3915887e: PLoS One, e71479-e71479(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Amyloid-β induced CA1 pyramidal cell loss in young adult rats is alleviated by systemic treatment with FGL, a neural cell adhesion molecule-derived mimetic peptide.
Author: Corbett,N.J., Gabbott,P.L., Klementiev,B., Davies,H.A., Colyer,F.M., Novikova,T., Stewart,M.G.
Abstract:
Increased levels of neurotoxic amyloid-β in the brain are a prominent feature of Alzheimer's disease. FG-Loop (FGL), a neural cell adhesion molecule-derived peptide that corresponds to its second fibronectin type III module, has been shown to provide neuroprotection against a range of cellular insults. In the present study impairments in social recognition memory were seen 24 days after a 5 mg/15 μl amyloid-β(25-35) injection into the right lateral ventricle of the young adult rat brain. This impairment was prevented if the animal was given a systemic treatment of FGL. Unbiased stereology was used to investigate the ability of FGL to alleviate the deleterious effects on CA1 pyramidal cells of the amyloid-β(25-35) injection. NeuN, a neuronal marker (for nuclear staining) was used to identify pyramidal cells, and immunocytochemistry was also used to identify inactive glycogen synthase kinase 3β (GSK3β) and to determine the effects of amyloid-β(25-35) and FGL on the activation state of GSK3β, since active GSK3β has been shown to cause a range of AD pathologies. The cognitive deficits were not due to hippocampal atrophy as volume estimations of the entire hippocampus and its regions showed no significant loss, but amyloid-β caused a 40% loss of pyramidal cells in the dorsal CA1 which was alleviated partially by FGL. However, FGL treatment without amyloid-β was also found to cause a 40% decrease in CA1 pyramidal cells. The action of FGL may be due to inactivation of GSK3β, as an increased proportion of CA1 pyramidal neurons contained inactive GSK3β after FGL treatment. These data suggest that FGL, although potentially disruptive in non-pathological conditions, can be neuroprotective in disease-like conditions.

E-Mail: nicola_j_corbett@rush.edu
Address: Open University, Department of Life, Health and Chemical Sciences, Milton Keynes, United Kingdom

3915900e: Front.Mol.Neurosci., 20-20(2013)
Link: [ PubMED ] [ DOI ]
Title: L-type CaV1.2 deletion in the cochlea but not in the brainstem reduces noise vulnerability: implication for CaV1.2-mediated control of cochlear BDNF expression.
Author: Zuccotti,A., Lee,S.C., Campanelli,D., Singer,W., Satheesh,S.V., Patriarchi,T., Geisler,H.S., Kopschall,I., Rohbock,K., Nothwang,H.G., Hu,J., Hell,J.W., Schimmang,T., Ruttiger,L., Knipper,M.
Abstract:
Voltage-gated L-type Ca2+ channels (L-VGCCs) like CaV1.2 are assumed to play a crucial role for controlling release of trophic peptides including brain-derived neurotrophic factor (BDNF). In the inner ear of the adult mouse, besides the well-described L-VGCC CaV1.3, CaV1.2 is also expressed. Due to lethality of constitutive CaV1.2 knock-out mice, the function of this ion channel as well as its putative relationship to BDNF in the auditory system is entirely elusive. We recently described that BDNF plays a differential role for inner hair cell (IHC) vesicles release in normal and traumatized condition. To elucidate a presumptive role of CaV1.2 during this process, two tissue-specific conditional mouse lines were generated. To distinguish the impact of CaV1.2 on the cochlea from that on feedback loops from higher auditory centers CaV1.2 was deleted, in one mouse line, under the Pax2 promoter (CaV1.2(Pax2)) leading to a deletion in the spiral ganglion neurons, dorsal cochlear nucleus, and inferior colliculus. In the second mouse line, the Egr2 promoter was used for deleting CaV1.2 (CaV1.2(Egr2)) in auditory brainstem nuclei. In both mouse lines, normal hearing threshold and equal number of IHC release sites were observed. We found a slight reduction of auditory brainstem response wave I amplitudes in the CaV1.2(Pax2) mice, but not in the CaV1.2(Egr2) mice. After noise exposure, CaV1.2(Pax2) mice had less-pronounced hearing loss that correlated with maintenance of ribbons in IHCs and less reduced activity in auditory nerve fibers, as well as in higher brain centers at supra-threshold sound stimulation. As reduced cochlear BDNF mRNA levels were found in CaV1.2(Pax2) mice, we suggest that a CaV1.2-dependent step may participate in triggering part of the beneficial and deteriorating effects of cochlear BDNF in intact systems and during noise exposure through a pathway that is independent of CaV1.2 function in efferent circuits.

Address: Molecular Physiology of Hearing, Hearing Research Center Tubingen, Department of Otolaryngology, University of Tubingen Tubingen, Germany

3915901e: Pharmacol.Rep., 666-671(2013)
Link: [ OpenAccess]
Title: Long-term inhibition of intestinal lipase by orlistat improves release of gut hormones increasing satiety in obese women.
Author: Olszanecka-Glinianowicz,M., Dabrowski,P., Kocelak,P., Janowska,J., Smertka,M., Jonderko,K., Chudek,J.
Abstract:
BACKGROUND: Reduced postprandial secretion of peptide YY (PYY), glucagon-like peptide-1 (GLP-1), cholecystokinin, and increased hunger was reported after a single dose of orlistat, an inhibitor of intestinal lipase. As yet, the influence of long-term therapy with orlistat on PYYand GLP-1 release has not been studied. Our study was aimed at assessing the influence of 8-week therapy with orlistat as a component of a weight loss program on pre-prandial circulating PYY and GLP-1 levels. METHODS: Forty obese women, without concomitant diseases, were randomly allocated to groups receiving orlistat or placebo during an 8-week weight management program. Body mass, body composition and plasma levels of PYY, GLP-1 and insulin (for QUICKI calculation) were determined prior to and at the end of therapy. RESULTS: Women treated with orlistat obtained significantly greater body and fat mass loss than those receiving placebo (9.0 +/- 3.1 vs. 5.9 +/- 3.2% and 21.9 +/- 10.9 vs. 7.4 +/- 15.6%, respectively). Only in those treated with orlistat a slight, but significant increase of the QUICKI was found (8.0 +/- 16.5 vs. -0.1 +/- 12.7 %, respectively). Weight loss was followed by a significant increase of plasma levels of PYY and GLP-1 in group treated with orlistat, and was about 2-times greater than receiving placebo. The increase was independent of body mass changes. CONCLUSION: The long-term inhibition of intestinal lipase by orlistat increases the pre-prandial levels of GLP-1 and PYY, independent of body mass changes. Therefore, it seems that long-term treatment with orlistat may exert hunger suppressing and insulin sensitizing incretin effect beyond weight reduction.

E-Mail: magols@esculap.pl
Address: Department of Pathophysiology, Medical University of Silesia, Katowice, Poland

3915902e: J.Rheumatol., 1650-1657(2013)
Link: [ PubMED ] [ DOI ]
Title: Five-year Favorable Outcome of Patients with Early Rheumatoid Arthritis in the 2000s: Data from the ESPOIR Cohort.
Author: Combe,B., Rincheval,N., Benessiano,J., Berenbaum,F., Cantagrel,A., Daures,J.P., Dougados,M., Fardellone,P., Fautrel,B., Flipo,R.M., Goupille,P., Guillemin,F., Le Loet,X., Logeart,I., Mariette,X., Meyer,O., Ravaud,P., Saraux,A., Schaeverbeke,T., Sibilia,J.
Abstract:
OBJECTIVE: To report the 5-year outcome of a large prospective cohort of patients with very early rheumatoid arthritis (RA), and to identify factors predictive of outcome. METHODS: Patients were recruited if they had early arthritis of < 6 months' duration, had a high probability of developing RA, and had never been prescribed disease-modifying antirheumatic drugs (DMARD) or steroids. Logistic regression analysis was used to determine factors that predict outcome. RESULTS: We included 813 patients from December 2002 to April 2005. Age was 48.1 +/- 12.6 years, delay before referral 103.1 +/- 52.4 days, 28-joint Disease Activity Score (DAS28) 5.1 +/- 1.3, Health Assessment Questionnaire (HAQ) 1.0 +/- 0.7; 45.8% and 38.7% had rheumatoid factor or antibodies to cyclic citrullinated peptide (anti-CCP), respectively; 22% had hand or foot erosions; 78.5% fulfilled the American College of Rheumatology/European League Against Rheumatism criteria for RA at baseline and 93.8% during followup. At 5 years, 573 patients were evaluated. The outcome was mild for most patients: disease activity (median DAS28 = 2.5) and HAQ disability (median 0.3) were well controlled over time; 50.6% achieved DAS28 remission and 64.7% low disease activity. Radiographic progression was low (2.9 Sharp unit/year) and only a few patients required joint surgery. Nevertheless, some patients developed new comorbidities. During the 5 years, 82.7% of patients had received at least 1 DMARD (methotrexate, 65.9%), 18.3% a biological DMARD, and about 60% prednisone at least once. Anti-CCP was the best predictor of remaining in the cohort for 5 years, of prescription of synthetic or biologic DMARD, and of radiographic progression. CONCLUSION: The 5-year outcome of an early RA cohort in the 2000s was described. Anti-CCP was a robust predictor of outcome. The generally good 5-year outcome could be related to early referral and early effective treatment, key processes in the management of early RA in daily practice.

Address: From the Lapeyronie Hospital, Montpellier I University, UMR 5535, EA2415, Montpellier

3915904e: J.Nucl.Med., 1789-1796(2013)
Link: [ PubMED ] [ DOI ]
Title: PulmoBind, an Adrenomedullin-Based Molecular Lung Imaging Tool.
Author: Letourneau,M., Nguyen,Q.T., Harel,F., Fournier,A., Dupuis,J.
Abstract:
Previous studies showed that adrenomedullin (AM) could be a promising agent for molecular imaging of the pulmonary circulation, with abundant specific binding sites at the pulmonary vascular endothelium. The purpose of this work was to design an AM-based compound that encompasses the desired imaging properties without posing safety issues for clinical applications. METHODS: AM analogs were synthesized through solid-phase peptide synthesis. They were evaluated for 99mTc labeling efficiency and in vivo lung uptake. Biodistribution and hemodynamic characteristics of the lead compound were determined in anesthetized dogs as well as by a dosimetric analysis. Lung perfusion was evaluated in the monocrotaline model of pulmonary arterial hypertension in rats. RESULTS: A cyclic AM (residues 22-52) analog encompassing a polyethylene glycol spacer and a tetrapeptide chelating moiety was found to possess the desired characteristics, with 90.7% +/- 0.3% (mean +/- SD) labeling efficiency, 40% lung uptake at 10 min after injection, and a favorable safety profile. Lung uptake of the 99mTc-labeled compound was markedly reduced in rats with pulmonary arterial hypertension. CONCLUSION: This lead compound could be a suitable clinical imaging agent for the molecular diagnosis of disorders of the pulmonary circulation.

Address: Laboratoire d'Etudes Moleculaires et Pharmacologiques des Peptides, INRS-Institut Armand-Frappier, Laval, Quebec, Canada

3915906e: J.Mol.Neurosci., 771-778(2013)
Link: [ PubMED ] [ DOI ]
Title: Beta Amyloid Peptide (25-35) Leading to Inflammation Through Toll-Like Receptors and the Anti-inflammatory Effect of Genistein in BV-2 Cells.
Author: Yu,H.L., Li,X.Y., Zhou,X., Yuan,L.H., Ma,W.W., Xi,Y.D., Zhao,X., Wu,J., Xiao,R.
Abstract:
Genistein, the main soy isoflavone component, has received much attention for its potential multifunction. Here, we reported that in BV-2 cells, genistein significantly inhibited β amyloid peptides 25-35 (Aβ25-35)-induced inflammatory response. The results indicated that Aβ25-35-stimulated BV-2 cells upregulated Toll-like receptors 2 and 4, Myd88, and IKK gene expression with the increasing expression of IL-6 and decreasing expression of TGF-β and IL-10. Further, inhibiting TLR4 expression with small interfering RNA prevented the inflammatory response induced by Aβ25-35, indicating the key role of TLRs in Aβ-mediated inflammation. Genistein pre-treated BV-2 cells showed less inflammatory response when exposed to Aβ25-35. These results suggested that Aβ induced BV-2 cells inflammation though TLRs and genistein has an anti-inflammatory effect in vitro.

E-Mail: yuhlzjl@ccmu.edu.cn
Address: School of Public Health, Capital Medical University, No.10 Xitoutiao, You An Men, Beijing, People's Republic of China,

3915907e: Crit.Care Med., e344-e351(2013)
Link: [ PubMED ] [ DOI ]
Title: Complement C5A Antagonist Treatment Improves the Acute Circulatory and Inflammatory Consequences of Experimental Cardiac Tamponade.
Author: Erces,D., Nogrady,M., Nagy,E., Varga,G., Vass,A., Suveges,G., Imai,M., Okada,N., Okada,H., Boros,M., Kaszaki,J.
Abstract:
OBJECTIVE: Cardiogenic shock often leads to splanchnic macro- and microcirculatory complications, and these events are linked to local and systemic inflammatory activation. Our aim was to investigate the consequences of complement C5a antagonist treatment on the early circulatory and inflammatory changes in a clinically relevant large animal model of cardiac tamponade. DESIGN AND SETTING: A randomized, controlled in vivo animal study in a university research laboratory. SUBJECTS: Anesthetized, ventilated, and thoracotomized Vietnamese mini pigs (24 +/- 3 kg). INTERVENTIONS: Group 1 (n = 6) served as sham-operated control. In group 2 (n = 7), cardiac tamponade was induced for 60 minutes by the administration of intrapericardial fluid, while the mean arterial pressure was kept in the interval 40 to 45 mm Hg. Group 3 (n = 6) was treated with a complement C5a antagonist compound (the peptide acetyl-peptide-A, 4 mg/kg) after 45 minutes of tamponade. MEASUREMENTS AND MAIN RESULTS: The macrohemodynamics, including the superior mesenteric artery flow, was monitored; the average red blood cell velocity in the small intestinal mucosa was determined by an intravital orthogonal polarization imaging technique. The whole blood superoxide production, the plasma level of high-mobility group box protein-1 and big-endothelin and the small intestinal myeloperoxidase activity were measured. One hundred eighty minutes after the relief of tamponade, the mean arterial pressure was decreased, while the plasma levels of superoxide, high-mobility group box protein-1, and big-endothelin, and the intestinal myeloperoxidase activity were increased. The administration of acetyl-peptide-A normalized the mean arterial pressure and preserved the cardiac output, while the superior mesenteric artery flow and mucosal average red blood cell velocity were increased significantly, and the plasma superoxide, high-mobility group box protein-1, big-endothelin, and intestinal myeloperoxidase levels were reduced. CONCLUSIONS: These results provide evidence that blockade of the C5a effects significantly influences the acute splanchnic macro- and microhemodynamic complications and decreases the potentially harmful inflammatory consequences of experimental cardiogenic shock.

Address: Szeged, Hungary. 3Department of Traumatology, University of Szeged, Szeged, Hungary. 4Department of Immunology, Nagoya City University School of Medicine, Nagoya, Japan. 5Choju Medical Institute, Fukushimura Hospital, Toyohashi, Japan

3915909e: Skin Pharmacol.Physiol., 47-55(2014)
Link: [ PubMED ] [ DOI ]
Title: Oral supplementation of specific collagen peptides has beneficial effects on human skin physiology: a double-blind, placebo-controlled study.
Author: Proksch,E., Segger,D., Degwert,J., Schunck,M., Zague,V., Oesser,S.
Abstract:
Various dietary supplements are claimed to have cutaneous anti-aging properties; however, there are a limited number of research studies supporting these claims. The objective of this research was to study the effectiveness of collagen hydrolysate (CH) composed of specific collagen peptides on skin biophysical parameters related to cutaneous aging. In this double-blind, placebo-controlled trial, 69 women aged 35-55 years were randomized to receive 2.5 g or 5.0 g of CH or placebo once daily for 8 weeks, with 23 subjects being allocated to each treatment group. Skin elasticity, skin moisture, transepidermal water loss and skin roughness were objectively measured before the first oral product application (t0) and after 4 (t1) and 8 weeks (t2) of regular intake. Skin elasticity (primary interest) was also assessed at follow-up 4 weeks after the last intake of CH (t3, 4-week regression phase). At the end of the study, skin elasticity in both CH dosage groups showed a statistically significant improvement in comparison to placebo. After 4 weeks of follow-up treatment, a statistically significantly higher skin elasticity level was determined in elderly women. With regard to skin moisture and skin evaporation, a positive influence of CH treatment could be observed in a subgroup analysis, but data failed to reach a level of statistical significance. No side effects were noted throughout the study.

Address: Department of Dermatology, University of Kiel, Germany

3915910e: J.Alzheimer's Dis., 185-200(2014)
Link: [ PubMED ] [ DOI ]
Title: Involvement of Insulin-Degrading Enzyme in Insulin- and Atrial Natriuretic Peptide-Sensitive Internalization of Amyloid-β Peptide in Mouse Brain Capillary Endothelial Cells.
Author: Ito,S., Ohtsuki,S., Murata,S., Katsukura,Y., Suzuki,H., Funaki,M., Tachikawa,M., Terasaki,T.
Abstract:
Cerebral clearance of amyloid-β peptide (Aβ), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aβ1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aβ1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAβ1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAβ1-40, suggesting that hAβ1-40 and hANP share a common elimination process. Internalization of [125I]hAβ1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAβ1-40, suggesting that there is no direct interaction between Npr-C and hAβ1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAβ1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAβ1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.

Address: Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai, Japan SORST of the Japan Science and Technology Agency, Japan Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan

3915911e: J.Alzheimer's Dis., 49-62(2014)
Link: [ PubMED ] [ DOI ]
Title: Picomolar Amyloid-β Peptides Enhance Spontaneous Astrocyte Calcium Transients.
Author: Lee,L., Kosuri,P., Arancio,O.
Abstract:
Amyloid-β (Aβ) peptides are constitutively produced in the brain throughout life via mechanisms that can be regulated by synaptic activity. Although Aβ has been extensively studied as the pathological plaque-forming protein species in Alzheimer's disease (AD), little is known about the normal physiological function(s) and signaling pathway(s). We previously discovered that physiologically-relevant, low picomolar amounts of Aβ can enhance synaptic plasticity and hippocampal-dependent cognition in mice. In this study, we demonstrated that astrocytes are cellular candidates for participating in this type of Aβ signaling. Using calcium imaging of primary astrocyte cultures, we observed that picomolar amounts of Aβ peptides can enhance spontaneous intracellular calcium transient signaling. After application of 200 pM Aβ42 peptides, the frequency and amplitude averages of spontaneous cytosolic calcium transients were significantly increased. These effects were dependent on α7 nicotinic acetylcholine receptors (α7-nAChRs), as the enhancement effects were blocked by a pharmacological α7-nAChR inhibitor and in astrocytes from an α7 deficient mouse strain. We additionally examined evoked intercellular calcium wave signaling but did not detect significant picomolar Aβ-induced alterations in propagation parameters. Overall, these results indicate that at a physiologically-relevant low picomolar concentration, Aβ peptides can enhance spontaneous astrocyte calcium transient signaling via α7-nAChRs. Since astrocyte-mediated gliotransmission has been previously found to have neuromodulatory roles, Aβ peptides may have a normal physiological function in regulating neuron-glia signaling. Dysfunction of this signaling process may underlie glia-based aspects of AD pathogenesis.

Address: Columbia University, New York, NY, USA Department of Pathology and Cell Biology, Columbia University, New York, NY, USA Center for Neurobiology and Behavior, Columbia University, New York, NY, USA

3915912e: J.Alzheimer's Dis., 39-48(2014)
Link: [ PubMED ] [ DOI ]
Title: Comparison of Two Different Methods for Measurement of Amyloid-β Peptides in Cerebrospinal Fluid after BACE1 Inhibition in a Dog Model.
Author: Borghys,H., Jacobs,T., Van Broeck,B., Dillen,L., Dhuyvetter,D., Gijsen,H., Mercken,M.
Abstract:
Beta-secretase is the first cleavage enzyme of amyloid-β protein precursor (AβPP) in the amyloidogenic pathway, leading to the formation of the plaque forming Amyloid-β (Aβ)1-42 peptide. BACE (β-site AβPP cleaving enzyme) 1 inhibition is therefore considered to be a promising disease modifying therapy for Alzheimer's disease. An early assessment of the in vivo activity of BACE inhibitors was done in dogs since AβPP processing is the same as in humans and this species easily enables longitudinal cerebrospinal fluid (CSF) sampling. Aβ changes in CSF compared to baseline are used to evaluate target engagement of the compounds. Levels of Aβ1-37, Aβ1-38, Aβ1-40, and Aβ1-42 in CSF are measured with immunoassay (Mesoscale electrochemiluminescence technology) and with an ultra high-performance liquid chromatography mass spectrometry (UPLC-MS/MS). Two experimental BACE inhibitors were evaluated. With the immunoassay, a dose dependent decrease is observed for all four Aβ peptides. Measurements with the UPLC-MS/MS are in line with the immunoassay for Aβ1-37, Aβ1-38, and Aβ1-40, however, for Aβ1-42, differences are sometimes observed when comparing to changes seen in the other peptides with UPLC-MS/MS and with immunoassay results. Generally lower concentrations are measured with immunoassay. The reason for these differences is still unknown. Aβ1-42 is more prone to form aggregates compared to the other peptides. One hypothesis could be that while the immunoassay only measures free Aβ, bound and aggregated Aβ peptides are at least partially dissolved with the UPLC-MS/MS method, since acetonitrile is added to the CSF samples. This increases variability in the concentration of Aβ peptide measured with UPLC-MS/MS, especially for Aβ1-42, potentially masking the compound effect on Aβ1-42 levels.

Address: Janssen Research & Development, Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium

3915913e: J.Alzheimer's Dis., 75-83(2014)
Link: [ PubMED ] [ DOI ]
Title: Increased Susceptibility to Amyloid-β Toxicity in Rat Brain Microvascular Endothelial Cells under Hyperglycemic Conditions.
Author: Carvalho,C., Katz,P.S., Dutta,S., Katakam,P.V., Moreira,P.I., Busija,D.W.
Abstract:
We hypothesized that hyperglycemia-induced mitochondrial dysfunction and oxidative stress are closely associated with amyloid-β peptide (Aβ) toxicity in endothelial cells. Brain microvascular endothelial cells from rat (RBMEC) and mice (MBMEC) were isolated from adult Sprague-Dawley rats and homozygous db/db (Leprdb/Leprdb) and heterozygous (Dock7m/Leprdb) mice, and cultured under normo- and hyperglycemic conditions for 7 d followed by 24 h exposure to Aβ1-40. Some experiments were also performed with two mitochondrial superoxide (O2*-) scavengers, MitoTempo and Peg-SOD. Cell viability was measured by the Alamar blue assay and mitochondrial membrane potential (ΔPsim) by confocal microscopy. Mitochondrial O2*- and hydrogen peroxide (H2O2) production was assessed by fluorescence microscopy and H2O2 production was confirmed by microplate reader. Hyperglycemia or Aβ1-40 alone did not affect cell viability in RBMEC. However, the simultaneous presence of high glucose and Aβ1-40 reduced cell viability and ΔPsim, and enhanced mitochondrial O2*- and H2O2 production. MitoTempo and PEG-SOD prevented Aβ1-40 toxicity. Interestingly, MBMEC presented a similar pattern of alterations with db/db cultures presenting higher susceptibility to Aβ1-40. Overall, our results show that high glucose levels increase the susceptibility of brain microvascular endothelial cells to Aβ toxicity supporting the idea that hyperglycemia is a major risk factor for vascular injury associated with AD.

Address: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Department of Life Sciences - Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal

3915914e: J.Alzheimer's Dis., 713-733(2013)
Link: [ PubMED ] [ DOI ]
Title: Intracellular Localization of Amyloid-β Peptide in SH-SY5Y Neuroblastoma Cells.
Author: Zheng,L., Calvo-Garrido,J., Hallbeck,M., Hultenby,K., Marcusson,J., Cedazo-Minguez,A., Terman,A.
Abstract:
Amyloid-β peptide (Aβ), the main component of Alzheimer's disease (AD) senile plaques, has been found to accumulate within the lysosomal compartment of AD neurons. We have previously shown that in differentiated SH-SY5Y neuroblastoma cells cultured under normal conditions, the majority of Aβ is localized extralysosomally, while oxidative stress significantly increases intralysosomal Aβ content through activation of macroautophagy. It is, however, not clear which cellular compartments contain extralysosomal Aβ in intact SH-SY5Y cells, and how oxidative stress influences the distribution of extralysosomal Aβ. Using confocal laser scanning microscopy and immunoelectron microscopy, we showed that in differentiated neuroblastoma cells cultured under normal conditions Aβ (Aβ40, Aβ42, and Aβ oligomers) is colocalized with both membrane-bound organelles (endoplasmic reticulum, Golgi complexes, multivesicular bodies/late endosomes, lysosomes, exocytotic vesicles and mitochondria) and non-membrane-bound cytosolic structures. Neuroblastoma cells stably transfected with AβPP Swedish KM670/671NL double mutation showed enlarged amount of Aβ colocalized with membrane compartments. Suppression of exocytosis by 5 nM tetanus toxin resulted in a significant increase of the amount of cytosolic Aβ as well as Aβ colocalized with exocytotic vesicles, endoplasmic reticulum, Golgi complexes, and lysosomes. Hyperoxia increased Aβ localization in the endoplasmic reticulum, Golgi apparatus, mitochondria, and lysosomes, but not in the secretory vesicles. These results indicate that in SH-SY5Y neuroblastoma cells intracellular Aβ is not preferentially localized to any particular organelle and, to a large extent, is secreted from the cells. Challenging cells to hyperoxia, exocytosis inhibition, or Aβ overproduction increased intracellular Aβ levels but did not dramatically changed its localization pattern.

Address: KI-Alzheimer Disease Research Center, NVS, Novum, Karolinska Institutet, Stockholm, Sweden

3915915e: J.Alzheimer's Dis., 735-746(2013)
Link: [ PubMED ] [ DOI ]
Title: Tetrahydrohyperforin Induces Mitochondrial Dynamics and Prevents Mitochondrial Ca2+ Overload after Aβ and Aβ-AChE Complex Challenge in Rat Hippocampal Neurons.
Author: Zolezzi,J.M., Carvajal,F.J., Rios,J.A., Ordenes,D., Silva-Alvarez,C., Godoy,J.A., Inestrosa,N.C.
Abstract:
St. John's wort has been the subject of studies focused on its therapeutic properties against several diseases, including Alzheimer's disease (AD). Amyloid β-peptide (Aβ), a critical peptide in AD, has been linked to the mitochondrial dysfunction often observed in this disease. Despite many efforts to prevent Aβ levels from increasing in AD, less has been done regarding the mitochondrial component. Therefore, we studied the effects of tetrahydrohyperforin (THH) on mitochondrial dysfunction of hippocampal neurons, challenged with Aβ oligomers (Aβo) and Aβo-AChE complexes. We show that THH prevents mitochondrial calcium overload and induces the modulation of fusion-fission events, arresting mitochondrial dysfunction. Moreover, our results suggest that the modulation of mitochondrial dynamics probably occurs through a peroxisome proliferator-activated receptor γ co-activator 1α-mediated mechanism, inducing mitochondrial fusion-fission protein expression. Our results offer further explanation for the effects observed for THH and the beneficial effects of this ethno-botanical drug in AD.

Address: Centro de Envejecimiento y Regeneracion (CARE), Santiago, Chile

3915916e: J.Alzheimer's Dis., 637-648(2013)
Link: [ PubMED ] [ DOI ]
Title: Subcutaneous administration of liraglutide ameliorates Alzheimer-associated tau hyperphosphorylation in rats with type 2 diabetes.
Author: Yang,Y., Zhang,J., Ma,D., Zhang,M., Hu,S., Shao,S., Gong,C.X.
Abstract:
Background/Objective: Type 2 diabetes increases the risk for developing Alzheimer's disease (AD), a progressive neurodegenerative disorder. Brain insulin resistance contributes to the pathogenesis of AD, and abnormal hyperphosphorylation of tau protein is crucial to neurodegeneration. Here we studied whether liraglutide, an agonist of glucagon-like peptide-1 (GLP-1) and a new anti-diabetic drug, can promote brain insulin signaling and inhibit tau hyperphosphorylation in the brains of type 2 diabetic rats. Methods: Type 2 diabetic rats were treated with subcutaneous administration of liraglutide (0.2 mg/kg body weight) or, as a control, saline twice a day for up to four weeks. Blood, cerebrospinal fluid (CSF), and brain tissue (n = 7 each group) were collected for analyses after liraglutide or saline administration for one, two, three, and four weeks. Results: We found decreased CSF insulin, hyperphosphorylation of tau at AD-relevant phosphorylation sites, and decreased phosphorylation of protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) in the brain, which indicated decreased insulin signaling leading to overactivation of GSK-3β, a major tau kinase, in type 2 diabetic rats. Liraglutide treatment not only ameliorated hyperglycemia and peripheral insulin resistance, but also reversed these brain abnormalities in a time-dependent manner. Conclusion: Our results indicated that subcutaneous administration of liraglutide restores both peripheral and brain insulin sensitivity and ameliorates tau hyperphosphorylation in rats with type 2 diabetes. These findings support the potential use of liraglutide for the prevention and treatment of AD in individuals with type 2 diabetes.

Address: Department of Endocrinology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, People's Republic of China

3915917e: J.Alzheimer's Dis., 367-377(2013)
Link: [ PubMED ] [ DOI ]
Title: Upregulation and expression patterns of the angiogenic transcription factor ets-1 in Alzheimer's disease brain.
Author: Jantaratnotai,N., Ling,A., Cheng,J., Schwab,C., McGeer,P.L., McLarnon,J.G.
Abstract:
Immunohistochemical staining has been used to determine expression patterns of the angiogenic transcription factor, Ets-1, in the brains of Alzheimer's disease (AD) individuals. Brain tissue from non-demented controls showed little expression of Ets-1 whereas in AD brain tissue, Ets-1 was ubiquitously expressed in cortex and hippocampus. Double immunostaining with von Willerbrand factor demonstrated prominent Ets-1 intravascular immunoreactivity (ir) in AD cortical microvessels. In addition, Ets-1 also exhibited extravascular expression characterized by a diffuse pattern of Ets-1 ir in AD brain. Double staining also showed Ets-1 colocalization in microvasculature with the potent angiogenic agent, vascular endothelial growth factor (VEGF). Cell-associated tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine with pro-angiogenic activity, was primarily associated with diffuse extravascular Ets-1 ir. Clusters of HLA-DR positive microglia, resident immune cells of brain which release TNF-α, were also localized with diffuse Ets-1. Intravascular Ets-1 ir was maximally co-localized with soluble amyloid-β peptide (Aβ), Aβ1-40, in microvasculature whereas diffuse extravascular Ets-1 ir appeared in proximity to Aβ plaques in brain parenchyma. Similar overall results were obtained for patterns of Ets-1 staining in AD hippocampal tissue. This work provides novel findings on expression of the angiogenic transcription factor, Ets-1, in vascular remodeling and its association with pro-angiogenic factors, reactive microglia, and Aβ deposition in AD brain.

Address: Department of Pharmacology, Faculty of Science, Mahidol University, Phayathai, Bangkok, Thailand

3915918e: J.Alzheimer's Dis., 379-387(2013)
Link: [ PubMED ] [ DOI ]
Title: Neprilysin-like activity correlates with CSF-Tau and phospho-tau in patients with Alzheimer's disease.
Author: Sorensen,K.C., Simonsen,A.H., Holmetoft,U.B., Hasselbalch,S.G., Heegaard,N.H.
Abstract:
A relation between amyloid-β peptide (Aβ) accumulation and neprilysin (NEP), an Aβ degrading enzyme, has been proposed but studies of NEP and the levels of the pathological hallmarks of Alzheimer's disease (AD), Aβ and tau, in cerebrospinal fluid (CSF) are scarce. In this study, we measured the level and enzyme activity of NEP in serum and CSF, using a sandwich enzyme-linked immunosorbent assay and a fluorescence resonance energy transfer assay, respectively, in patients with AD, frontotemporal dementia (FTD), Creutzfeldt-Jakob disease (CJD), and depression. Results were correlated with the levels of CSF AD biomarkers Aβ42, hyperphosphorylated tau (p-tau), and total tau (t-tau). In serum, we found no differences in NEP-like activity or concentration between the groups and there were no correlations between NEP and AD biomarkers. In CSF, no influence of age or gender on NEP levels or enzyme activity was seen. However, NEP concentration was lower and the specific activity was higher in FTD compared to AD. Aβ42 levels in CSF did not correlate with NEP concentration or activity in the AD, CJD, or depression groups, but NEP-like activity and Aβ42 levels correlated significantly in the FTD group. In AD and depression, the NEP-like activity in CSF correlated with levels of p-tau, and, in the AD group, it also was correlated with t-tau levels. Our results suggest that the relation between the specific activity of NEP and t-tau and p-tau is a characteristic trait of AD. The correlation between NEP concentration and Aβ42 in FTD is unexpected and warrants further investigation.

Address: Department of Clinical Biochemistry, Immunology & Genetics, Statens Serum Institut, Copenhagen, Denmark

3915919e: Nat.Commun., 2217-2217(2013)
Link: [ PubMED ] [ DOI ]
Title: Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum.
Author: Wu,Y., Yuan,L., Guo,X., Holding,D.R., Messing,J.
Abstract:
Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.

Address: Waksman Institute of Microbiology, Rutgers University, 190 Frelinghuysen Road, Piscataway, New Jersey 08854, USA

3915920e: Oncology, 137-144(2013)
Link: [ PubMED ] [ DOI ]
Title: Cardiotoxicity and cancer therapy: treatment-related cardiac morbidity in patients presenting with symptoms suggestive of heart or lung disease.
Author: Wieshammer,S., Dreyhaupt,J., Muller,D., Momm,F., Jakob,A., Freund,U.
Abstract:
Objectives: Cardiac injury is one of the complications of cancer treatment. This study aimed to investigate the relationships between the types of radiotherapy of the chest (RT), chemotherapy (CT), cancer surgery (CS) and endocrine therapy (ET), and the presence of heart disease, and their associations with the serum level of N-terminal pro-B-type natriuretic peptide (NT-proBNP). Methods: A consecutive series of 374 patients with cancer who were referred because of symptoms suggestive of heart or lung disease prospectively underwent a diagnostic workup. Results: The prevalence of heart disease was 36.9%. RT administered before 1995 (n = 19) was associated with both increased odds of heart disease [adjusted odds ratio 10.3, 95% confidence interval 3.1-34.0] and higher ln-transformed NT-proBNP values (p < 0.01) compared to the control group (no RT or RT for right-sided breast cancer from 1995 onwards; n = 311). Anthracycline-treated patients (n = 54) had higher adjusted values for ln(NT-proBNP) compared to the control group (no CT; n = 243; p < 0.01) but no increased odds of heart disease. Conclusions: While pre-1995 RT and anthracycline-containing CT were associated with cardiac effects, there was no evidence that RT using modern cardioprotective techniques, CT in the absence of anthracyclines, CS or ET had detrimental effects on the heart.

Address: Department of Cardiology, Pulmonology and Critical Care Medicine, Offenburg Hospital, Offenburg, Germany

3915921e: Transl.Res., 297-308(2013)
Link: [ PubMED ] [ DOI ]
Title: Clinical impact of aspartyl aminopeptidase expression and activity in colorectal cancer.
Author: Larrinaga,G., Perez,I., Ariz,U., Sanz,B., Beitia,M., Errarte,P., Etxezarraga,C., Candenas,M.L., Pinto,F.M., Lopez,J.I.
Abstract:
Aspartyl aminopeptidase (ASP; EC 3.4.11.21) is a widely distributed and abundant cytosolic enzyme that regulates bioactive peptides such as angiotensin II. It has been demonstrated that the expression and activity of this enzyme is modified in tissue and serum of patients with several types of cancer. However, the involvement of ASP in the neoplastic development and survival of patients with colorectal cancer (CRC) has not been analyzed to date. The activity and messenger RNA expression of ASP in tumor tissue (n = 71) and plasma (n = 40) of patients with CRC was analyzed prospectively using fluorometric and quantitative real-time polymerase chain reaction methods. Data obtained from tumor tissue were compared with those from the surrounding normal mucosa. Classic pathologic parameters (grade, stage, nodal invasion, distant metastases and perineural, lymphatic, and vascular invasion) were stratified following ASP data and analyzed for 5-year survival. ASP was upregulated in CRC tissues, and greater activity correlated significantly with the absence of lymph node metastases and with better overall survival. Inversely, greater plasmatic ASP activity was associated with worse overall and disease-free survival. Data suggest that ASP is involved in colorectal neoplasia and point to this enzyme as a potential useful diagnostic tool in clinical practice.

Address: Department of Nursing I, School of Nursing, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain

3915926e: J.Reprod.Med., 305-311(2013)
Link:
Title: Metabolic hormones in follicular fluid in women undergoing in vitro fertilization.
Author: Varnagy,A., Bodis,J., Kovacs,G.L., Sulyok,E., Rauh,M., Rascher,W.
Abstract:
OBJECTIVE: To determine the concentrations of metabolic hormones in follicular fluid (FF) and to find clinical correlates of these biochemical parameters. STUDY DESIGN: FF was obtained from 108 women by ultrasonography-guided transvaginal puncture following controlled ovarian hyperstimulation. FF insulin, leptin, adiponectin and resistin levels were measured by enzyme-linked immunosorbent assay, and ghrelin was analyzed with radioimmunoassay. RESULTS: It was demonstrated that oocyte number correlated negatively with FF leptin (r = -0.190, p < 0.050) and insulin (r = -0.209, p < 0.031) and positively with resistin (r = 0.236, p < 0.014). After adjustments for confounding hormone parameters, resistin remained a positive (p < 0.000) predictor and insulin (p < 0.039) and adiponectin (p < 0.033), negative predictors of oocyte number. When the embryo number was considered, FF leptin proved to be a strong negative (p < 0.012) whereas resistin proved to be a positive outcome predictor (p < 0.004). CONCLUSION: In women undergoing in vitro fertilization (IVF), FF metabolic hormones may be involved in regulating ovarian function and in determining fertilization outcome. Resistin appears to have beneficial effects on the outcome of IVF, while leptin, insulin, adiponectin and ghrelin appear to have adverse or no effects.

E-Mail: akos.varnagy@aok.pte.hu
Address: Department of Obstetrics and Gynecology, Institute of Laboratory Medicine, Faculty of Medicine, University of Pecs, Hungary

3915930e: J.Neurosci., 13270-13285(2013)
Link: [ PubMED ] [ DOI ]
Title: ERK2-mediated phosphorylation of Par3 regulates neuronal polarization.
Author: Funahashi,Y., Namba,T., Fujisue,S., Itoh,N., Nakamuta,S., Kato,K., Shimada,A., Xu,C., Shan,W., Nishioka,T., Kaibuchi,K.
Abstract:
Axon formation is one of the most important events in neuronal polarization and is regulated by signaling molecules involved in cytoskeletal rearrangement and protein transport. We previously found that Partition-defective 3 (Par3) is associated with KIF3A (kinesin-2) and is transported into the nascent axon in a KIF3A-dependent fashion. Par3 interacts with the Rac-specific guanine nucleotide-exchange factors (GEFs) Tiam1/2, which activate Rac1, and participates in axon formation in cultured hippocampal neurons. However, the regulatory mechanism of the Par3-KIF3A interaction is poorly understood, and the role of Par3 in neuronal polarization in vivo remains elusive. Here, we found that extracellular signal-regulated kinase 2 (ERK2) directly interacts with Par3, that ERK2 phosphorylates Par3 at Ser-1116, and that the phosphorylated Par3 accumulates at the axonal tips in a manner dependent upon ERK2 activity. The phosphorylation of Par3 by ERK2 inhibited the interaction of Par3 with KIF3A but not with the other Par3 partners, including Par6 and aPKC. The phosphomimic mutant of Par3 (Par3-S1116D) showed less binding activity with the KIF3s and slower transport in the axons. The knockdown of Par3 by RNA interference impaired neuronal polarization, which was rescued with RNAi-resistant Par3, but not with the phosphomimic Par3 mutant, in cultured rat hippocampal neurons and mouse cortical projection neurons in vivo. These results suggest that ERK2 phosphorylates Par3 and inhibits its binding with KIF3A, thereby controlling Par3 transport and neuronal polarity.

Address: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan

3915935e: Blood, 3054-3061(2013)
Link: [ PubMED ] [ DOI ]
Title: A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates.
Author: Cooke,K.S., Hinkle,B., Salimi-Moosavi,H., Foltz,I., King,C., Rathanaswami,P., Winters,A., Steavenson,S., Begley,C.G., Molineux,G., Sasu,B.J.
Abstract:
Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Address: Department of Oncology

3915936e: Mol.Oral Microbiol., 452-466(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of gingipain-specific I-A -restricted CD4 T cells following mucosal colonization with Porphyromonas gingivalis in C57BL/6 mice.
Author: Bittner-Eddy,P.D., Fischer,L.A., Costalonga,M.
Abstract:
Chronic periodontitis is associated with Porphyromonas gingivalis infection. Although virulence factors of P. gingivalis are hypothesized to contribute to the pathogenesis of periodontitis, it is unclear whether the local CD4+ T-cell-mediated response they elicit prevents or contributes to periodontal bone destruction. We hypothesize that major histocompatibility complex class II I-Ab -binding peptides existing in Kgp and RgpA are presented to CD4+ T cells during P. gingivalis oral colonization. The protein sequences of gingipains RgpA and Kgp, and OMP40 and OMP41 of P. gingivalis were scanned using an I-Ab -binding matrix. From this analysis we identified 53 candidate peptides that had the potential to engage the peptide-binding groove of the I-Ab molecule of C57BL/6 mice. An ELISpot-based screen revealed those peptide-primed effector/memory CD4+ T cells that could be re-stimulated in vitro with P. gingivalis or the peptide itself to produce interleukin-17A or interferon-γ. Two immunodominant peptides, Kgp467-477 (pKgp) and RgpA1054-1064 /Kgp1074-1084 (pR/Kgp) were identified and engineered to be displayed on I-Ab molecular tetramers. Peptide pR/Kgp is conserved across all sequenced P. gingivalis strains. C57BL/6 mice were orally inoculated with P. gingivalis strain 53977 and cervical lymph node cells were stained with phycoerythrin-conjugated pKgp::I-Ab and pR/Kgp::I-Ab tetramers. We found that only pR/Kgp::I-Ab bound with the desired specificity to gingipain-specific CD4+ T cells. The pR/Kgp::I-Ab tetramer complex will allow the identification of effector/memory CD4+ T cells specific for two virulence factors of P. gingivalis strains associated with periodontal disease.

Address: Division of Periodontology, Department of Developmental and Surgical Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN, USA

3915939e: J.Exp.Bot., 4343-4360(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: A type III ACC synthase, ACS7, is involved in root gravitropism in Arabidopsis thaliana.
Author: Huang,S.J., Chang,C.L., Wang,P.H., Tsai,M.C., Hsu,P.H., Chang,I.F.
Abstract:
Ethylene is an important plant hormone that regulates developmental processes in plants. The ethylene biosynthesis pathway is a highly regulated process at both the transcriptional and post-translational level. The transcriptional regulation of these ethylene biosynthesis genes is well known. However, post-translational modifications of the key ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) are little understood. In vitro kinase assays were conducted on the type III ACS, AtACS7, fusion protein and peptides to determine whether the AtACS7 protein can be phosphorylated by calcium-dependent protein kinase (CDPK). AtACS7 was phosphorylated at Ser216, Thr296, and Ser299 by AtCDPK16 in vitro. To investigate further the function of the ACS7 gene in Arabidopsis, an acs7-1 loss-of-function mutant was isolated. The acs7-1 mutant exhibited less sensitivity to the inhibition of root gravitropism by treatment with the calcium chelator ethylene glycol tetraacetic acid (EGTA). Seedlings were treated with gradient concentrations of ACC. The results showed that a certain concentration of ethylene enhanced the gravity response. Moreover, the acs7-1 mutant was less sensitive to inhibition of the gravity response by treatment with the auxin polar transport inhibitor 1-naphthylphthalamic acid, but exogenous ACC application recovered root gravitropism. Altogether, the results indicate that AtACS7 is involved in root gravitropism in a calcium-dependent manner in Arabidopsis.

Address: Institute of Plant Biology, National Taiwan University, Taipei, Taiwan

3915944e: BioEssays, 847-852(2013)
Link: [ PubMED ] [ DOI ]
Title: A new molecular explanation for age-related neurodegeneration: The Tyr682 residue of amyloid precursor protein.
Author: Matrone,C.
Abstract:
Emerging evidence supports the role for the intracellular domains of amyloid precursor protein (APP) in the physiology and function of APP. In this short report, I discuss the hypothesis that mutation of Tyr682 on the Y682 ENPTY687 C-terminal motif of APP may be directly or indirectly associated with alterations in APP functioning and activity, leading to neuronal defects and deficits. Mutation of Tyr682 induces an early and progressive age-dependent cognitive and locomotor decline that is associated with a loss of synaptic connections, a decrease in cholinergic tone, and defects in NGF signaling. These findings support a model in which APP-C-terminal domain exerts a pathogenic function in neuronal development and decline, and suggest that Tyr682 potentially could modulate the properties of APP metabolites in humans.

Address: Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark

3915949e: Fungal Genet.Biol., 105-115(2013)
Link: [ PubMED ] [ DOI ]
Title: Genes involved in protein glycosylation determine the activity and cell internalization of the antifungal peptide PAF26 in Saccharomyces cerevisiae.
Author: Harries,E., Carmona,L., Munoz,A., Ibeas,J.I., Read,N.D., Gandia,M., Marcos,J.F.
Abstract:
We have previously characterized the synthetic hexapeptide PAF26 as a cell-penetrating and non-lytic antifungal peptide that is active against Saccharomyces cerevisiae and filamentous fungi. Numerous cell wall (CW) proteins are glycosylated in fungi and many of these play important roles in fungal pathogenesis. In this study, we screened a collection of S. cerevisiae deletion mutants for protein glycosylation genes whose deletion altered the sensitivity to PAF26. Increased tolerance to PAF26 was observed in mutants with the following disrupted genes: PMT1-6, EOS1, ALG5, MNN1, MNN4 and MNN5. Significantly, genes coding for protein O-mannosyltransferase 2 (Pmt2p), which is responsible for the addition of the first mannosyl residue of O-linked carbohydrates, and for Eos1p, an enzyme involved in N-linked glycosylation of proteins, showed resistance to PAF26 and defects in CW integrity. Microscopic studies on the S. cerevisiae Δeos1 deletion mutant demonstrated a blockage of peptide internalization by cells. Protoplasts lacking CWs interacted with the peptide, but were more resistant to peptide killing than cells possessing CWs due to a blockage in PAF26 internalization. Interestingly, protoplasts obtained from Δeos1 behaved similarly to those of the parental strain. Collectively, these observations demonstrate that the CW is a positive factor that determines the internalization of the PAF26, and that Eos1p exerts its activity through the glycosylation of specific protein(s) involved in peptide internalization.

Address: Food Science Department, Instituto de Agroquimica y Tecnologia de Alimentos (IATA), Consejo Superior de Investigaciones Cientificas (CSIC), Avda Agustin Escardino 7, 46980 Paterna, Valencia, Spain

3915951e: Coll.Antropol., 601-606(2013)
Link:
Title: Analysis of purity profiles of generic lisinopril tablets marketed in Croatia.
Author: Benkovic,G., Sokolic,M., Cudina,B., Zulj,R.T., Tomic,S.
Abstract:
In view of an increasing number of generic drugs emerging, a comparative study was performed including the approved lisinopril preparations in the form of tablets marketed in Croatia, to compare purity profiles of generic drugs versus the original medicinal product. Several batches of each individual medicinal product at different stages of their shelf life were analyzed. Impurities were determined by means of high performance liquid chromatography (HPLC). Impurity profiles were demonstrated to be specific for each individual drug. Original drug, as compared to its generic copies, had the lowest values and also the lowest variability of all the tested parameters--type, total number and content of impurities--suggesting that its manufacturing process is to certain degree better controlled compared to other manufacturers. A characteristic impurity C appearing in all the assessed preparations has the lowest levels in the original drug, whereas the amount of the highest unknown impurity does not exceed 0.10% in any of the analyzed preparations. Although the original drug stands out from all the generic preparations with its purity, it can be generally concluded that, as regarding impurities levels, all the analyzed medicinal products are within the ranges of specification limits; accordingly, it is therefore not expected that, in case of lisinopril tablets, administration of the original drug as compared to any of its generic drugs, will be safer for the patient.

E-Mail: goran.benkovic@halmed.hr
Address: Agency for Medicinal Products and Medical Devices of Croatia, Zagreb, Croatia

3915953e: J.Gen.Physiol., 191-206(2013)
Link: [ PubMED ] [ DOI ]
Title: Interactions among DIV voltage-sensor movement, fast inactivation, and resurgent Na current induced by the NaVβ4 open-channel blocking peptide.
Author: Lewis,A.H., Raman,I.M.
Abstract:
Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the NaVβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed NaV1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of NaVβ4 (the "β4 peptide"). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; "CW" channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.

Address: Interdepartmental Biological Sciences Program and 2 Department of Neurobiology, Northwestern University, Evanston, IL 60208, USA

3915954e: J.Exp.Med., 1807-1821(2013)
Link: [ PubMED ] [ DOI ]
Title: Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide-MHC.
Author: Hoerter,J.A., Brzostek,J., Artyomov,M.N., Abel,S.M., Casas,J., Rybakin,V., Ampudia,J., Lotz,C., Connolly,J.M., Chakraborty,A.K., Gould,K.G., Gascoigne,N.R.
Abstract:
Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide-MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4-MHCII interaction is generally weaker than CD8-MHCI.

Address: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA

3915956e: J.Clin.Endocrinol.Metab., 3920-3925(2013)
Link: [ PubMED ] [ DOI ]
Title: Isolated prolactin deficiency associated with serum autoantibodies against prolactin-secreting cells.
Author: Iwama,S., Welt,C.K., Romero,C.J., Radovick,S., Caturegli,P.
Abstract:
Context: Isolated prolactin (PRL) deficiency is a rare entity of unknown etiology manifesting as failure of puerperal lactogenesis. Objective: The aim of the study was to determine the cause of isolated PRL deficiency in an affected woman. Design and Setting: We examined genetic and autoimmune causes of isolated PRL deficiency at academic medical centers. Patient: The patient was a 39-year-old woman with puerperal alactogenesis after two deliveries and undetectable PRL. The other pituitary axes, serum calcium levels, and cranial magnetic resonance imaging were normal. Intervention: Recombinant human PRL (r-hPRL) was administered to the patient. Main Outcome Measures: We measured the sequencing of candidate genes and immunofluorescence analysis of autoantibodies directed against pituitary endocrine cells. Results: There were no rare sequence variants in the genes encoding for PRL, putative PRL-releasing peptide, putative PRL-releasing peptide receptor, or in other genes important for lactotroph lineage development (POU1F1, PROP1, LHX3, LHX4, HESX1, OTX2, and LSD1). The patient serum, on the contrary, contained autoantibodies that specifically recognized a subset of PRL-secreting cells but not PRL itself or any other pituitary cells or hormones. The mother was able to lactate fully after 17 days of treatment with r-hPRL 60 μg/kg every 12 hours, but alactogenesis resumed after treatment was completed. Conclusions: These studies report a new autoimmune etiology for women with isolated PRL deficiency and puerperal alactogenesis.

E-Mail: pcat@jhmi.edu
Address: MD, MPH, Johns Hopkins Pathology, Ross 656, 720 Rutland Avenue, Baltimore, Maryland 21205

3915960e: Antimicrob.Agents Chemother., 5315-5319(2013)
Link: [ PubMED ] [ DOI ]
Title: In Vitro and In Vivo Activities of Antibiotic PM181104.
Author: Mahajan,G., Thomas,B., Parab,R., Patel,Z.E., Kuldharan,S., Yemparala,V., Mishra,P.D., Ranadive,P., D'Souza,L., Pari,K., Sivaramkrishnan,H.
Abstract:
Drug resistance has become a global threat that, if not addressed, may return us to the preantibiotic era. A way to overcome the problem of growing incidence of global antibiotic resistance is to introduce compounds belonging to classes that are new to the clinic. During a screening of the marine microbial extract library for new antibiotics, one of the extracts showed promising antibacterial activity against Gram-positive organisms. Bioactivity-guided isolation and characterization of active metabolites led to the discovery of a novel thiazolyl cyclic-peptide antibiotic, PM181104. It was isolated and characterized from a marine sponge-associated actinobacterium strain of the genus Kocuria (MTCC 5269). The compound exhibited a potent in vitro antibacterial activity against a broad range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The MIC values evaluated for the compound were found to be in the single-digit nanomolar range. In in vivo studies of PM181104 in a BALB/c murine septicemia model, the compound displayed 100% effective dose (ED100) values of 2.5 and 5.0 mg/kg of body weight against MRSA and 10.0 mg/kg against VRE. In this report, in vitro and in vivo studies of PM181104 are described.

Address: Piramal Enterprises Limited, Goregaon, Mumbai, India

3915961e: Mol.Cancer Ther., 2100-2109(2013)
Link: [ PubMED ] [ DOI ]
Title: PI3K Stimulates DNA Synthesis and Cell-Cycle Progression via Its p55PIK Regulatory Subunit Interaction with PCNA.
Author: Wang,G., Cao,X., Lai,S., Luo,X., Feng,Y., Xia,X., Yen,P.M., Gong,J., Hu,J.
Abstract:
Previously, we have shown that p55PIK, an isoform of class IA phosphoinositide 3-kinase (PI3K), specifically interacts with important cell-cycle regulators, such as retinoblastoma (Rb), to promote cell-cycle progression. Here, we used the glutathione S-transferase pull-down assay to identify other p55PIK-interacting proteins besides Rb in a Rb-deficient cell line and found that p55PIK interacted with proliferation cell nuclear antigen (PCNA), which plays a key role in coordinating both initiation of the leading strand DNA replication and discontinuous lagging strand synthesis. Overexpression of p55PIK increased, and knockdown decreased, DNA synthesis and DNA replication by modulating the binding of DNA polymerase δ (Polδ) to PCNA. Moreover, a cell-permeable peptide containing the N-terminal-binding domain of p55PIK (TAT-N24) disrupted the p55PIK-PCNA interaction in cancer cells, and also inhibited the DNA synthesis and tumor growth in cell culture and in vivo. Altogether, our results show that the p55PIK-PCNA interaction is important in regulating DNA synthesis and contributes to tumorigenesis. Furthermore, the p55PIK-PCNA interaction provides a potential new target for anticancer drug development.

E-Mail: jbhu@tjh.tjmu.edu.cn
Address: Corresponding Authors: Junbo Hu, Cancer Research Institute, Tongji Hospital, Huazhong University of Science and Technology, 1095 Jiefang Road, Wuhan 430030, China

3915962e: Mol.Cancer Ther., 1981-1993(2013)
Link: [ PubMED ] [ DOI ]
Title: A Urokinase Receptor-Derived Peptide Inhibiting VEGF-Dependent Directional Migration and Vascular Sprouting.
Author: Bifulco,K., Longanesi-Cattani,I., Liguori,E., Arra,C., Rea,D., Masucci,M.T., De Rosa,M., Pavone,V., Stoppelli,M.P., Carriero,M.V.
Abstract:
The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration, and uPAR88-92 is the minimal sequence required to induce cell motility. We previously showed that soluble forms of uPAR elicit angiogenic responses through their uPAR88-92 chemotactic sequence and that the synthetic peptide SRSRY exerts similar effects. By a drug design approach, based on the conformational analysis of the uPAR88-92 sequence, we developed peptides (pERERY, RERY, and RERF) that potently inhibit signaling triggered by uPAR88-92. In this study, we present evidence that these peptides are endowed also with a clear-cut antiangiogenic activity, although to different extents. The most active, RERF, prevents tube formation by human endothelial cells exposed to SRSRY. RERF also inhibits VEGF-triggered endothelial cell migration and cord-like formation in a dose-dependent manner, starting in the femtomolar range. RERF prevents F-actin polymerization, recruitment of αvβ3 integrin at focal adhesions, and αvβ3/VEGFR2 complex formation in endothelial cells exposed to VEGF. At molecular level, the inhibitory effect of RERF on VEGF signaling is shown by the decreased amount of phospho-FAK and phospho-Akt in VEGF-treated cells. In vivo, RERF prevents VEGF-dependent capillary sprouts originating from the host vessels that invaded angioreactors implanted in mice and neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Consistently, RERF reduced the growth and vascularization rate of tumors formed by HT1080 cells injected subcutaneously in the flanks of nude mice, indicating that RERF is a promising therapeutic agent for the control of diseases fuelled by excessive angiogenesis such as cancer.

E-Mail: m.carriero@istitutotumori.na.it
Address: Corresponding Author: Maria Vincenza Carriero, Department of Experimental Oncology-National Cancer Institute of Naples, via M. Semmola, Naples 80131, Italy

3915963e: Neurosci.Lett., 146-150(2013)
Link: [ PubMED ] [ DOI ]
Title: Intrathecal adrenomedullin modulates acute inflammatory pain in the rat formalin test.
Author: Sugimoto,Y., Shiraishi,S., Yasuda,T., Hamada,H., Kawamoto,M.
Abstract:
Adrenomedullin (AM), a member of the calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pronociceptive mediator. This study was undertaken to investigate the role of AM in acute inflammatory pain induced by formalin injection in rats. Interestingly Cerebrospinal fluid (CSF) levels of AM increased 45min after formalin injection and a selective AM receptor antagonist, AM22-52, administered intrathecally (i.t.) decreased phase 2 flinching in a dose-dependent manner but not phase 1 flinching during the formalin test. This anti-hyperalgesic effect of i.t. AM22-52 lasted for 4h or more. AM in the CSF contributes to the modulation of acute inflammatory pain in the formalin test, and blocking downstream signaling effects of the AM receptor has the potential to relieve pain associated with acute inflammation.

Address: Department of Anesthesiology and Critical Care, Division of Clinical Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

3915965e: Neurobiol.Dis., 183-193(2013)
Link: [ PubMED ] [ DOI ]
Title: Pharmacological blockade of IL-1β/IL-1 receptor type 1 axis during epileptogenesis provides neuroprotection in two rat models of temporal lobe epilepsy.
Author: Noe,F.M., Polascheck,N., Frigerio,F., Bankstahl,M., Ravizza,T., Marchini,S., Beltrame,L., Bandero,C.R., Loscher,W., Vezzani,A.
Abstract:
We studied whether pharmacological blockade of the IL-1β-mediated signaling, rapidly activated in forebrain by epileptogenic injuries, affords neuroprotection in two different rat models of status epilepticus (SE). As secondary outcome, we measured treatment's effect on SE-induced epileptogenesis. IL-1β signaling was blocked by systemic administration of two antiinflammatory drugs, namely human recombinant IL-1 receptor antagonist (anakinra), the naturally occurring and clinically used competitive IL-1 receptor type 1 antagonist, and VX-765 a specific non-peptide inhibitor of IL-1β cleavage and release. Antiinflammatory drugs were given 60min after antiepileptic (AED) drug-controlled SE induced by pilocarpine, or 180min after unrestrained electrical SE, for 7days using a protocol yielding therapeutic drug levels in brain. This drug combination significantly decreased both IL-1β expression in astrocytes and cell loss in rat forebrain. Neuroprotection and the antiinflammatory effect were more pronounced in the electrical SE model. Onset of epilepsy, and frequency and duration of seizures 3months after electrical SE were not significantly modified. Transcriptomic analysis in the hippocampus showed that the combined treatment did not affect the broad inflammatory response induced by SE during epileptogenesis. In particular, the treatment did not prevent the induction of the complement system and Toll-like receptors, both contributing to cell loss and seizure generation. We conclude that the IL-1β signaling represents an important target for reducing cell loss after SE. The data highlight a new class of clinically tested agents affording neuroprotection after a delayed post-injury intervention. Earlier blockade of this rapid onset inflammatory pathway during SE, or concomitant treatment with antiinflammatory drugs targeting additional components of the broad inflammatory response to SE, or co-treatment with AEDs, is likely to be required for optimizing beneficial outcomes.

Address: Department of Neuroscience, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy

3915967e: Part.Fibre Toxicol., 39-39(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Asbestos and erionite prime and activate the NLRP3 inflammasome that stimulates autocrine cytokine release in human mesothelial cells.
Author: Hillegass,J.M., Miller,J.M., MacPherson,M.B., Westbom,C.M., Sayan,M., Thompson,J.K., Macura,S.L., Perkins,T.N., Beuschel,S.L., Alexeeva,V., Pass,H.I., Steele,C., Mossman,B.T., Shukla,A.
Abstract:
BACKGROUND: Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear. RESULTS: We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model. CONCLUSIONS: These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.

Address: Department of Pathology, University of Vermont College of Medicine, Burlington, VT, USA

3915971e: J.Drug Target., 801-808(2013)
Link: [ PubMED ] [ DOI ]
Title: Cellular uptake on N- and C-termini conjugated FITC of Rath cell penetrating peptides and its consequences for gene-expression profiling in U-937 human macrophages and HeLa cervical cancer cells.
Author: Kuo,J.H., Lin,C.W.
Abstract:
Rath peptide has been introduced as a delivery vector that transports various membrane-impermeable cargoes in a non-covalent fashion. In this paper, we present a study on Rath peptide conjugated with fluorescein-5-isothiocynate (FITC) differing in its N- and C-termini. We conducted cellular toxicity and uptake experiments in U-937 and HeLa cells to analyze biocompatibility profiles and translocation efficiencies of Rath peptide with FITC serving as both a cargo and a fluorescent marker. We found that the conjugation of FITC on Rath peptide at N-terminus (FITC-Rath) led to more rapid cellular uptake in U-937 cells and significantly higher cellular uptake in HeLa cells than that which occurred at C-terminus. From DNA microarray analysis, FITC-Rath induced gene expression changes in both U-937 and HeLa cells. Five overlapping regulated genes were identified, and this overlap indicated that FITC-Rath displayed some degree of generality regarding gene responses in the two cell lines used. A real-time quantitative reverse transcriptase-polymerase chain reaction was used to confirm which regulated genes were affected by FITC-Rath. Cell communication, signal transduction, cell surface receptor signaling pathway, signal transducer activity and cellular process, were identified as overlapping biological themes. These data provide useful information on molecular mechanisms for using Rath-based delivery systems.

Address: Department of Pharmacy, Chia Nan University of Pharmacy and Science, Jen-Te , Tainan , Taiwan

3915972e: Clin.Exp.Pharmacol.Physiol., 715-723(2013)
Link: [ PubMED ] [ DOI ]
Title: Haploinsufficiency of endogenous parathyroid hormone-related peptide impairs bone fracture healing.
Author: Wang,Y.H., Qiu,Y., Han,X.D., Xiong,J., Chen,Y.X., Shi,H.F., Karaplis,A.
Abstract:
Previous studies have demonstrated that endogenous parathyroid hormone-related peptide (PTHrP) plays a central role in the physiological regulation of bone formation. However, it is unclear whether endogenous PTHrP plays an important function in enhancing bone fracture healing. To determine whether endogenous PTHrP haploinsufficiency impaired bone fracture healing, closed mid-diaphyseal femur fractures were created in 8-week-old wild-type and Pthrp(+/-) mice. Callus tissue properties were analysed 1, 2 and 4 weeks after fracture by radiography, histology, histochemistry, immunohistochemistry and molecular biology. The size of the calluses was reduced 2 weeks after fracture, and the fracture repairs were poor 4 weeks after fractures, in Pthrp(+/-) compared with wild-type mice. Cartilaginous callus areas were reduced 1 week after fracture, but were increased 2 weeks after fracture in Pthrp(+/-) mice. There was a reduction in the number of ostoblasts, alkaline phosphatase (ALP)-positive areas, Type I collagen immunopositive areas, mRNA levels of ALP, Runt-related transcription factor 2 (Runx2) and Type I collagen, Runx2 and insulin-like growth factor-1 protein levels, the number of osteoclasts and the surface in callus tissues in Pthrp(+/-) compared with wild-type mice. These results demonstrate that endogenous PTHrP haploinsufficiency impairs the fracture repair process by reducing cartilaginous and bony callus formation, with downregulation of osteoblastic gene and protein expression and a reduction in endochondral bone formation, osteoblastic bone formation and osteoclastic bone resorption. Together, the results indicate that endogenous PTHrP plays an important role in fracture healing.

Address: Department of Orthopaedic Surgery, The Affiliated Drum Tower Hospital of Nanjing, University Medical School, Nanjing, China

3915977e: Clin.Cancer Res., 5434-5443(2013)
Link: [ PubMED ] [ DOI ]
Title: In Vivo Imaging of Prostate Cancer Using [68Ga]-Labeled Bombesin Analog BAY86-7548.
Author: Kahkonen,E., Jambor,I., Kemppainen,J., Lehtio,K., Gronroos,T.J., Kuisma,A., Luoto,P., Sipila,H.J., Tolvanen,T., Alanen,K., Silen,J., Kallajoki,M., Roivainen,A., Schafer,N., Schibli,R., Dragic,M., Johayem,A., Valencia,R., Borkowski,S., Minn,H.
Abstract:
PURPOSE: A novel [(68)Ga]-labeled DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 peptide (BAY86-7548) having high affinity to bombesin receptor subtype II to detect primary and metastatic prostate carcinoma using positron emission tomography/computed tomography (PET/CT) was synthesized and evaluated for prostate cancer. EXPERIMENTAL DESIGN: In this first human study with BAY86-7548, 14 men scheduled for radical prostatectomy (n = 11) or with biochemical recurrence after surgery or hormonal therapy (n = 3) were enrolled. The patients received an intravenous injection of BAY86-7548 followed by over 60-minute dynamic imaging of prostate gland (n = 10) and/or subsequent whole-body imaging (n = 14). The visual assessment of PET/CT images included evaluation of intraprostatic (12 subsextants) and pelvic nodal uptake of BAY86-7548 in 11 surgical patients and detection of potential metastatic foci in all patients. In patients with biochemical recurrence, results were compared with those of either [(11)C]-acetate (n = 2) or [18F]-fluoromethylcholine (n = 1) PET/CT. RESULTS: We found a sensitivity, specificity, and accuracy of 88%, 81% and 83%, respectively, for detection of primary PCa and sensitivity of 70% for metastatic lymph nodes using histology as gold standard. BAY86-7548 correctly detected local recurrence in prostate bed and showed nodal relapse in accordance with [(11)C]-acetate PET/CT in 2 patients with biochemical relapse. In the third hormone refractory patient, BAY86-7548 failed to show multiple bone metastases evident on [18F]-fluoromethylcholine PET/CT. CONCLUSION: BAY86-7548 PET/CT is a promising molecular imaging technique for detecting intraprostatic prostate cancer.

Address: Department of Surgery, Division of Urology, Departments of Clinical Physiology and Nuclear Medicine, Oncology and Radiotherapy, and Pathology, Turku University Hospital

3915978e: Circ.Heart Fail., 1067-1076(2013)
Link: [ PubMED ] [ DOI ]
Title: Global proteomics and pathway analysis of pressure-overload-induced heart failure and its attenuation by mitochondrial-targeted peptides.
Author: Dai,D.F., Hsieh,E.J., Chen,T., Menendez,L.G., Basisty,N.B., Tsai,L., Beyer,R.P., Crispin,D.A., Shulman,N.J., Szeto,H.H., Tian,R., MacCoss,M.J., Rabinovitch,P.S.
Abstract:
BACKGROUND: We investigated the protective effects of mitochondrial-targeted antioxidant and protective peptides, Szeto-Schiller (SS) 31 and SS20, on cardiac function, proteomic remodeling, and signaling pathways. METHODS AND RESULTS: We applied an improved label-free shotgun proteomics approach to evaluate the global proteomics changes in transverse aortic constriction (TAC)-induced heart failure and the associated signaling pathway changes using ingenuity pathway analysis. We found that 538 proteins significantly changed after TAC, which mapped to 53 pathways. The top pathways were in the categories of actin cytoskeleton, mitochondrial function, intermediate metabolism, glycolysis/gluconeogenesis, and citrate cycle. Concomitant treatment with SS31 ameliorated the congestive heart failure phenotypes and mitochondrial damage induced by TAC, in parallel with global attenuation of mitochondrial proteome changes, with an average of 84% protection of mitochondrial and 69% of nonmitochondrial protein changes. This included significant amelioration of all the ingenuity pathway analysis noted above. SS20 had only modest effects on heart failure and this tracked with only partial attenuation of global proteomics changes; furthermore, actin cytoskeleton pathways were significantly protected in SS20, whereas mitochondrial and metabolic pathways essentially were not. CONCLUSIONS: This study elucidates the signaling pathways significantly changed in pressure-overload-induced heart failure. The global attenuation of TAC-induced proteomic alterations by the mitochondrial-targeted peptide SS31 suggests that perturbed mitochondrial function may be an upstream signal to many of the pathway alterations in TAC and supports the potential clinical application of mitochondrial-targeted peptide drugs for the treatment heart failure.

Address: Departments of Pathology, Genome Sciences, Anesthesiology, Environmental Health and Biostatistics, University of Washington, Seattle, WA

3915982e: Cancer Chemother.Pharmacol., 669-682(2013)
Link: [ PubMED ] [ DOI ]
Title: The association between the expression of solute carrier transporters and the prognosis of pancreatic cancer.
Author: Mohelnikova-Duchonova,B., Brynychova,V., Hlavac,V., Kocik,M., Oliverius,M., Hlavsa,J., Honsova,E., Mazanec,J., Kala,Z., Melichar,B., Soucek,P.
Abstract:
OBJECTIVES: The aim of this study was to investigate the prognostic significance of fourteen anticancer drug-relevant solute carrier transporters (SLCs) in pancreatic cancer in the context of clinical-pathological characteristics and the KRAS mutation status of tumors. METHODS: Tumors and non-neoplastic pancreatic tissues were obtained from 32 histologically verified patients with pancreatic ductal adenocarcinoma. The transcript profile of SLCs was assessed using quantitative real-time PCR. KRAS mutations in exon 2 were assessed by high-resolution melting analysis and confirmed by sequencing. RESULTS: SLC22A3 and SLC22A18 were upregulated and SLC22A1, SLC22A2, SLC22A11, SLC28A1, SLC28A3 and SLC29A1 were downregulated when compared with non-neoplastic pancreatic tissues. Moreover, significantly lower levels of SLC22A1, SLC22A11 and SLC29A1 were found in tumors with angioinvasion. There was also a significantly higher transcript level of SLC28A1 in tumors with regional lymph nodes affected by metastasis. The study found that a high expression of SLC28A1 was significantly associated with poor overall survival in unselected patients. In contrast, a high expression of SLC22A3 or SLC29A3 was significantly associated with longer overall survival in patients treated with nucleoside analogs. Protein expression of SLC22A1, SLC22A3 and SLC29A3 in tumor tissues of patients with pancreatic carcinoma was observed by immunoblotting for the first time. Finally, SLC levels were not found to be associated with KRAS mutation status in exon 2. CONCLUSIONS: This study identified a number of associations of transcript levels of SLCs with prognosis of pancreatic cancer patients.

E-Mail: D.Beatrice@seznam.cz
Address: Toxicogenomics Unit, National Institute of Public Health, Srobarova 48, Prague 10, Czech Republic

3915983e: Mol.Cell.Biochem., 231-242(2013)
Link: [ PubMED ] [ DOI ]
Title: Response of rat lung tissue to short-term hyperoxia: a proteomic approach.
Author: Spelten,O., Wetsch,W.A., Wrettos,G., Kalenka,A., Hinkelbein,J.
Abstract:
An inspiratory oxygen fraction of 1.0 is often required to avoid hypoxia both in many pre- and in-hospital situations. On the other hand, hyperoxia may lead to deleterious consequences (cell growth inhibition, inflammation, and apoptosis) for numerous tissues including the lung. Whereas clinical effects of hyperoxic lung injury are well known, its impact on the expression of lung proteins has not yet been evaluated sufficiently. The aim of this study was to analyze time-dependent alterations of protein expression in rat lung tissue after short-term normobaric hyperoxia (NH). After approval of the local ethics committee for animal research, N = 36 Wistar rats were randomized into six different groups: three groups with NH with exposure to 100 % oxygen for 3 h and three groups with normobaric normoxia (NN) with exposure to room air (21 % oxygen). After the end of the experiments, lungs were removed immediately (NH0 and NN0), after 3 days (NH3 and NN3) and after 7 days (NH7 and NN7). Lung lysates were analyzed by two-dimensional gel electrophoresis (2D-GE) followed by peptide mass fingerprinting using mass spectrometry. Statistical analysis was performed with Δ 2D (DECODON GmbH, Greifswald, Germany; ANOVA, Bonferroni correction, p < 0.01). Biological functions of differential regulated proteins were studied using functional network analysis (Ingenuity Pathways Analysis, IPA). pO2 was significantly higher in NH-groups compared to NN-groups (581 +/- 28 vs. 98 +/- 12 mmHg; p < 0.01), all other physiological parameters did not differ. Expression of 14 proteins were significantly altered: two proteins were up-regulated and 12 proteins were down-regulated. Even though NH was comparatively short termed, significant alterations in lung protein expression could be demonstrated up to 7 days after hyperoxia. The identified proteins indicate an association with cell growth inhibition, regulation of apoptosis, and approval of structural cell integrity.

E-Mail: oliver.spelten@uk-koeln.de
Address: Department of Anesthesiology and Intensive Care Medicine, University Hospital of Cologne, 50937, Cologne, Germany,

3915987e: Cardiovasc.Res., 297-306(2013)
Link: [ PubMED ] [ DOI ]
Title: Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation.
Author: Rodriguez-Moyano,M., Diaz,I., Dionisio,N., Zhang,X., Avila-Medina,J., Calderon-Sanchez,E., Trebak,M., Rosado,J.A., Ordonez,A., Smani,T.
Abstract:
AIMS: Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca2+)-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca2+ entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. METHODS AND RESULTS: We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca2+ mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca2+ concentration ([Ca2+]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd3+-sensitive current with similar features of the Ca2+ release-activated Ca2+ current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca2+/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca2+/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca2+ influx, CREB activation and VSMCs proliferation. CONCLUSION: Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.

Address: Group of Cardiovascular Physiopathology Lab 113, Department of Medical Physiology and Biophysic, Institute of Biomedicine of Seville, Hospital of Virgen del Rocio/CSIC/University of Sevilla, Avenida Manuel Siurot s/n, Sevilla 41013, Spain

3915988e: Free Radic.Biol.Med., 595-606(2013)
Link: [ PubMED ] [ DOI ]
Title: Lymphocyte mitochondria: toward identification of peripheral biomarkers in the progression of Alzheimer disease.
Author: Sultana,R., Baglioni,M., Cecchetti,R., Cai,J., Klein,J.B., Bastiani,P., Ruggiero,C., Mecocci,P., Butterfield,D.A.
Abstract:
Alzheimer disease (AD) is an age-related neurodegenerative condition. AD is histopathologically characterized by the presence of three main hallmarks: senile plaques (rich in amyloid-β peptide), neuronal fibrillary tangles (rich in phosphorylated tau protein), and synapse loss. However, definitive biomarkers for this devastating disease in living people are still lacking. In this study, we show that levels of oxidative stress markers are significantly increased in the mitochondria isolated from lymphocytes of subjects with mild cognitive impairment (MCI) compared to cognitively normal individuals. Further, an increase in mitochondrial oxidative stress in MCI is associated with MMSE score, vitamin E components, and β-carotene. Further, a proteomics approach showed that alterations in the levels of thioredoxin-dependent peroxide reductase, myosin light polypeptide 6, and ATP synthase subunit β might be important in the progression and pathogenesis of AD. Increased understanding of oxidative stress and protein alterations in easily obtainable peripheral tissues will be helpful in developing biomarkers to combat this devastating disorder.

Address: Department of Chemistry, Center of Membrane Sciences, Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40506-0055, USA

3915989e: Mol.Cell.Endocrinol., 168-174(2013)
Link: [ PubMED ] [ DOI ]
Title: Urotensin II exerts antiapoptotic effect on NRK-52E cells through prostacyclin-mediated peroxisome proliferator-activated receptor α and Akt activation.
Author: Hsu,Y.H., Chen,T.H., Chen,Y.C., Cheng,C.Y., Sue,Y.M., Chen,J.R., Chen,C.H.
Abstract:
Urotensin II (UII) is a cyclic vasoactive peptide which is mainly expressed in kidneys. Although elevated plasma UII levels are associated with renal impairment, the influence of UII on renal injury is unclear. In this study, we monitored the influence of UII on gentamicin-induced apoptosis in rat tubular cells (NRK-52E). We found that UII significantly reduced gentamicin-induced apoptosis and apoptotic signals. Blocking endogenous UII secretion caused cells to be more susceptible to gentamicin. In gentamicin-treated mice, UII also expressed protective effect on renal tubular cells. UII was also found to induce prostacyclin (PGI2) production, which caused peroxisomal proliferator-activated receptor α (PPARα) activation as revealed by both PGI2 synthase siRNA transfection and piroxicam treatment. Blockage of PPARα by siRNA transfection inhibited UII-induced Akt phosphorylation and the antiapoptotic effect of UII. Our results suggest that UII can protect renal tubular cells from gentamicin-induced apoptosis through PGI2-mediated PPARα and Akt activation.

Address: Division of Nephrology, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan

3915990e: J.Vet.Cardiol., 197-204(2013)
Link: [ PubMED ] [ DOI ]
Title: Utility of N-terminal pro-brain natriuretic peptide for assessing hemodynamic significance of patent ductus arteriosus in dogs undergoing ductal repair.
Author: Hariu,C.D., Saunders,A.B., Gordon,S.G., Norby,B., Miller,M.W.
Abstract:
OBJECTIVE: Determine if plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) correlates with markers of hemodynamically significant patent ductus arteriosus (PDA) in dogs. ANIMALS: Ten dogs with PDA and 30 healthy dogs of similar ages. METHODS: Prospective case series with control population. Dogs with PDA were initially evaluated with thoracic radiographs, transthoracic echocardiography, pulmonary capillary wedge pressure (PCWP) and NT-proBNP. Following ductal occlusion, NT-proBNP and echocardiography were repeated within 24 h and at day 90. PCWP was repeated at day 90. Correlation between NT-proBNP and hemodynamic measurements was assessed, and accuracy of NT-proBNP for identifying PDA severity was estimated. RESULTS: NT-proBNP was significantly higher (median; absolute range) in dogs with PDA (895; 490-7118 pmol/L) than controls (663; 50-1318 pmol/L) (p = 0.025). NT-proBNP decreased significantly 90 days post-ductal closure (597; 154-1858 pmol/L) (p = 0.013). Left atrial and ventricular size decreased significantly within 24 h and at day 90 as did PCWP (day 90 only). NT-proBNP correlated with vertebral heart size (VHS) and indexed left ventricular systolic diameter (iLVIDs); concentrations >/= 1224 pmol/L distinguished dogs with elevated VHS and iLVIDs. CONCLUSIONS: NT-proBNP is elevated in dogs with PDA, decreases following PDA closure and correlates with select radiographic and echocardiographic markers of cardiac remodeling.

E-Mail: drhariu@gcvs.com
Address: Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 4474 TAMU, College Station, TX 77843-4474, USA. Electronic address:

3915991e: Int.J.Pharm., 195-201(2013)
Link: [ PubMED ] [ DOI ]
Title: A liposomal delivery system that targets liver endothelial cells based on a new peptide motif present in the ApoB-100 sequence.
Author: Akhter,A., Hayashi,Y., Sakurai,Y., Ohga,N., Hida,K., Harashima,H.
Abstract:
Liver dysfunction is associated with a variety of liver diseases, including viral or alcoholic hepatitis, fibrosis, cirrhosis, and portal hypertension. A targeted drug delivery system would be very useful in the treatment of these diseases. We herein describe the development of a system comprised of a new peptide-lipid conjugate for the efficient delivery of molecules to LEC. The RLTRKRGLK sequence (3359-3367), which mediates the association of LDL with arterial CSPG and an LDL receptor, was utilized as a ligand for achieving this goal. The peptide modified PEG-LPs (RLTR-PEG-LPs) were efficiently taken up by primary liver endothelial cells (liver ECs) and other types of cells. In vivo biodistribution and confocal microscopy analysis showed that RLTR-PEG-LPs became widely accumulated in LECs within a short time. Distribution of RLTR-PEG-LPs was greatly reduced with a pretreatment of unlabeled RLTR-PEG-LPs, not cationic LPs, indicating that the sequence is important for LECs. The findings indicate that a reverse sequence of RLTR (KLGR) modified PEG-LPs (KLGR-PEG-LP) did the same pattern compared with RLTR-PEG-LPs, suggesting that the RKR or RXXR sequence might be essential for LECs targeting. Collectively RLTR-PEG-LPs and KLGR-PEG-LPs have the potential for delivering drugs to LECs.

Address: Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan

3916003e: Brain Res., 44-55(2013)
Link: [ PubMED ] [ DOI ]
Title: Involvement of cocaine- and amphetamine-regulated transcript peptide in the hyperphagic and body weight promoting effects of allopregnanolone in rats.
Author: Nakhate,K.T., Subhedar,N.K., Bharne,A.P., Singru,P.S., Kokare,D.M.
Abstract:
Allopregnanolone (ALLO), a γ-aminobutyric acid (GABA) type A receptor active neurosteroid, elicits hyperphagic response in rodents. Since GABA-A receptors are present on the peptidergic neurons in the hypothalamus, we were interested in finding out if ALLO and neuropeptide cocaine- and amphetamine-regulated transcript (CART) interact and influence feeding behavior. While subcutaneous ALLO treatment, for a period of 7 days, produced a significant increase in food intake and body weight, pretreatment with subthreshold dose of CART (intracerebroventricular) attenuated both the effects. On the other hand, subcutaneous administration of dehydroepiandrosterone sulfate (DHEAS; GABA-A inhibitor neurosteroid) for a period of 7 days resulted in a significant reduction in food intake and body weight. These effects of DHEAS were potentiated by intracerebroventricular pretreatment with subeffective dose of CART. The brains of ALLO-treated rats were processed for the immunohistochemical analysis of CART immunoreactive elements. ALLO treatment resulted in a significant reduction in CART immunoreactivity in the hypothalamic arcuate, paraventricular and lateral nuclei, and nucleus accumbens shell. The results of the present study suggest that ALLO and CART might interact in the brain, and influence food intake and body weight. However, further investigations are needed to clarify the precise mechanisms by which ALLO modulate feeding behavior.

Address: Rungta College of Pharmaceutical Sciences and Research, Rungta Educational Campus, Kohka-Kurud Road, Bhilai 490024, Chhattisgarh, India

3916008e: Neurobiol.Aging, 88-95(2014)
Link: [ PubMED ] [ DOI ]
Title: L-type Ca2+ currents at CA1 synapses, but not CA3 or dentate granule neuron synapses, are increased in 3xTgAD mice in an age-dependent manner.
Author: Wang,Y., Mattson,M.P.
Abstract:
Abnormal neuronal excitability and impaired synaptic plasticity might occur before the degeneration and death of neurons in Alzheimer's disease (AD). To elucidate potential biophysical alterations underlying aberrant neuronal network activity in AD, we performed whole-cell patch clamp analyses of L-type (nifedipine-sensitive) Ca2+ currents (L-VGCC), 4-aminopyridine-sensitive K+ currents, and AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) and NMDA (N-methyl-D-aspartate) currents in CA1, CA3, and dentate granule neurons in hippocampal slices from young, middle-age, and old 3xTgAD mice and age-matched wild type mice. 3xTgAD mice develop progressive widespread accumulation of amyloid β-peptide, and selective hyperphosphorylated tau pathology in hippocampal CA1 neurons, which are associated with cognitive deficits, but independent of overt neuronal degeneration. An age-related elevation of L-type Ca2+ channel current density occurred in CA1 neurons in 3xTgAD mice, but not in wild type mice, with the magnitude being significantly greater in older 3xTgAD mice. The NMDA current was also significantly elevated in CA1 neurons of old 3xTgAD mice compared with in old wild type mice. There were no differences in the amplitude of K+ or AMPA currents in CA1 neurons of 3xTgAD mice compared with wild type mice at any age. There were no significant differences in Ca2+, K+, AMPA, or NMDA currents in CA3 and dentate neurons from 3xTgAD mice compared with wild type mice at any age. Our results reveal an age-related increase of L-VGCC density in CA1 neurons, but not in CA3 or dentate granule neurons, of 3xTgAD mice. These findings suggest a potential contribution of altered L-VGCC to the selective vulnerability of CA1 neurons to tau pathology in the 3xTgAD mice and to their degeneration in AD patients.

E-Mail: wangyu@grc.nia.nih.gov
Address: Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD, USA. Electronic address:

3916009e: Metab.Clin.Exp., 1543-1552(2013)
Link: [ PubMED ] [ DOI ]
Title: Maladaptive immune and inflammatory pathways lead to cardiovascular insulin resistance.
Author: Aroor,A.R., McKarns,S., Demarco,V.G., Jia,G., Sowers,J.R.
Abstract:
Insulin resistance is a hallmark of obesity, the cardiorenal metabolic syndrome and type 2 diabetes mellitus (T2DM). The progression of insulin resistance increases the risk for cardiovascular disease (CVD). The significance of insulin resistance is underscored by the alarming rise in the prevalence of obesity and its associated comorbidities in the Unites States and worldwide over the last 40-50years. The incidence of obesity is also on the rise in adolescents. Furthermore, premenopausal women have lower CVD risk compared to men, but this protection is lost in the setting of obesity and insulin resistance. Although systemic and cardiovascular insulin resistance is associated with impaired insulin metabolic signaling and cardiovascular dysfunction, the mechanisms underlying insulin resistance and cardiovascular dysfunction remain poorly understood. Recent studies show that insulin resistance in obesity and diabetes is linked to a metabolic inflammatory response, a state of systemic and tissue specific chronic low grade inflammation. Evidence is also emerging that there is polarization of macrophages and lymphocytes towards a pro-inflammatory phenotype that contributes to progression of insulin resistance in obesity, cardiorenal metabolic syndrome and diabetes. In this review, we provide new insights into factors, such as, the renin-angiotensin-aldosterone system, sympathetic activation and incretin modulators (e.g., DPP-4) and immune responses that mediate this inflammatory state in obesity and other conditions characterized by insulin resistance.

Address: Division of Endocrinology, Diabetes and Metabolism, Diabetes Cardiovascular Center, University of Missouri, Columbia, MO, USA

3916014e: Biomaterials, 8478-8490(2013)
Link: [ PubMED ] [ DOI ]
Title: Bioimaging and pulmonary applications of self-assembled Flt1 peptide-hyaluronic acid conjugate nanoparticles.
Author: Kim,H., Park,H.T., Tae,Y.M., Kong,W.H., Sung,D.K., Hwang,B.W., Kim,K.S., Kim,Y.K., Hahn,S.K.
Abstract:
Despite wide exploitation of corticosteroid drugs for the treatment of asthma, the poor therapeutic effect on a neutrophilic subtype of asthma prohibits the full recovery of asthma patients. In this work, dexamethasone (Dexa) was loaded in Flt1 peptide-hyaluronic acid (HA) conjugate nanoparticles to overcome the limitation of corticosteroid resistance for the treatment of neutrophilic pulmonary inflammation. Flt1 peptide-HA conjugates are self-assembled to nanoparticles because of hydrophobic Flt1 peptide conjugated to HA by benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) chemistry. In vitro bioimaging showed efficient internalization of Flt1 peptide-HA conjugate nanoparticles into lung epithelial cells by HA-receptor mediated endocytosis. Also, ex vivo imaging for the biodistribution in ICR mice revealed long-term retention of Flt1 peptide-HA conjugate nanoparticles in deep lung tissues possibly due to mucoadhesive property of HA. On the basis of bioimaging results for pulmonary drug delivery applications, we prepared Dexa-loaded Flt1 peptide-HA conjugate nanoparticles. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed the formation of nanoparticles, which reduced cytokine levels of lipopolysaccharide (LPS)-stimulated cells more efficiently than free Dexa. Furthermore, according to the bronchoalveolar lavage (BAL) cellularity and histological analysis, Dexa loaded Flt1 peptide-HA conjugate nanoparticles showed remarkable therapeutic effects in both eosinophilic and neutrophilic asthma model mice.

Address: Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, Republic of Korea

3916017e: Eur.J.Obstet.Gynecol.Reprod.Biol., 198-201(2013)
Link: [ PubMED ] [ DOI ]
Title: Leptin promotes human endometrial carcinoma cell proliferation by enhancing aromatase (P450arom) expression and estradiol formation.
Author: Liu,L., Wang,L., Zheng,J., Tang,G.
Abstract:
OBJECTIVE: Leptin, a multifunctional peptide hormone encoded by the obese (ob) gene, plays an important role in modulating lipid metabolism and energy equilibrium. Leptin reportedly acts as a cell growth factor and enhances the proliferation of various tumors. We investigated the effect of leptin on aromatase (P450arom) expression and estradiol (E2) formation in a model of endometrial carcinoma. STUDY DESIGN: We established a co-culture model of endometrial fibroblasts and the Ishikawa endometrial carcinoma cell line. P450arom mRNA and protein expression were measured with RT-PCR and immunoblotting, respectively, before and after leptin treatment. The effect of leptin on estradiol formation in endometrial carcinoma cells was also detected with a radioimmunological method. RESULTS: P450arom mRNA expression was increased in co-cultures treated with 100 ng/ml leptin (P<0.01). Estradiol synthesis was induced when androstenedione was added to the culture medium, and significantly higher estradiol concentration was observed in co-cultures treated with 100 ng/ml leptin, CONCLUSIONS: Leptin is an important component of the microenvironment and stimulates endometrial carcinoma cell proliferation via enhancing P450arom expression and estradiol synthesis.

E-Mail: clearsky315@yahoo.com.cn
Address: Department of Obstetrics and Gynecology, First Affiliated Hospital, Liaoning Medical College, Jinzhou, Liaoning, China. Electronic address:

3916020e: Int.J.Cardiol., 4178-4183(2013)
Link: [ PubMED ] [ DOI ]
Title: Histone deacetylase inhibition improved cardiac functions with direct antifibrotic activity in heart failure.
Author: Kao,Y.H., Liou,J.P., Chung,C.C., Lien,G.S., Kuo,C.C., Chen,S.A., Chen,Y.J.
Abstract:
BACKGROUND: Histone deacetylases (HDACs), important epigenetic regulatory enzymes, can reduce cardiac hypertrophy and cardiac fibrosis. However, the mechanisms underlying the antifibrotic activity of HDAC inhibitors remain unclear. The purposes of this study were to evaluate the effects of an HDAC inhibitor on systolic heart failure (HF) and investigate the potential mechanisms. METHODS: Echocardiographic, histologic, atrial natriuretic peptide (ANP), and Western blot measurements were performed in HF rats (isoproterenol 100mg/kg, subcutaneous injection) with and without orally administered (100mg/kg for 7 consecutive days) MPT0E014 (a novel HDAC inhibitor). Western blot, migration and proliferation assays were carried out on primary isolated cardiac fibroblasts with and without MPT0E014 (0.1 and 1μM) for 24h. RESULTS: MPT0E014-treated HF rats (n=6) had better fraction shortening (48+/-2 vs. 33+/-4%, p=0.006) and smaller left ventricular end diastolic diameter (4.6+/-0.2 vs. 5.6+/-0.3mm, p=0.031) and systolic diameter (2.4+/-0.2 vs. 3.9+/-0.3mm, p=0.006) than HF (n=7) rats. MPT0E014-treated HF rats had lower ANP, cardiac fibrosis, and angiotensin II type I receptor (AT1R), transforming growth factor (TGF)-β, and CaMKIIδ protein levels compared to HF rats. MPT0E014 (at 1μM, but not 0.1μM) decreased the migration and proliferation of cardiac fibroblasts. MPT0E014 (0.1 and 1μM) decreased expression of the AT1R and TGF-β. CONCLUSIONS: MPT0E014 improved cardiac contractility and attenuated structural remodeling in isoproterenol-induced dilated cardiomyopathy. The direct antifibrotic activity may have contributed to these beneficial effects.

Address: Department of Medical Education and Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

3916022e: Expert Opin.Investig.Drugs, 1425-1436(2013)
Link: [ PubMED ] [ DOI ]
Title: New insights into the role of aminopeptidases in the treatment for both preeclampsia and preterm labor.
Author: Mizutani,S., Tsunemi,T., Mizutani,E., Hattori,A., Tsujimoto,M., Kobayashi,H.
Abstract:
Introduction: Evidence elucidating the pathophysiology and pharmacology of conventional drugs, β-2 stimulants and magnesium sulfate, on safety and effectiveness for preeclampsia and preterm labor are rarely found. Both compounds pass through the placental barrier and could exert their adverse effects on the fetus. Exposure to these agents could be problematic long after the birth, and possibly result in diseases such as autism and cardiomyopathy. Since 1970 the possible roles of placental aminopeptidases, which degrade peptide hormones, in preeclampsia and preterm labor have been studied. Areas covered: Many studies reveal that the fetus secretes peptide hormones, such as angiotensin II, vasopressin, and oxytocin, under hypoxia (stress) during the course of its growth, suggesting the critical effects these hormones have during pregnancy. The roles of placental aminopeptidases, the enzymes which degrade fetal hormones without passing through the placental barrier, were clarified. A first-step production system for recombinant aminopeptidases was established, by which engineered recombinant aminopeptidases were used for further experiments testing expected efficacy on controlling the level of hormones. Expert opinion: The authors conclude that both aminopeptidase A and placental leucine aminopeptidase could be potentially safe and effective drugs for patients and their babies in the treatment of preeclampsia and preterm labor.

Address: Daiya Building Ladys' Clinic , 1F, No.2, 3-15-1, Meieki, Nakamura-ku, Nagoya, 450-0002 , Japan

3916029e: Chronobiol.Int., 1144-1159(2013)
Link: [ PubMED ] [ DOI ]
Title: Chronobiology of the renin-angiotensin-aldosterone system in dogs: relation to blood pressure and renal physiology.
Author: Mochel,J.P., Fink,M., Peyrou,M., Desevaux,C., Deurinck,M., Giraudel,J.M., Danhof,M.
Abstract:
The renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in the regulation of blood pressure and volume homeostasis. Its contribution to the development of cardiovascular diseases has long been recognized. Extensive literature has shown that peptides of the RAAS oscillate with a circadian periodicity in humans, under strong influence of posture, sleep, and age. Although observations of time-variant changes in the renin cascade are available in dogs, no detailed chronobiological investigation has been conducted so far. The present studies were designed to explore the circadian variations of plasma renin activity (RA) and urinary aldosterone-to-creatinine ratio (UA:C) in relation to blood pressure (BP), sodium (UNa, UNa,fe), and potassium (UK, UK,fe) renal handling. Data derived from intensive blood and urine sampling, as well as continuous BP monitoring, were collected throughout a 24-h time period, and analyzed by means of nonlinear mixed-effects models. Differences between the geometric means of day and night observations were compared by parametric statistics. Our results show that variables of the renin cascade, BP, and urinary electrolytes oscillate with significant day-night differences in dogs. An approximately 2-fold (1.6-3.2-fold) change between the average day and night measurements was found for RA (p < 0.001), UA:C (p = 0.01), UK,fe (p = 0.01), and UNa (p = 0.007). Circadian variations in BP, albeit small (less than 10 mm Hg), were statistically significant (p < 0.01) and supported by the model-based analysis. For all variables but UNa and UNa,fe, the levels were higher at night than during the day. The data also indicate that blood pressure oscillates in parallel to the RAAS, such that, as opposed to healthy humans, BP does not drop at night in dogs. The postprandial decrease in RA is assumed to be related to body fluid volume expansion secondary to water and sodium intake, whereas the reduction of UA:C reflects aldosterone-stimulated secretion by the renin-angiotensin II pathway. UNa and UNa,fe peaked in the afternoon, about 7-8 h after food intake, which is consistent with the "impulse-response pattern" of sodium excretion described in previous publications. Finally, UK and UK,fe mirrored aldosterone-mediated potassium secretion in the kidney tubules. To describe the circadian variations of the various variables, two different mathematical representations were applied. A cosine model with a fixed 24-h period was found to fit the periodic variations of RA, UA:C, UK, UK,fe, and BP well, whereas changes in UNa and UNa,fe were best characterized by a surge model. The use of nonlinear mixed effects allowed estimation of population characteristics that can influence the periodicity of the RAAS. Specifically, sodium intake was found to interact with the tonic and the phasic secretion of renin, suggesting that varying feeding time could also impact the chronobiology of the renin cascade. (Author correspondence: jonathan.mochel@novartis.com).

Address: Department of Pharmacology, Leiden-Academic Centre for Drug Research , Leiden , The Netherlands

3916030e: Expert Opin.Drug Deliv., 1497-1513(2013)
Link: [ PubMED ] [ DOI ]
Title: pH- and ion-sensitive polymers for drug delivery.
Author: Yoshida,T., Lai,T.C., Kwon,G.S., Sako,K.
Abstract:
Introduction: Drug delivery systems (DDSs) are important for effective, safe, and convenient administration of drugs. pH- and ion-responsive polymers have been widely employed in DDS for site-specific drug release due to their abilities to exploit specific pH- or ion-gradients in the human body. Areas covered: Having pH-sensitivity, cationic polymers can mask the taste of drugs and release drugs in the stomach by responding to gastric low pH. Anionic polymers responsive to intestinal high pH are used for preventing gastric degradation of drug, colon drug delivery and achieving high bioavailability of weak basic drugs. Tumor-targeted DDSs have been developed based on polymers with imidazole groups or poly(β-amino ester) responsive to tumoral low pH. Polymers with pH-sensitive chemical linkages, such as hydrazone, acetal, ortho ester and vinyl ester, pH-sensitive cell-penetrating peptides and cationic polymers undergoing pH-dependent protonation have been studied to utilize the pH gradient along the endocytic pathway for intracellular drug delivery. As ion-sensitive polymers, ion-exchange resins are frequently used for taste-masking, counterion-responsive drug release and sustained drug release. Polymers responding to ions in the saliva and gastrointestinal fluids are also used for controlled drug release in oral drug formulations. Expert opinion: Stimuli-responsive DDSs are important for achieving site-specific and controlled drug release; however, intraindividual, interindividual and intercellular variations of pH should be considered when designing DDSs or drug products. Combination of polymers and other components, and deeper understanding of human physiology are important for development of pH- and ion-sensitive polymeric DDS products for patients.

Address: Drug Delivery, Pharmaceutical Research and Technology Labs, Astellas Pharma, Inc. , 180 Ozumi, Yaizu, Shizuoka 425-0072 , Japan +81 54 627 6861

3916032e: Clin.Cardiol., 542-547(2013)
Link: [ PubMED ] [ DOI ]
Title: Predictors of 30-day readmission in patients hospitalized with decompensated heart failure.
Author: Hernandez,M.B., Schwartz,R.S., Asher,C.R., Navas,E.V., Totfalusi,V., Buitrago,I., Lahoti,A., Novaro,G.M.
Abstract:
BACKGROUND: Heart failure (HF) is the leading cause of hospitalizations and readmissions in the United States. Approximately one-third of patients admitted for HF are readmitted within 3 months; however, there are few markers that can identify those at highest risk for readmission. The purpose of this study was to identify clinical and laboratory markers associated with hospital readmission in decompensated HF. HYPOTHESIS: Clinical and laboratory markers are associated with readmission rates in decompensated HF. METHODS: Clinical and laboratory data from 412 patients admitted with HF were analyzed using a multivariable logistic regression analysis to find predictors of HF readmission by 30 days. RESULTS: HF readmission rates at 30 days were lowest in those with at least 2 of the following discharge criteria: net fluid reduction >1.3 L (odds ratio [OR]: 0.27, P = 0.019), serum sodium level >135 (OR: 0.46, P = 0.034), and N-terminal brain natriuretic peptide level reduction >23% (OR: 0.11, P = 0.048). In multivariate analysis, those patients meeting >/=2 criteria had a very low risk of 30-day readmission (OR: 0.10, 95% confidence interval: 0.01-0.68, P = 0.019) compared to patients who failed to meet 2 criteria. CONCLUSIONS: A negative fluid balance, normal serum sodium, and net reduction in N-terminal brain natriuretic peptide level during hospitalization may be important indices to target to help reduce the likelihood of HF readmission within 30 days.

Address: Department of Internal Medicine, Cleveland Clinic Florida, Weston, Florida

3916033e: J.Biol.Rhythms, 239-248(2013)
Link: [ PubMED ] [ DOI ]
Title: E and M circadian pacemaker neurons use different PDF receptor signalosome components in drosophila.
Author: Duvall,L.B., Taghert,P.H.
Abstract:
We used real-time imaging to detect cAMP levels in neurons of intact fly brains to study the mechanisms of circadian pacemaker synchronization by the neuropeptide pigment dispersing factor (PDF) in Drosophila. PDF receptor (PDF-R) is expressed by both M (sLNv) and E (LNd) pacemaker subclasses and is coupled to G(sα) in both cases. We previously reported that PDF-R in M pacemakers elevates cAMP levels by activating the ortholog of mammalian adenylate cyclase 3 (AC3) but that AC3 disruptions had no effect on E pacemaker sensitivity to PDF. Here, we show that PDF-R in E pacemakers activates a different AC isoform, AC78C, an ortholog of mammalian AC8. Knockdown of AC78C by transgenic RNAi substantially reduces, but does not completely abrogate, PDF responses in these E pacemakers. The knockdown effect is intact when restricted to mature stages, suggesting a physiological and not a development role for AC78C in E pacemakers. The AC78C phenotype is rescued by the overexpression of AC78C but not by overexpression of the rutabaga AC. AC78C overexpression does not disrupt PDF responses in these E pacemakers, and neither AC78C knockdown nor its overexpression disrupted locomotor rhythms. Finally, knockdown of 2 AKAPs, nervy and AKAP200, partially reduces LNd PDF responses. These findings begin to identify the components of E pacemaker PDF-R signalosomes and indicate that they are distinct from PDF-R signalosomes in M pacemakers: we propose they contain AC78C and at least 1 other AC.

Address: Department of Anatomy & Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA

3916034e: Euro Surveill., 20548-20548(2013)
Link:
Title: First report of IMI-1-producing colistin-resistant Enterobacter clinical isolate in Ireland, March 2013.
Author: Boo,T.W., O'Connell,N., Power,L., O'Connor,M., King,J., McGrath,E., Hill,R., Hopkins,K.L., Woodford,N.
Abstract:
We report the first case in Ireland of an IMI-1 carbapenemase-producing Enterobacter asburiae, which was resistant to both colistin and fosfomycin. The circumstances under which this isolate was acquired were unclear. Several reports of IMI-producing Enterobacter spp. have emerged in recent years, and colistin resistance in Enterobacteriaceae is also increasingly reported. Laboratories should be aware of the unusual antibiograms of IMI-producing isolates.

E-Mail: teck.boo@hse.ie
Address: Department of Medical Microbiology, Galway University Hospitals, HSE West, Ireland

3916036e: J.Clin.Endocrinol.Metab., E1575-E1582(2013)
Link: [ PubMED ] [ DOI ]
Title: Parallel Variations of Insulin-Like Peptide 3 (INSL3) and Antimullerian Hormone (AMH) in Women With the Polycystic Ovary Syndrome According to Menstrual Cycle Pattern.
Author: Pelusi,C., Fanelli,F., Pariali,M., Zanotti,L., Gambineri,A., Pasquali,R.
Abstract:
Context: Antimullerian hormone (AMH) and insulin-like factor 3 (INSL3) represent ovarian functional markers of granulosa and theca cells, respectively. Objective: We conducted a prospective study to investigate AMH and INSL3 plasma levels in 3 groups of women with polycystic ovary syndrome (PCOS) classified according to menstrual cyclicity pattern and their relationship with ovarian morphology and hormonal levels. Design and Participants: AMH and INSL3 were measured in a cohort of 57 patients with PCOS, divided into 3 groups according to menstrual status: eumenorrheic (PCOS-E, n = 15), oligomenorrheic (PCOS-O, n = 25), and amenorrheic (PCOS-A, n = 17). Clinical and endocrine characteristics and ovarian morphology were compared among the groups. Twenty-seven age- and weight-matched women without hyperandrogenism were included as controls. Results: According to the menstrual pattern, the women with PCOS-A and PCOS-O had higher INSL3 levels with respect to the control women (P = .025 and P = .004, respectively) and higher but not significant INSL3 levels compared with those of the women with PCOS-E. AMH levels were significantly higher in women with PCOS-A and PCOS-O with respect to those in women with PCOS-E (P < .001 and P < .001, respectively) and control women (P < .001 and P < .001, respectively). Interestingly, a significant positive correlation was found between INSL3 and AMH blood levels in all women with PCOS (R = 0.43; P = .002) and across the groups (R = 0.41; P < .001). Conclusions: INSL3 and AMH levels are significantly correlated with each other in women with PCOS, and they are significantly increased, particularly in the presence of amenorrhea and oligomenorrhea. INSL3 and AMH may reflect a dysfunction of PCOS thecal and granulosa cells, which are responsible for the increased androgen production and chronic anovulation of this condition.

E-Mail: renato.pasquali@unibo.it
Address: Department of Medical and Surgical Science, Division of Endocrinology, University Alma Mater Studiorum, Sant'Orsola-Malpighi Hospital, Via Massarenti 9, 40138 Bologna, Italy

3916037e: Endocrinology, 4249-4258(2013)
Link: [ PubMED ] [ DOI ]
Title: Evidence that Orphanin FQ Mediates Progesterone Negative Feedback in the Ewe.
Author: Nestor,C.C., Coolen,L.M., Nesselrod,G.L., Valent,M., Connors,J.M., Hileman,S.M., Cheng,G., Lehman,M.N., Goodman,R.L.
Abstract:
Orphanin FQ (OFQ), a member of the opioid family, is found in many areas of the hypothalamus and, when given centrally OFQ inhibits episodic LH secretion in rodents and sheep. Because GnRH neurons are devoid of the appropriate receptors to mediate steroid negative feedback directly, neurons that release OFQ may be involved. Using immunocytochemistry, we first determined that most OFQ neurons in the arcuate nucleus (ARC) and other hypothalamic regions of luteal phase ewes contained both estrogen receptor α and progesterone (P) receptor. Given a similar high degree of steroid receptor colocalization in other ARC subpopulations, we examined whether OFQ neurons of the ARC contained those other neuropeptides and neurotransmitters. OFQ did not colocalize with kisspeptin, tyrosine hydroxylase, or agouti-related peptide, but all ARC OFQ neurons coexpressed proopiomelanocortin. To test for a role for endogenous OFQ, we examined the effects of an OFQ receptor antagonist, [Nphe1,Arg14,Lys15]Nociceptin-NH2 (UFP-101) (30 nmol intracerebroventricular/h), on LH secretion in steroid-treated ewes in the breeding season and ovary-intact ewes in anestrus. Ovariectomized ewes with luteal phase concentrations of P and estradiol showed a significant increase in LH pulse frequency during infusion of UFP-101 (4.5 +/- 0.5 pulses/6 h) compared with saline infusion (2.6 +/- 0.4 pulses/6 h), whereas ewes implanted with only estradiol did not. Ovary-intact anestrous ewes displayed no significant differences in LH pulse amplitude or frequency during infusion of UFP-101. Therefore, we conclude that OFQ mediates, at least in part, the negative feedback action of P on GnRH/LH pulse frequency in sheep.

E-Mail: rgoodman@hsc.wvu.edu
Address: Department of Physiology and Pharmacology, PO Box 9229, West Virginia University, Morgantown, West Virginia 26506

3916038e: J.Control.Release, 144-151(2013)
Link: [ PubMED ] [ DOI ]
Title: A depot-forming glucagon-like peptide-1 fusion protein reduces blood glucose for five days with a single injection.
Author: Amiram,M., Luginbuhl,K.M., Li,X., Feinglos,M.N., Chilkoti,A.
Abstract:
Peptide drugs are an exciting class of pharmaceuticals for the treatment of a variety of diseases; however, their short half-life dictates multiple and frequent injections causing undesirable side effects. Herein, we describe a novel peptide delivery system that seeks to combine the attractive features of prolonged circulation time with a prolonged release formulation. This system consists of glucagon-like peptide-1, a type-2 diabetes drug fused to a thermally responsive, elastin-like-polypeptide (ELP) that undergoes a soluble-insoluble phase transition between room temperature and body temperature, thereby forming an injectable depot. We synthesized a set of GLP-1-ELP fusions and verified their proteolytic stability and potency in vitro. Significantly, a single injection of depot forming GLP-1-ELP fusions reduced blood glucose levels in mice for up to 5days, 120 times longer than an injection of the native peptide. These findings demonstrate the unique advantages of using ELPs to release peptide-ELP fusions from a depot combined with enhanced systemic circulation to create a tunable peptide delivery system.

Address: Department of Biomedical Engineering, Duke University, Durham 27708, USA

3916040e: Biol.Blood Marrow Transplant., 1525-1529(2013)
Link: [ PubMED ] [ DOI ]
Title: Reciprocal expression of enteric antimicrobial proteins in intestinal graft-versus-host disease.
Author: Eriguchi,Y., Uryu,H., Nakamura,K., Shimoji,S., Takashima,S., Iwasaki,H., Miyamoto,T., Shimono,N., Hashimoto,D., Akashi,K., Ayabe,T., Teshima,T.
Abstract:
We recently demonstrated that expression of α-defensins, the major antimicrobial peptides produced by Paneth cells, was severely suppressed in mice with graft-versus-host disease (GVHD). In this study, we found that antibacterial lectin, regenerating islet-derived IIIγ (RegIIIγ) was upregulated in villous enterocytes, thus demonstrating the reciprocal control of enteric antimicrobial proteins in GVHD. Upregulation of RegIIIγ was mediated by a mechanism independent upon radiation-induced intestinal tract damage. MyD88-mediated signaling in intestinal epithelium was required for RegIIIγ upregulation in GVHD and antibiotic therapy downregulated RegIIIγ expression. These results suggest that MyD88-mediated sensing of the intestinal microbes disregulated in GVHD induces RegIIIγ upregulation in GVHD and argue a role for RegIIIγ in the pathogenesis of GVHD.

Address: Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Science, Fukuoka, Japan

3916041e: J.Neuroendocrinol., 876-886(2013)
Link: [ PubMED ] [ DOI ]
Title: Kisspeptin neurones do not directly signal to RFRP-3 neurones but RFRP-3 may directly modulate a subset of hypothalamic kisspeptin cells in mice.
Author: Poling,M.C., Quennell,J.H., Anderson,G.M., Kauffman,A.S.
Abstract:
The neuropeptides kisspeptin (encoded by Kiss1) and RFamide-related peptide-3 (also known as GnIH; encoded by Rfrp) are potent stimulators and inhibitors, respectively, of reproduction. Whether kisspeptin or RFRP-3 might act directly on each other's neuronal populations to indirectly modulate reproductive status is unknown. To examine possible interconnectivity of the kisspeptin and RFRP-3 systems, we performed double-label in situ hybridisation (ISH) for the RFRP-3 receptors, Gpr147 and Gpr74, in hypothalamic Kiss1 neurones of adult male and female mice, as well as double-label ISH for the kisspeptin receptor, Kiss1r, in Rfrp-expressing neurones of the hypothalamic dorsal-medial nucleus (DMN). Only a very small proportion (5-10%) of Kiss1 neurones of the anteroventral periventricular region expressed Gpr147 or Gpr74 in either sex, whereas higher co-expression (approximately 25%) existed in Kiss1 neurones in the arcuate nucleus. Thus, RFRP-3 could signal to a small, primarily arcuate, subset of Kiss1 neurones, a conclusion supported by the finding of approximately 35% of arcuate kisspeptin cells receiving RFRP-3-immunoreactive fibre contacts. By contrast to the former situation, no Rfrp neurones co-expressed Kiss1r in either sex, and Tacr3, the receptor for neurokinin B (NKB; a neuropeptide co-expressed with arcuate kisspeptin neurones) was found in <10% of Rfrp neurones. Moreover, kisspeptin-immunoreactive fibres did not readily appose RFRP-3 cells in either sex, further excluding the likelihood that kisspeptin neurones directly communicate to RFRP-3 neurones. Lastly, despite abundant NKB in the DMN region where RFRP-3 soma reside, NKB was not co-expressed in the majority of Rfrp neurones. Our results suggest that RFRP-3 may modulate a small proportion of kisspeptin-producing neurones in mice, particularly in the arcuate nucleus, whereas kisspeptin neurones are unlikely to have any direct reciprocal actions on RFRP-3 neurones.

Address: Department of Reproductive Medicine, University of California San Diego, La Jolla, CA, USA

3916043e: J.Biomed.Nanotechnol., 1345-1353(2013)
Link: [ PubMED ] [ DOI ]
Title: A tissue engineered renovascular graft composed of proteins, polymers, smooth muscle and endothelial cells for renal artery stenosis.
Author: Yin,H., Wang,X.H., Zhu,X.D., Han,H., Guo,W.Y., Ful,Z.R.
Abstract:
Endarterectomy and bypass surgery to treat renal artery stenosis are increasingly shunned these days due to high risks of complications during and after the surgery. Striving to find a sound alternative solution, we pioneered the construction of a tissue engineered renovascular graft that could immediately restore the normal blood flow to kidneys and sustain renal functions without suffering restenosis after the surgery. A highly porous scaffold was first constructed by electrospinning polycaprolactone, poliglecaprone, gelatin and elastin, giving the vast majority of non-woven fibers in the scaffold a diameter below 1200 nm. To recapitulate the anatomical and functional signatures of renal arteries, a bi-layer vasculature comprising a smooth muscle layer topped by an endothelial layer was built on the scaffold. The vasculature witnessed a sustained proliferation for up to 10 days in vitro and robustly secreted prostacyclin and endothelin-1, evidencing that the vasculature was functionally comparable to native renal arteries. After 30 days as a renovascular graft in mice, the luminal diameter of the graft remained clear without a restenosis and an increased confluence of the endothelial layer was observed. The tensile test confirmed that the renovascular graft was mechanically superior to native renal arteries and retained this advantage within 30 days in vivo. Also, this renovascular graft sustained renal functions as evidenced by normal levels of serum creatinine, urine creatinine and serum urea nitrogen and the lack of edema in the kidney cortex. These results demonstrate that this renovascular graft holds a great therapeutic promise for renal artery stenosis.

Address: Department of Surgery, Transplant Section, Shanghai Changzheng Hospital, Shanghai Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China

3916044e: J.Reprod.Infertil., 50-55(2013)
Link: [ PubMED ] [ OpenAccess]
Title: Investigation of apelin expression in endometriosis.
Author: Ozkan,Z.S., Cilgin,H., Simsek,M., Cobanoglu,B., Ilhan,N.
Abstract:
BACKGROUND: Apelin is a mitogenic peptide; it has functions in vessel formation and cell proliferation. In this study we aimed to evaluate the serum and tissue levels and local expression pattern of apelin in eutopic and ectopic endometrium from patients with and without endometriosis and to compare the proliferative and secretory phase differences. METHODS: Thirty women with endometriosis and 15 women without endometriosis undergoing surgery for benign indications as control group were included in the study. Serum and tissue concentrations and proliferative and secretory phase expression patterns of apelin were evaluated in the ectopic and eutopic endometrium using immunoassay and immunohistochemistry methods. The results were compared with Mann-Whitney U test. The p-values smaller than 0.05 were considered as statistically significant. RESULTS: Apelin expression was detected in eutopic and ectopic endometrium of women with endometriosis and endometrium of control group. Intense immunoreactivity of apelin was observed in glandular cells of eutopic and ectopic endometrial tissues of women with endometriosis and endometrium of control group during secretory phase (p<0.01). In both groups, tissue concentrations of apelin were higher than of the serum (p=0.03) but, there were no significant differences between the two groups for tissue and serum concentrations of apelin. CONCLUSION: Apelin expression showed cyclic changes in eutopic and ectopic endometrium. Its expression may be related to menstrual changes of angiogenesis in endometrium of women.

Address: Department of Obstetrics and Gynecology, Firat University School of Medicine, Elazig, Turkey

3916046e: J.Pharmacol.Exp.Ther., 529-539(2013)
Link: [ PubMED ] [ DOI ]
Title: Bimodal concentration-response of nicotine involves the nicotinic acetylcholine receptor, transient receptor potential vanilloid type 1, and transient receptor potential ankyrin 1 channels in mouse trachea and sensory neurons.
Author: Kichko,T.I., Lennerz,J., Eberhardt,M., Babes,R.M., Neuhuber,W., Kobal,G., Reeh,P.W.
Abstract:
High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 μM --nicotine, a maxiμm at 100 μM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pHe-dependent, - five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 μM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200-fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded.

Address: Institute of Physiology and Pathophysiology (T.I.K., J.L., M.E., R.M.B., P.W.R.) and Institute of Anatomy I (W.N.), Friedrich-Alexander-University, Erlangen, Germany

3916047e: Am.J.Physiol.R, 804-810(2013)
Link: [ PubMED ] [ DOI ]
Title: Neuropeptide W increases mean arterial pressure as a result of behavioral arousal.
Author: Pate,A.T., Yosten,G.L., Samson,W.K.
Abstract:
Neuropeptide W (NPW), an endogenous ligand for G protein-coupled receptors NPBWR1 (GPR7) and NPBWR2 (GPR8), has been detected in neurons in limbic and reticular activating system areas known to be important in arousal, as well as hypothalamic nuclei known to be important in food and water intake and the neuroendocrine response to stress. In rat, central administration of NPW increased mean arterial pressure (MAP) and behaviors associated with locomotion and grooming. We hypothesized that the NPW-induced increase in MAP was secondary to those increases in physical activity. Since peptides that stimulate arousal have been shown to increase sympathetic activity (e.g., orexin), we tested the ability of the mixed α1- and α2-adrenergic antagonist, phentolamine, to block the NPW-23-induced rise in MAP. Phentolamine pretreatment abrogated the NPW-induced MAP increase. However, we noticed the animals no longer exhibited NPW-associated behavioral arousal when pretreated with phentolamine. Anesthesia also blocked the NPW-induced increase in MAP, although the animals still were able to respond with an increase in MAP to centrally administered ANG II. Additionally, pretreatment with an orexin type 1 receptor antagonist significantly reduced the behavioral action of NPW-23 and completely blocked the peptide's action to increase MAP, suggesting that orexin neurons are downstream targets of NPW. Our results suggest that NPW increased MAP secondary to increased behavioral arousal.

Address: Department of Pharmacological and Physiological Science, Saint Louis University, St. Louis, Missouri

3916055e: Oncol.Res., 419-425(2013)
Link: [ PubMED ] [ DOI ]
Title: Therapeutic effect of oncolytic adenovirus expressing relaxin in radioresistant oral squamous cell carcinoma.
Author: Lee,S.Y., Park,H.R., Rhee,J., Park,Y.M., Kim,S.H.
Abstract:
Radioresistance is one of the main determinants of treatment outcome in oral squamous cell carcinoma (OSCC), and treatment of radioresistant OSCC is difficult due to cross resistance to other conventional treatments. We aimed to identify whether genetically modified oncolytic adenovirus expressing relaxin (RLX), which affects collagen metabolism, can effectively inhibit growth of the radioresistant OSCC. Therapeutic effect of oncolytic adenovirus was compared between radiosensitive and radioresistant OSCC cell lines in vitro and in vivo, and spread of adenovirus throughout the tumor mass was verified by immunohistochemistry (IHC). Oncolytic adenovirus effectively killed cancer cells and there was no significant difference in the cytotoxic effect between radiosensitive and radioresistant OSCC cell lines. In animal experiments, the adenovirus significantly reduced the size of tumor, and there was no significant difference between radiosensitive and radioresistant OSCC. In IHC, RLX expressing adenovirus showed better proliferation and eliminated collagens more effectively compared to RLX nonexpressing adenovirus. These findings suggested that genetically modified oncolytic adenovirus can effectively inhibit growth of the radioresistant OSCC and might be a new therapeutic option in radioresistant OSCC.

Address: Department of Otolaryngology-Head and Neck Surgery, Chung-Ang University College of Medicine, Seoul, Korea

3916056e: Oncol.Res., 411-417(2013)
Link: [ PubMED ] [ DOI ]
Title: Inhibition of gastric cancer cell growth in vivo by overexpression of adeno-associated virus-mediated survivin mutant C84A.
Author: Weng,Y., Fei,B., Chi,A.L., Cai,M.
Abstract:
Survivin is overexpressed in most of human cancer cells and tissues. Its overexpression is associated with apoptosis inhibition, drug resistance, and poor prognosis. In this study, we investigated the effect of adeno-associated virus (AAV)-mediated survivin mutant Cys84Ala [rAAV-Sur-Mut(C84A)] on gastric cancer growth. Sur-Mut(C84A) was subcloned into the AAV expression vector pAM/CAG to generate recombinant (r)AAV-Sur-Mut(C84A) virus. Cell survival was determined by the MTT method. Apoptosis was measured by FACS analysis and TUNEL. Tumor growth was assessed using a xenograft mouse model. Results showed that treatment of rAAV-Sur-Mut(C84A) virus significantly reduced cell survival, induced apoptosis, and sensitized gastric cancer cells to 5-fluorouracil in vitro. Furthermore, treatment of rAAV-Sur-Mut(C84A) virus markedly induced apoptosis and inhibited gastric cancer growth in vivo. Moreover, rAAV-Sur-Mut(C84A) treatment strongly enhanced the antitumor activity of 5-fluorouracil. Our results suggest that the combination of rAAV-Sur-Mut(C84A) with chemotherapy may be a promising strategy for gastric cancer therapy.

Address: Department of Thoracic and Cardiovascular Surgery, No. 4 People's Hospital of Wuxi City, Wuxi City, PR China

3916057e: Oncol.Res., 383-392(2013)
Link: [ PubMED ] [ DOI ]
Title: CD40 signal regulates CXCR4 mediating ovarian carcinoma cell migration: implications for extrapelvic metastastic factors.
Author: Qu,Q.X., Huang,Q., Xu,J., Duan,L.T., Zhu,Y.B., Zhang,X.G.
Abstract:
Ovarian carcinomas are highly invasive, especially in the peritoneal cavity. SDF-1α and its receptor, CXCR4, play a crucial role in migration of cancer cells. Here, SDF-1α directed HO8910 cell migration, but not SKOV3 cells. After being educated to express CXCR4 in vivo or by treating with sCD40L, SDF-1α reexhibited the ability of directing SKOV3 cell migration, which could be antagonized by CXCR4-neutralizing antibody. Furthermore, concomitant expression of CXCR4/CD40 in ovarian carcinoma tissues had stronger correlation with pelvic metastasis than did each alone. It is suggest that SDF-1α acts through CXCR4 to induce ovarian cancer cell migration, which could be facilitated by CD40 activation. Simultaneously examining the expression of CXCR4 and CD40 will provide valuable diagnosis of pelvic metastasis for ovarian carcinomas.

Address: Clinical Immunology Institute, The First Affiliated Hospital of Soochow University, Suzhou, China

3916061e: Drug Metab.Dispos., 1867-1874(2013)
Link: [ PubMED ] [ DOI ]
Title: Impact of peptide transporter 1 on the intestinal absorption and pharmacokinetics of valacyclovir after oral dose escalation in wild-type and PepT1 knockout mice.
Author: Yang,B., Hu,Y., Smith,D.E.
Abstract:
The primary objective of this study was to determine the in vivo absorption properties of valacyclovir, including the potential for saturable proton-coupled oligopeptide transporter 1 (PepT1)-mediated intestinal uptake, after escalating oral doses of prodrug within the clinical dose range. A secondary aim was to characterize the role of PepT1 on the tissue distribution of its active metabolite, acyclovir. [3H]Valacyclovir was administered to wild-type (WT) and PepT1 knockout (KO) mice by oral gavage at doses of 10, 25, 50, and 100 nmol/g. Serial blood samples were collected over 180 minutes, and tissue distribution studies were performed 20 minutes after a 25-nmol/g oral dose of valacyclovir. We found that the C(max) and area under the curve (AUC)(0)-(1)(8)(0) of acyclovir were 4- to 6-fold and 2- to 3-fold lower, respectively, in KO mice for all four oral doses of valacyclovir. The time to peak concentration of acyclovir was 3- to 10-fold longer in KO compared with WT mice. There was dose proportionality in the C(max) and AUC(0)-(1)(8)(0) of acyclovir in WT and KO mice over the valacyclovir oral dose range of 10-100 nmol/g (i.e., linear absorption kinetics). No differences were observed in the peripheral tissue distribution of acyclovir once these tissues were adjusted for differences in perfusing drug concentrations in the systemic circulation. In contrast, some differences were observed between genotypes in the concentrations of acyclovir in the distal intestine. Collectively, the findings demonstrate a critical role of intestinal PepT1 in improving the rate and extent of oral absorption for valacyclovir. Moreover, this study provides definitive evidence for the rational development of a PepT1-targeted prodrug strategy.

Address: Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan

3916062e: ASN Neuro, e00121-e00121(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Neuroendocrine and cardiac metabolic dysfunction and NLRP3 inflammasome activation in adipose tissue and pancreas following chronic spinal cord injury in the mouse.
Author: Bigford,G.E., Bracchi-Ricard,V.C., Keane,R.W., Nash,M.S., Bethea,J.R.
Abstract:
CVD (cardiovascular disease) represents a leading cause of mortality in chronic SCI (spinal cord injury). Several component risk factors are observed in SCI; however, the underlying mechanisms that contribute to these risks have not been defined. Central and peripheral chronic inflammation is associated with metabolic dysfunction and CVD, including adipokine regulation of neuroendocrine and cardiac function and inflammatory processes initiated by the innate immune response. We use female C57 Bl/6 mice to examine neuroendocrine, cardiac, adipose and pancreatic signaling related to inflammation and metabolic dysfunction in response to experimentally induced chronic SCI. Using immuno-histochemical, -precipitation, and -blotting analysis, we show decreased POMC (proopiomelanocortin) and increased NPY (neuropeptide-Y) expression in the hypothalamic ARC (arcuate nucleus) and PVN (paraventricular nucleus), 1-month post-SCI. Long-form leptin receptor (Ob-Rb), JAK2 (Janus kinase)/STAT3 (signal transducer and activator of transcription 3)/p38 and RhoA/ROCK (Rho-associated kinase) signaling is significantly increased in the heart tissue post-SCI, and we observe the formation and activation of the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome in VAT (visceral adipose tissue) and pancreas post-SCI. These data demonstrate neuroendocrine signaling peptide alterations, associated with central inflammation and metabolic dysfunction post-SCI, and provide evidence for the peripheral activation of signaling mechanisms involved in cardiac, VAT and pancreatic inflammation and metabolic dysfunction post-SCI. Further understanding of biological mechanisms contributing to SCI-related inflammatory processes and metabolic dysfunction associated with CVD pathology may help to direct therapeutic and rehabilitation countermeasures.

Address: *The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, U.S.A

3916063e: Ren.Fail., 1216-1222(2013)
Link: [ PubMED ] [ DOI ]
Title: Role of B-type natriuretic peptide as a marker of mortality in acute kidney injury patients treated with continuous renal replacement therapy.
Author: Jeong,E.G., Nam,H.S., Lee,S.M., An,W.S., Kim,S.E., Son,Y.K.
Abstract:
OBJECTIVES: Acute kidney injury (AKI) treated with continuous renal replacement therapy (CRRT) is associated with poor outcome. Plasma B-type natriuretic peptide (BNP) is a biomarker related to fluid volume overload, and is elevated in AKI patients. The purpose of the study was to assess whether BNP levels at the time of starting CRRT could be used as a predictor of mortality in patients with AKI receiving CRRT. METHODS: We conducted a prospective observational cohort study enrolling 149 patients with AKI receiving CRRT. The primary outcome was mortality during CRRT. RESULTS: The median BNP level of 84 (56.3%) patients who expired was significantly higher than that of those who survived (1812.5 vs. 475.0 pg/mL; p = 0.01). Receiver operating characteristic curves demonstrated BNP levels as a predictor of mortality during CRRT with an area under the curve of 0.77 (p = 0.000), and the optimal threshold for BNP was 1054 pg/mL. Patients with BNP levels above 1054 pg/mL had a significantly higher mortality (76.6 vs. 34.7%; p = 0.01). CONCLUSION: Elevated BNP level is associated with mortality in patients with AKI receiving CRRT.

Address: Department of Internal Medicine, Dong-A University College of Medicine , Busan , Korea

3916067e: Am.J.Transplant., 2664-2671(2013)
Link: [ PubMED ] [ DOI ]
Title: Metabolic assessment prior to total pancreatectomy and islet autotransplant: utility, limitations and potential.
Author: Lundberg,R., Beilman,G.J., Dunn,T.B., Pruett,T.L., Chinnakotla,S.C., Radosevich,D.M., Robertson,R.P., Ptacek,P., Balamurugan,A.N., Wilhelm,J.J., Hering,B.J., Sutherland,D.E., Moran,A., Bellin,M.D.
Abstract:
Islet autotransplant (IAT) may ameliorate postsurgical diabetes following total pancreatectomy (TP), but outcomes are dependent upon islet mass, which is unknown prior to pancreatectomy. We evaluated whether preoperative metabolic testing could predict islet isolation outcomes and thus improve assessment of TPIAT candidates. We examined the relationship between measures from frequent sample IV glucose tolerance tests (FSIVGTT) and mixed meal tolerance tests (MMTT) and islet mass in 60 adult patients, with multivariate logistic regression modeling to identify predictors of islet mass >/=2500 IEQ/kg. The acute C-peptide response to glucose (ACRglu) and disposition index from FSIVGTT correlated modestly with the islet equivalents per kilogram body weight (IEQ/kg). Fasting and MMTT glucose levels and HbA1c correlated inversely with IEQ/kg (r values -0.33 to -0.40, p </= 0.05). In multivariate logistic regression modeling, normal fasting glucose (<100 mg/dL) and stimulated C-peptide on MMTT >/=4 ng/mL were associated with greater odds of receiving an islet mass >/=2500 IEQ/kg (OR 0.93 for fasting glucose, CI 0.87-1.0; OR 7.9 for C-peptide, CI 1.75-35.6). In conclusion, parameters obtained from FSIVGTT correlate modestly with islet isolation outcomes. Stimulated C-peptide >/=4 ng/mL on MMTT conveyed eight times the odds of receiving >/=2500 IEQ/kg, a threshold associated with reasonable metabolic control postoperatively.

Address: Departments of Surgery, University of Minnesota and University of Minnesota Amplatz Children's Hospital, Minneapolis, MN

3916070e: Trop.Gastroenterol., 18-24(2013)
Link: [ PubMED ] [ DOI ]
Title: Insulin resistance and metabolic syndrome in nonobese Indian patients with non-alcoholic fatty liver disease.
Author: Bhat,G., Baba,C.S., Pandey,A., Kumari,N., Choudhuri,G.
Abstract:
BACKGROUND: Insulin resistance has been recognized as a major factor in the development of non-alcoholic fatty liver disease (NAFLD). The association between insulin resistance and NAFLD, as a risk factor independent of obesity has been less well established. This study aims to determine presence of insulin resistance and components of metabolic syndrome in non-obese patients with NAFLD. METHODS: 150 patients (mean age 42.25 _ 10.50 y; 115 (76%) male, 35 (24%) female) diagnosed with NAFLD participated in the study. We measured body mass index (BMI), waist circumference (WC), waist hip ratio (WHR), fasting lipid profile, fasting glucose, fasting insulin, and liver function. Insulin resistance was calculated using the homeostasis model of assessment (HOMA) formula. Insulin resistance was arbitrarily considered altered when it was >1.64. RESULTS: 120 (80%) of the 150 patients were pbese (BMI >23) according to the Asia Pacific criteria. 40 (30%) had metabolic syndrome. 97.5% (117/120) had insulin resistance with mean HOMA-insulin resistance (IR) of 10.9+/-5.3. Thirty (20%) were non-obese; of these, 7 had central obesity (WC > 90 cm for men, > 80 cm for women). Twenty-three (15.3%) patients were lean NAFLD with BMI 21.6+/-1.5, WC 82.9+/-4.7 (BMI< 23, WC <90 cm in men and < 80 cm in women) 80% of these 23 (18/23) had insulin resistance with mean HOMA-IR of 3.4+/-1.9. Only 4 (17%) did not have any component of metabolic syndrome. Conclusion: Insulin resistance often associated with metabolic syndrome is common and plays a key role amongst lean Indian patients with non-alcoholic fatty liver disease.

Address: Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow

3916071e: Mol.Vis., 1723-1733(2013)
Link: [ PubMED ] [ OpenAccess]
Title: LDLR-/-ApoB100/100 mice with insulin-like growth factor II overexpression reveal a novel form of retinopathy with photoreceptor atrophy and altered morphology of the retina.
Author: Kinnunen,K., Heinonen,S.E., Kalesnykas,G., Laidinen,S., Uusitalo-Jarvinen,H., Uusitalo,H., Yla-Herttuala,S.
Abstract:
PURPOSE: The aim of this study was to characterize the ocular morphology of low-density lipoprotein receptor-deficient apolipoprotein B-100-only mice, where overexpression of insulin-like growth factor II (IGF-II) has been shown to induce glucose intolerance and increase atherosclerotic lesion progression and calcification. METHODS: Fifteen-month-old mice were examined on a normal chow diet and after 3 months of a high-fat Western diet. IGF-II-negative LDLR(-/-)ApoB(100/100) littermates and C57Bl/6J mice served as controls. In vivo color images of the fundi were obtained, and eyes were processed either for retinal flat mounts for assessment of neovascularization or for paraffin-embedded samples for immunohistochemical analyses. RESULTS: IGF-II overexpression and the resulting prediabetic phenotype did not induce microvascular damage when assessed in fundus photographs and retinal whole mounts, and the number of capillaries in IGF-II/LDLR(-/-)ApoB(100/100) mice was not significantly different from LDLR(-/-)ApoB(100/100) mice. However, morphology of the inner nuclear, outer plexiform, and outer nuclear layers was altered in the IGF-II/LDLR(-/-)ApoB(100/100) mice. Moreover, photoreceptor atrophy and thinning of the outer nuclear layer were present. Caspase-3 staining was positive in the photoreceptor inner segment. In addition, retinas of the IGF-II/LDLR(-/-)ApoB(100/100) mice displayed reduced rhodopsin positivity, consistent with the decreased number of photoreceptor cells. CONCLUSIONS: This study reports a novel form of retinopathy with photoreceptor atrophy and abundant changes in retinal morphology in a mouse model of prediabetes and atherosclerosis.

Address: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland

3916072e: Mol.Vis., 1684-1693(2013)
Link: [ PubMED ] [ OpenAccess]
Title: Hydrogen saline prevents selenite-induced cataract in rats.
Author: Yang,C.X., Yan,H., Ding,T.B.
Abstract:
PURPOSE: The aim of this study was to investigate the potential antioxidative effect and mechanism for the protective effects of hydrogen saline on selenite-induced cataract in rats. METHODS: Sprague-Dawley rat pups were divided into the following groups: control (Group A), selenite induced (Group B), and selenite plus hydrogen saline treated (Group C). Rat pups in Groups B and C received a single subcutaneous injection of sodium selenite (25 mumol/kg bodyweight) on postnatal day 12. Group C also received an intraperitoneal injection of H2 saline (5 ml/kg bodyweight) daily from postnatal day 8 to postnatal day 17. The development of cataract was assessed weekly by slit-lamp examination for 2 weeks. After sacrifice, extricated lenses were analyzed for activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, levels of malondialdehyde, reduced glutathione (GSH), and total sulfhydryl contents. RESULTS: The magnitude of lens opacification in Group B was significantly higher than in Group A (p<0.05), while Group C had less opacification than Group B (p<0.05). Compared with Group B, the mean activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, levels of GSH, and total sulfhydryl contents were higher, whereas the level of malondialdehyde was lower following treatment with hydrogen saline(p<0.05). CONCLUSIONS: This is an initial report showing that hydrogen saline can prevent selenite-induced cataract in rats. It acts via maintaining antioxidant enzymes and GSH, protecting the sulfhydryl group, and inhibiting lipid peroxidation.

Address: Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China

3916075e: J.Infect.Dis., 1474-1481(2013)
Link: [ PubMED ] [ DOI ]
Title: Immunomodulatory Effects of Vitamin D on Innate and Adaptive Immune Responses to Streptococcus pneumoniae.
Author: Olliver,M., Spelmink,L., Hiew,J., Meyer-Hoffert,U., Henriques-Normark,B., Bergman,P.
Abstract:
Background. Streptococcus pneumoniae forms part of the normal nasopharyngeal flora but can also cause a broad spectrum of inflammatory diseases. Vitamin D has potent effects on human immunity, including induction of antimicrobial peptides and suppression of T-cell proliferation, but its ability to modulate the immune response to pneumococci is unknown. Methods. Monocyte-derived dendritic cells (DCs) were stimulated with pneumococcal peptidoglycan (PGN) in the presence or absence of vitamin D. Expression of maturation markers, cytokines, pattern recognition receptors, and antimicrobial peptides were measured with flow cytometry, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Stimulated DCs were cocultured with autologous T-helper cells, and concentrations of T-helper (Th) 1-, Th17-, and regulatory T-cell-related cytokines were measured with enzyme-linked immunosorbent assay. Results. Vitamin D enhanced DC maturation and expression of the migration marker C-C chemokine receptor type 7 (CCR7) in PGN-stimulated cells. It also enhanced expression of key pattern recognition receptors (Toll-like receptor 2, Nucleotide-binding oligomerization domain-containing protein 2 [Nod2]) and induced a synergistic up-regulation of the inflammatory mediator IL-1β and the β-defensin Human Beta Defensin 3 (hBD-3). Furthermore, vitamin D skewed the DC-mediated T-helper response to PGN from an inflammatory Th1/Th17 phenotype toward a regulatory T-cell phenotype. Conclusion. Vitamin D modulates key elements of innate immunity while dampening adaptive immune responses in DCs after pneumococcal challenge, which may have implications for prevention and treatment of pneumococcus-induced inflammation.

Address: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

3916077e: Clin.Cancer Res., 5351-5360(2013)
Link: [ PubMED ] [ DOI ]
Title: Merkel Polyomavirus-Specific T Cells Fluctuate with Merkel Cell Carcinoma Burden and Express Therapeutically Targetable PD-1 and Tim-3 Exhaustion Markers.
Author: Afanasiev,O.K., Yelistratova,L., Miller,N., Nagase,K., Paulson,K., Iyer,J.G., Ibrani,D., Koelle,D.M., Nghiem,P.
Abstract:
PURPOSE: The persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins in Merkel cell carcinoma (MCC) provides a unique opportunity to characterize immune evasion mechanisms in human cancer. We isolated MCPyV-specific T cells and determined their frequency and functional status. EXPERIMENTAL DESIGN: Multiparameter flow cytometry panels and HLA/peptide tetramers were used to identify and characterize T cells from tumors (n = 7) and blood (n = 18) of patients with MCC and control subjects (n = 10). PD-1 ligand (PD-L1) and CD8 expression within tumors were determined using mRNA profiling (n = 35) and immunohistochemistry (n = 13). RESULTS: MCPyV-specific CD8 T cells were detected directly ex vivo from the blood samples of 7 out of 11 (64%) patients with MCPyV-positive tumors. In contrast, 0 of 10 control subjects had detectable levels of these cells in their blood (P < 0.01). MCPyV-specific T cells in serial blood specimens increased with MCC disease progression and decreased with effective therapy. MCPyV-specific CD8 T cells and MCC-infiltrating lymphocytes expressed higher levels of therapeutically targetable PD-1 and Tim-3 inhibitory receptors compared with T cells specific to other human viruses (P < 0.01). PD-L1 was present in 9 of 13 (69%) MCCs and its expression was correlated with CD8-lymphocyte infiltration. CONCLUSIONS: MCC-targeting T cells expand with tumor burden and express high levels of immune checkpoint receptors PD-1 and Tim-3. Reversal of these inhibitory pathways is therefore a promising therapeutic approach for this virus-driven cancer.

Address: Departments of Medicine/Dermatology, Pathology, Medicine, Laboratory Medicine, Global Health, University of Washington

3916079e: Circ.Cardiovasc.Interv., 452-459(2013)
Link: [ PubMED ] [ DOI ]
Title: Effects of endothelial dysfunction on residual platelet aggregability after dual antiplatelet therapy with aspirin and clopidogrel in patients with stable coronary artery disease.
Author: Fujisue,K., Sugiyama,S., Ono,T., Matsuzawa,Y., Akiyama,E., Sugamura,K., Matsubara,J., Kurokawa,H., Kaikita,K., Iwashita,S., Sumida,H., Hokimoto,S., Oniki,K., Nakagawa,K., Matsui,K., Ogawa,H.
Abstract:
BACKGROUND: Dual antiplatelet therapy with aspirin and clopidogrel is widely used in patients with coronary stents. High residual platelet reactivity (high RPR) after dual antiplatelet therapy is associated with increased cardiovascular events. Endothelial function could affect platelet reactivity in vivo. We hypothesized that endothelial dysfunction could be associated with high RPR after dual antiplatelet therapy in patients with stable coronary artery disease. METHODS AND RESULTS: We screened patients with stable coronary artery disease for cytochrome P450 (CYP) 2C19 genotypes and enrolled 103 patients who lacked CYP2C19*2 or *3 loss-of-function allele to minimize the effect of this gene on high RPR. All patients received aspirin (100 mg/d) and clopidogrel (75 mg/d for long-term treatment or a loading dose of 300 mg) before the following tests. Platelet aggregability was assessed as P2Y12 reaction unit using the VerifyNow System. High RPR was defined as P2Y12 reaction unit >/= 230. Peripheral endothelial function was expressed as reactive hyperemia index using reactive hyperemia peripheral arterial tonometry. Fifty-three patients exhibited high RPR. High RPR patients were significantly older, had higher levels of B-type natriuretic peptide, and were predominantly hypertensive compared with non-high RPR patients. Reactive hyperemia index was significantly lower in high RPR patients (0.46 +/- 0.15) compared with non-high RPR patients (0.61 +/- 0.18; P<0.001). Linear regression analysis demonstrated significant negative correlation between reactive hyperemia index and P2Y12 reaction unit (r=-0.32; P=0.001). Multivariable logistic regression analysis identified reactive hyperemia index as an independent and significant determinant of high RPR (odds ratio, 0.55; 95% confidence interval, 0.39-0.78; P=0.001). CONCLUSIONS: In patients with stable coronary artery disease, endothelial function was significantly impaired in high RPR patients. Endothelial dysfunction is independently correlated with high RPR after dual antiplatelet therapy. CLINICAL TRIAL REGISTRATION URL: http://www.umin.ac.jp/ctr. Unique identifier: UMIN000008239.

Address: Department of Cardiovascular Medicine, Graduate School of Medical Sciences

3916080e: Toxicol.Appl.Pharmacol., 591-597(2013)
Link: [ PubMED ] [ DOI ]
Title: Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway.
Author: Liang,Q.H., Liu,Y., Wu,S.S., Cui,R.R., Yuan,L.Q., Liao,E.Y.
Abstract:
Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus-pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways.

Address: Institute of Metabolism and Endocrinology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, People's Republic of China

3916082e: J.Ethnopharmacol., 707-712(2013)
Link: [ PubMED ] [ DOI ]
Title: Fuzi-Lizhong pill compensates hypothyroid-hypothermia via ghrelin release.
Author: Zhao,X., Yang,S., Zhang,W., Zu,C., Tang,B., Zhang,B., Li,G., Su,L., Cai,D.
Abstract:
ETHNOPHARMACOLOGICAL RELEVANCE: Fuzi-Lizhong pill (FLZ) is a traditional Chinese medicine for treating patients with Spleen Yang deficient syndrome. Ghrelin, a peptide with 28 amino acid residues, plays multiple roles in thermogenesis. This study aims to explore FLZ regulating ghrelin to compensate hypothermia in rats with hypothyroid and indigestion. MATERIALS AND METHODS: In litter-matched rats, hypothermia was developed with both thyroidectomy at d1 and interscapular brown adipose (IBA) removal at d42, indigestion was induced with both high fat diet and fasting-feeding cycle from d56; the littermates with hypothermia and indigestion were administrated with FLZ from d70. Adaptive thermogenesis, thyroid hormones, metabolites, ghrelin dynamics were measured at d98. RESULTS: The results showed that plasma ghrelin levels were inversely correlated with the gastric ghrelin levels and adaptive thermogenesis in rats undergone both thyroidectomy and IBA removal. Fatty diet and FLZ enhanced the increase of plasma ghrelin of hypothyroid rats. These were supported by the changes of plasma thyroid related hormones, plasma metabolites, gastric ghrelin mRNA and protein, and the effects of fatty diet or FLZ. CONCLUSIONS: Our results suggest that more ghrelin release compensate chronic hypothermia in rats with both hypothyroidism and indigestion. It could explain the mechanisms of FLZ in relieving chronic hypothermia.

Address: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China

3916084e: Diabetes, 3807-3816(2013)
Link: [ PubMED ] [ DOI ]
Title: Elastin-derived peptides are new regulators of insulin resistance development in mice.
Author: Blaise,S., Romier,B., Kawecki,C., Ghirardi,M., Rabenoelina,F., Baud,S., Duca,L., Maurice,P., Heinz,A., Schmelzer,C.E., Tarpin,M., Martiny,L., Garbar,C., Dauchez,M., Debelle,L., Durlach,V.
Abstract:
Although it has long been established that the extracellular matrix acts as a mechanical support, its degradation products, which mainly accumulate during aging, have also been demonstrated to play an important role in cell physiology and the development of cardiovascular and metabolic diseases. In the current study, we show that elastin-derived peptides (EDPs) may be involved in the development of insulin resistance (IRES) in mice. In chow-fed mice, acute or chronic intravenous injections of EDPs induced hyperglycemic effects associated with glucose uptake reduction and IRES in skeletal muscle, liver, and adipose tissue. Based on in vivo, in vitro, and in silico approaches, we propose that this IRES is due to interaction between the insulin receptor (IR) and the neuraminidase-1 subunit of the elastin receptor complex triggered by EDPs. This interplay was correlated with decreased sialic acid levels on the β-chain of the IR and reduction of IR signaling. In conclusion, this is the first study to demonstrate that EDPs, which mainly accumulate with aging, may be involved in the insidious development of IRES.

Address: Formations de Recherche en Evolution CNRS 3481, Matrice Extracellulaire et Dynamique Cellulaire, Universite de Reims Champagne Ardenne, UFR Sciences Exactes et Naturelles, Reims, France

3916091e: Exp.Biol.Med., 678-686(2013)
Link: [ PubMED ] [ DOI ]
Title: Erythropoietin promotes the repair effect of acute kidney injury by bone-marrow mesenchymal stem cells transplantation.
Author: Liu,N., Han,G., Cheng,J., Huang,J., Tian,J.
Abstract:
Bone-marrow mesenchymal stem cells (BMSCs) transplantation is effective for acute kidney injury (AKI) repair but with limited efficiency. In the present study, BMSCs marked by bromodeoxyuridine (BrdU) were transplanted to the AKI mouse model with erythropoietin (EPO) being subcutaneously injected. The blood urea nitrogen (BUN) and serum creatinine (Scr) levels, pathological changes, distribution of BMSCs, expressions of the cytokeratin 18 (CK18) and the stromal cell-derived factor 1 (SDF-1) in the nephridial tissues were measured. The directional migration of BMSCs to the AKI microenvironment in vitro was also tested. The results showed that BMSCs transplantation or EPO injection alone decreased the BUN and Scr levels and the acute tubular necrosis (ATN) scoring in varied degrees. The combination of these decreased the above indicators' levels significantly. BrdU+ cells (BMSCs) were observed in the AKI nephridial tissues, and CK18 expressed in the cytoplasm of these cells. EPO injection increased the proportion of BrdU+ cells with the enhanced expression of SDF-1 in the AKI nephridial tissues. EPO increased the migrating number of BMSCs to the AKI microenvironment in vitro, and additional anti-SDF-1 treatment with SDF-1 antibody neutralized this effect. Our results showed that EPO increased the number of the transplanted BMSCs in the injured nephridial tissues and enhanced the AKI repair effect of BMSCs transplantation. The enhanced kidney-homing efficiency for BMSCs mediated by the SDF-1/CXCR4 pathway is one of the possible mechanisms for EPO performance.

Address: Department of Nephrology, 455th hospital of PLA, Shanghai 200052, China

3916102e: J.Am.Coll.Cardiol., 1203-1212(2013)
Link: [ PubMED ] [ DOI ]
Title: N-Terminal Pro-B-Type Natriuretic Peptide and Its Relationship With Cardiac Function in Adults With Congenital Heart Disease.
Author: Eindhoven,J.A., Van den Bosch,A.E., Ruys,T.P., Opic,P., Cuypers,J.A., McGhie,J.S., Witsenburg,M., Boersma,E., Roos-Hesselink,J.W.
Abstract:
OBJECTIVES: The aim of this study was to determine the value of N-terminal pro-B-type natriuretic peptide (NT-proBNP) in adults with congenital heart disease (CHD) and investigate its relationship with ventricular function and exercise capacity. BACKGROUND: NT-proBNP may detect early deterioration in cardiac function. METHODS: In this cross-sectional study, extensive echocardiography, exercise testing, and NT-proBNP measurements were performed on the same day in consecutive adult patients with CHD. RESULTS: In total, 475 patients were included in this study (mean age of 34 +/- 12 years, 57% male, 90% New York Heart Association class I). The median NT-proBNP level was 15.1 pmol/l (interquartile range [IQR]: 7.1 to 31.3 pmol/l), and the NT-proBNP level was >14 pmol/l in 53% of patients. The highest NT-proBNP levels were observed in patients with Fontan circulation (36.1 pmol/l [IQR: 14.4 to 103.8 pmol/l]) and a systemic right ventricle (RV) (31.1 pmol/l [IQR: 21.8 to 56.0 pmol/l]), and the lowest values were seen in patients with aortic coarctation (7.3 pmol/l [IQR: 2.8 to 19.5 pmol/l]). NT-proBNP levels correlated with age (r = 0.39, p < 0.001) and were higher in women (median of 21.7 vs. 10.4 pmol/l; p < 0.001). In patients with aortic stenosis or aortic coarctation, NT-proBNP levels correlated with diastolic function parameters of E/E' ratio (r = 0.40, p < 0.001) and left atrial dimension (r = 0.36, p < 0.001). In patients with a systemic RV, NT-proBNP levels correlated with RV annulus diameter (r = 0.31, p = 0.024). In patients with tetralogy of Fallot, the strongest correlations were observed with left atrial dimension (r = 0.46, p < 0.001) and left ventricular ejection fraction (r = 0.37, p < 0.001). NT-proBNP levels were associated with exercise capacity (n = 198) (maxiμm workload: β = -0.08, p = 0.021) and peak oxygen uptake (β = -0.012, p = 0.011) in a multivariable regression model adjusted for age and sex. CONCLUSIONS: NT-proBNP levels in adults with CHD clearly differ by diagnosis and are related to echocardiographic parameters and exercise capacity. Disease-specific correlations contribute to the understanding of the main hemodynamic problems per diagnosis. Follow-up data are needed to elucidate the additional prognostic value.

Address: Department of Cardiology, Thoraxcenter, Erasmus Medical Center, Rotterdam, the Netherlands

3916103e: Neurosci.Res., 8-15(2013)
Link: [ PubMED ] [ DOI ]
Title: Expression variations of chromogranin A and α1,2,4 GABAARs in discrete limbic and brainstem areas rescue cardiovascular alterations.
Author: Avolio,E., Facciolo,R.M., Alo,R., Mele,M., Carelli,A., Canonaco,A., Mosciaro,L., Talani,G., Biggio,G., Sanna,E., Mahata,S.K., Canonaco,M.
Abstract:
Recent interferences of hemodynamic functions via modified brain neuronal mechanisms have proven to be major causes of dementia and sleeping disorders. In this work, cerebral expression differences of the neuroactive vesicular chromogranin A (CgA) and distinct α GABAAR subunits were detected in the facultative hibernating hamster. In particular, damaged neuronal fields of hypotensive torpor (TORP) state were correlated to elevated CgA and GABAAR α1, α4 mRNA levels in the paraventricular hypothalamic nucleus (PVN), central amygdalar nucleus (CeA) plus solitary tractus nucleus (NTS). Conversely, few neurodegeneration signals of hypertensive arousal (AROU) state, accounted for mostly lower CgA levels in the same areas. This state also provided increased α2-containing sites in amygdala, hippocampal and NTS neurons together with elevated α4-containing receptors in the periventricular hypothalamic nucleus (Pe). Interestingly in our hibernating model, CgA appeared to preferentially feature inhibitory neurosignals as indicated by preliminary perfusion of amygdalar sites with its highly specific antihypertensive derived peptide (catestatin) promoting GABA-dependent sIPSCs. Overall, evident neuronal damages plus altered expression capacities of CgA and α1-, α2-, α4-GABAARs in CeA, Pe, PVN as well as NTS during both hibernating states corroborate for the first time key molecular switching events guaranteeing useful cardiovascular rescuing abilities of neurodegenerative disorders.

Address: Comparative Neuroanatomy Laboratory, University of Calabria, Ponte P. Bucci 4B, 87030 Arcavacata di Rende, Cosenza, Italy

3916108e: J.Pak.Med.Assoc., 374-379(2013)
Link:
Title: Effect of cholecalciferol and levo carnitine on plasma glucose, plasma insulin and insulin resistance in type 2 diabetic rats.
Author: Anwar,M.K., Hussain,M.M., Khan,M.A., Ahmad,T.
Abstract:
OBJECTIVE: To compare the effects of combined and individual supplementation of cholecalciferol and levo carnitine on plasma glucose, plasma insulin and insulin resistance in type 2 diabetic rats. METHODS: The randomised controlled trial was conducted at the Department of Physiology, Army Medical College, Rawalpindi, between October 2010 and April 2011. It comprised 80 healthy Sprague Dawley rats who were divided into four groups (n = 20 each). Rats were fed high-fat diet for 2 weeks followed by an intraperitoneal injection of streptozocin to induce type 2 diabetes mellitus. group I served as diabetic control; group II was given cholecalciferol; group III; levo carnitine; and group IV was administered cholecalciferol and levo carnitine together. After 6 days of supplementation, terminal intracardiac blood extraction was done and samples were analysed for fasting plasma glucose and plasma insulin. Insulin resistance was calculated by homeostatic model assessment for insulin resistance. SPSS 17.0 was used for statistical analysis. RESULTS: Fasting plasma glucose levels were significantly decreased (p < 0.001) in the combined supplementation group compared to the diabetic control and individual supplementation groups. Combined supplementation showed a significant increase in fasting plasma insulin levels when compared with diabetic control and levo carnitine groups (p < 0.001), and the effect of combined supplementation on ameliorating insulin resistance was significantly better (p < 0.001) as compared to the individual supplementation of cholecalciferol and levo carnitine. CONCLUSIONS: The combined supplementation of cholecalciferol and levo carnitine for 6 days markedly improved the glycaemic control, insulin secretion and insulin resistance in type 2 diabetic rats on high-fat diet A prolonged supplementation by both the compounds along with caloric restriction may yield a more promising outcome.

E-Mail: khalid.1613@hotmail.com
Address: Department of Physiology, Army Medical College, Rawalpindi, Pakistan

3916109e: Ir.Med.J., 148-149(2013)
Link:
Title: Atypical melanocytic naevi following melanotan injection.
Author: Reid,C., Fitzgerald,T., Fabre,A., Kirby,B.
Abstract:
Melanotan is a synthetic analogue of α melanocyte stimulating hormone (a-MSH) that stimulates melanogenesis. It is sold on the internet and tanning salons as a quick 'tanning jab'. We report a patient who developed multiple new onset atypical naevi within one week of receiving two Melanotan injections. This case highlights the potential risk of Melanotan in stimulating dysplastic naevi or possibly malignant melanoma.

E-Mail: C.Reid@st-vincents.ie
Address: Dermatology Department, St Vincent's University Hospital, Elm Park, Dublin 4

3916116e: Am.J.Physiol.H, 1050-1056(2013)
Link: [ PubMED ] [ DOI ]
Title: Endogenous ACh tonically stimulates ANP secretion in rat atria.
Author: Kim,H.Y., Cho,K.W., Xu,D.Y., Kang,D.G., Lee,H.S.
Abstract:
Exogenous acetylcholine (ACh) is known to stimulate atrial natriuretic peptide (ANP) secretion concomitantly with a decrease in atrial pulse pressure. However, the role of intrinsic ACh in the regulation of ANP secretion remains unknown. Recently, it was shown that nonneuronal and neuronal ACh is present in the cardiac atria. From this finding we hypothesize that endogenously released ACh is involved in the regulation of ANP secretion in an autocrine or paracrine manner in the atria. Experiments were performed in isolated beating rat atria. ANP was measured using radioimmunoassay. To increase the availability of the ACh in the extracellular space of the atrium, its degradation was inhibited with an inhibitor of acetylcholinesterase. Acetylcholinesterase inhibition with physostigmine increased ANP secretion concomitantly with a decrease in atrial dynamics in a concentration-dependent manner. Inhibitors of M2 muscarinic ACh receptor (mAChR), methoctramine, and ACh-activated K+ (KACh+) channels, tertiapin-Q, abolished the physostigmine-induced changes. The effects were not observed in the atria from rats treated with pertussis toxin. Furthermore, the physostigmine-induced effects were attenuated by an inhibitor of high-affinity choline transporter, hemicholinium-3, which is a rate-limiting step of ACh synthesis. Inhibitors of the mAChR signaling pathway and ACh synthesis also attenuated the basal levels of ANP secretion and accentuated atrial dynamics. These findings suggest that endogenously released ACh tonically stimulates ANP secretion from atrial cardiomyocytes via activation of M2 mAChR-Gi/o-KACh+ channel signaling. It is also suggested that the ACh-ANP signaling is implicated in cardiac physiology and pathophysiology.

Address: Hanbang Body-fluid Research Center & College of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, Republic of Korea

3916117e: Mol.Nutr.Food Res., 2061-2070(2013)
Link: [ PubMED ] [ DOI ]
Title: Allergenic relevance of nonspecific lipid transfer proteins 2: Identification and characterization of Api g 6 from celery tuber as representative of a novel IgE-binding protein family.
Author: Vejvar,E., Himly,M., Briza,P., Eichhorn,S., Ebner,C., Hemmer,W., Ferreira,F., Gadermaier,G.
Abstract:
SCOPE: Apium graveolens represents a relevant food allergen source linked with severe systemic reactions. We sought to identify an IgE-binding nonspecific lipid transfer protein (nsLTP) in celery tuber. METHODS AND RESULTS: A low molecular weight protein exclusively present in celery tuber was purified and designated Api g 6. The entire protein sequence was obtained by MS and classified as member of the nsLTP2 family. Api g 6 is monomeric in solution with a molecular mass of 6936 Da. The α-helical disulfide bond-stabilized structure confers tremendous thermal stability (Tm > 90 degrees C) and high resistance to gastrointestinal digestion. Endolysosomal degradation demonstrated low susceptibility and the presence of a dominant peptide cluster at the C-terminus. Thirty-eight percent of A. graveolens allergic patients demonstrated IgE reactivity to purified natural Api g 6 in ELISA and heat treatment did only partially reduce its allergenic activity. No correlation in IgE binding and limited cross-reactivity was observed with Api g 2 and Art v 3, nsLTP1 from celery stalks and mugwort pollen. CONCLUSION: Api g 6, a novel nsLTP2 from celery tuber represents the first well-characterized allergen in this protein family. Despite similar structural and physicochemical features as nsLTP1, immunological properties of Api g 6 are distinct which warrants its inclusion in molecule-based diagnosis of A. graveolens allergy.

Address: Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg, Salzburg, Austria

3916127e: J.Endocrinol., 59-68(2013)
Link: [ PubMED ] [ DOI ]
Title: Optimal bone mechanical and material properties require a functional glucagon-like peptide-1 receptor.
Author: Mabilleau,G., Mieczkowska,A., Irwin,N., Flatt,P.R., Chappard,D.
Abstract:
Bone is permanently remodeled by a complex network of local, hormonal, and neuronal factors that affect osteoclast and osteoblast biology. Among these factors, a role for gastrointestinal hormones has been proposed based on the evidence that bone resorption dramatically falls after a meal. Glucagon-like peptide-1 (GLP1) is one of these gut hormones, and despite several reports suggesting an anabolic effect of GLP1, or its stable analogs, on bone mass, little is known about the effects of GLP1/GLP1 receptor on bone strength. In this study, we investigated by three-point bending, quantitative X-ray microradiography, microcomputed tomography, qBEI, and FTIRI bone strength and bone quality in male Glp1r knockout (Glp1r KO) mice when compared with control WT animals. Animals with a deletion of Glp1r presented with a significant reduction in ultimate load, yield load, stiffness, and total absorbed and post-yield energies when compared with WT animals. Furthermore, cortical thickness and bone outer diameter were significantly decreased in deficient animals. The mineral quantity and quality were not significantly different between Glp1r KO and WT animals. On the other hand, the maturity of the collagen matrix was significantly reduced in deficient animals and associated with lowered material properties. Taken together, these data support a positive effect of GLP1R on bone strength and quality.

Address: Groupe d'Etudes sur le Remodelage Osseux et les bioMateriaux (GEROM)-LHEA, Service Commun d'Imageries et d'Analyses Microscopiques (SCIAM), IRIS-IBS Institut de Biologie en Sante, LUNAM Universite, CHU d'Angers, 49933 Angers Cedex, France

3916128e: Int.J.Pharm., 40-47(2013)
Link: [ PubMED ] [ DOI ]
Title: Cytoplasm-responsive nanocarriers conjugated with a functional cell-penetrating peptide for systemic siRNA delivery.
Author: Tanaka,K., Kanazawa,T., Horiuchi,S., Ando,T., Sugawara,K., Takashima,Y., Seta,Y., Okada,H.
Abstract:
To develop a gene carrier for cancer therapy by systemic injection, we synthesized methoxypolyethylene glycol-polycaprolactone (MPEG-PCL) diblock copolymers conjugated with a cytoplasm-responsive cell-penetrating peptide (CPP), CH2R4H2C (C, Cys; H, His; R, Arg). The carrier/small interfering RNA (siRNA) complexes (N/P ratio of 20) had a particle size of approximately 50 nm and stabilized the siRNA against RNase. The cellular uptake ability of the carrier/FAM-siRNA complexes with fetal bovine serum was significantly higher than that of naked FAM-siRNA. In addition, the carrier/anti-vascular endothelial growth factor siRNA (siVEGF) complexes attained a significantly greater silencing effect than naked siVEGF with low cytotoxicity, resulting from higher uptake, early endosomal escape, and efficient release from the complexes in the cytoplasm. Furthermore, intravenous injection of MPEG-PCL-CH2R4H2C/siVEGF complexes had a significantly higher anti-tumor effect in S-180 tumor-bearing mice, which could be attributed to the rigid compaction of siRNA by ionic interactions and disulfide linkages in the CPP polymer micelles in the blood, as well as higher release following cleavage of the disulfide bonds in the reductive cytosol. Taken together, our data demonstrated that these cytoplasm-responsive polymer micelles conjugated with multi-functional CPP, could facilitate siVEGF delivery to tumor tissues after systemic injection and could exert an extremely strong anti-tumor effect.

Address: Laboratory of Pharmaceutics and Drug Delivery, Department of Pharmaceutical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

3916129e: Growth Horm.IGF Res., 187-192(2013)
Link: [ PubMED ] [ DOI ]
Title: The frontal cortex IGF system is down regulated in the term, intrauterine growth restricted fetal baboon.
Author: Xie,L., Antonow-Schlorke,I., Schwab,M., McDonald,T.J., Nathanielsz,P.W., Li,C.
Abstract:
OBJECTIVE: The IGF system exerts systemic and local actions during development. We previously demonstrated that fetal cerebral cortical IGF1 is reduced at 0.5 gestation in our IUGR baboon nonhuman primate model. We hypothesized that by term protein expression of several key IGF system stimulatory peptide pathway components and downstream nutrient signaling effectors of IGF, mammalian target of rapamycin (mTOR) and S6, would decrease, indicating reduced cellular nutrient uptake and protein synthesis. DESIGN: We fed 7 control baboons ad libitum while 6 baboons ate a globally reduced diet (70% of feed eaten by controls) from 0.16 gestation through pregnancy that produces IUGR. Fetuses were removed at Cesarean section at 0.9 gestation. Frontal cortex sections were stained for IGFI, IGFII, IGFRI, IGFR2, IGFBP2, 3, 5 and 6, and mTOR and ribosomal protein S6 and double stained with NeuN a neuron-specific nuclear antigen. RESULTS: All proteins stained neuronal cytoplasm except IGFRI which showed only glial cell cytoplasmic and blood vessel staining. IUGR fetuses showed decreased frontal cortical immunoreactive IGFI, IGFII, IGFRI, IGFBP2, 5 and 6, and mTOR and S6 (p < 0.05). IGFBP3 increased (p < 0.05) and IGFR2 was unchanged (p > 0.05). There were no differences between male and female fetal brains. CONCLUSIONS: When fetal nutrient availability is decreased, IUGR down regulates the IGF system and its mTOR signaling pathway in the fetal frontal cortex coincident with slowed growth. These findings emphasize the importance of the local tissue IGF system in fetal primate brain development.

Address: The University of Texas Health Science Center San Antonio, Center for Pregnancy and Newborn Research, Dept. OB/GYN, San Antonio, TX 78229, USA

3916130e: Physiol.Behav., 114-123(2013)
Link: [ PubMED ] [ DOI ]
Title: Lipids, CHOs, proteins: Can all macronutrients put a 'brake' on eating?
Author: Shin,H.S., Ingram,J.R., McGill,A.T., Poppitt,S.D.
Abstract:
The gastrointestinal (GI) tract and specifically the most distal part of the small intestine, the ileum, has become a renewed focus of interest for mechanisms targeting appetite suppression. The 'ileal brake' is stimulated when energy-containing nutrients are delivered beyond the duodenum and jejunum and into the ileum, and is named for the feedback loop which slows or 'brakes' gastric emptying and duodeno-jejunal motility. More recently it has been hypothesized that the ileal brake also promotes secretion of satiety-enhancing GI peptides and suppresses hunger, placing a 'brake' on food intake. Postprandial delivery of macronutrients to the ileum, other than unavailable carbohydrates (CHO) which bypass absorption in the small intestine en route to fermentation in the large bowel, is an uncommon event and hence this brake mechanism is rarely activated following a meal. However the ability to place a 'brake' on food intake through delivery of protected nutrients to the ileum is both intriguing and challenging. This review summarizes the current clinical and experimental evidence for activation of the ileal brake by the three food macronutrients, with emphasis on eating behavior and satiety as well as GI function. While clinical studies have shown that exposure of the ileum to lipids, CHOs and proteins may activate GI components of the ileal brake, such as decreased gut motility, gastric emptying and secretion of GI peptides, there is less evidence as yet to support a causal relationship between activation of the GI brake by these macronutrients and the suppression of food intake. The predominance of evidence for an ileal brake on eating comes from lipid studies, where direct lipid infusion into the ileum suppresses both hunger and food intake. Outcomes from oral feeding studies are less conclusive with no evidence that 'protected' lipids have been successfully delivered into the ileum in order to trigger the brake. Whether CHO or protein may induce the ileal brake and suppress food intake has to date been little investigated, although both clearly have GI mediated effects. This review provides an overview of the mechanisms and mediators of activation of the ileal brake and assesses whether it may play an important role in appetite suppression.

Address: Human Nutrition Unit, University of Auckland, Auckland, New Zealand

3916131e: Neuropharmacology, 164-171(2013)
Link: [ PubMED ] [ DOI ]
Title: Endogenous mammalian RF-amide peptides, including PrRP, kisspeptin and 26RFa, modulate nociception and morphine analgesia via NPFF receptors.
Author: Elhabazi,K., Humbert,J.P., Bertin,I., Schmitt,M., Bihel,F., Bourguignon,J.J., Bucher,B., Becker,J.A., Sorg,T., Meziane,H., Petit-Demouliere,B., Ilien,B., Simonin,F.
Abstract:
Mammalian RF-amide peptides are encoded by five different genes and act through five different G protein-coupled receptors. RF-amide-related peptides-1 and -3, neuropeptides AF and FF, Prolactin releasing peptides, Kisspeptins and RFa peptides are currently considered endogenous peptides for NPFF1, NPFF2, GPR10, GPR54 and GPR103 receptors, respectively. However, several studies suggest that the selectivity of these peptides for their receptors is low and indicate that expression patterns for receptors and their corresponding ligands only partially overlap. In this study, we took advantage of the cloning of the five human RF-amide receptors to systematically examine their affinity for and their activation by all human RF-amide peptides. Binding experiments, performed on membranes from CHO cells expressing GPR10, GPR54 and GPR103 receptors, confirmed their high affinity and remarkable selectivity for their cognate ligands. Conversely, NPFF1 and NPFF2 receptors displayed high affinity for all RF-amide peptides. Moreover, GTPγS and cAMP experiments showed that almost all RF-amide peptides efficiently activate NPFF1 and NPFF2 receptors. As NPFF is known to modulate morphine analgesia, we undertook a systematic analysis in mice of the hyperalgesic and anti morphine-induced analgesic effects of a representative set of endogenous RF-amide peptides. All of them induced hyperalgesia and/or prevented morphine analgesia following intracerebroventricular administration. Importantly, these effects were prevented by administration of RF9, a highly selective NPFF1/NPFF2 antagonist. Altogether, our results show that all endogenous RF-amide peptides display pain-modulating properties and point to NPFF receptors as essential players for these effects.

E-Mail: elhabazi@unistra.fr
Address: Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS, Universite de Strasbourg, Laboratory of Excellence Medalis, Illkirch, France. Electronic address:

3916133e: Am.J.Pathol., 1144-1155(2013)
Link: [ PubMED ] [ DOI ]
Title: A Glucose-6-Phosphate Isomerase Peptide Induces T and B Cell-Dependent Chronic Arthritis in C57BL/10 Mice: Arthritis without Reactive Oxygen Species and Complement.
Author: Pizzolla,A., Wing,K., Holmdahl,R.
Abstract:
Immunization with human glucose-6-phosphate isomerase (hG6PI) protein or with several of its peptides induces arthritis in DBA/1 mice. We investigated G6PI peptide-induced arthritis in C57BL/10 mice and the effect of oxidative burst on disease. To study the arthritogenicity of G6PI peptides and its immune dependency, we used genetically modified and congenic mice on the C57BL/10 background and in vitro T- and B-cell assays. hG6PI325-339 peptide induced arthritis in C57BL/10 mice. The disease was associated with major histocompatibility complex class II and was dependent on T cells, B cells, and complement C5. Th1 and Th17 cells primed with the hG6PI325-339 peptide cross-reacted with the murine G6PI protein. The severity of the disease increased in mice carrying a mutation in Ncf1 (Ncf1 */ *), which abolishes the NADPH oxidase 2 complex oxidative burst. Ncf1 */ * mice developed arthritis also on immunization with the mouse G6PI325-339 peptide and in the absence of C5. The antibody responses to the G6PI protein and peptides were minimal in both Ncf1 */ * and wild-type mice. Herein is described G6PI peptide as the first peptide to induce arthritis in C57BL/10 mice. The differences between the wild-type and Ncf1 */ * mice suggest that an alternative complement-independent arthritogenic pathway could be operative in the absence of oxidative burst.

Address: Division of Medical Inflammation Research, Department of Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

3916134e: Chem.Biol., 1002-1011(2013)
Link: [ PubMED ] [ DOI ]
Title: N-carbamoylation of 2,4-diaminobutyrate reroutes the outcome in padanamide biosynthesis.
Author: Du,Y.L., Dalisay,D.S., Andersen,R.J., Ryan,K.S.
Abstract:
Padanamides are linear tetrapeptides notable for the absence of proteinogenic amino acids in their structures. In particular, two unusual heterocycles, (S)-3-amino-2-oxopyrrolidine-1-carboxamide (S-Aopc) and (S)-3-aminopiperidine-2,6-dione (S-Apd), are found at the C-termini of padanamides A and B, respectively. Here we identify the padanamide biosynthetic gene cluster and carry out systematic gene inactivation studies. Our results show that padanamides are synthesized by highly dissociated hybrid nonribosomal peptide synthetase/polyketide synthase machinery. We further demonstrate that carbamoyltransferase gene padQ is critical to the formation of padanamide A but dispensable for biosynthesis of padanamide B. Biochemical investigations show that PadQ carbamoylates the rare biosynthetic precursor l-2,4-diaminobutyrate, generating l-2-amino-4-ureidobutyrate, the presumed precursor to the C-terminal residue of padanamide A. By contrast, the C-terminal residue of padanamide B may derive from glutamine. An unusual thioesterase-catalyzed cyclization is proposed to generate the S-Aopc/S-Apd heterocycles.

Address: Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada

3916135e: Chem.Biol., 1033-1043(2013)
Link: [ PubMED ] [ DOI ]
Title: Genome mining expands the chemical diversity of the cyanobactin family to include highly modified linear peptides.
Author: Leikoski,N., Liu,L., Jokela,J., Wahlsten,M., Gugger,M., Calteau,A., Permi,P., Kerfeld,C.A., Sivonen,K., Fewer,D.P.
Abstract:
Ribosomal peptides are produced through the posttranslational modification of short precursor peptides. Cyanobactins are a growing family of cyclic ribosomal peptides produced by cyanobacteria. However, a broad systematic survey of the genetic capacity to produce cyanobactins is lacking. Here we report the identification of 31 cyanobactin gene clusters from 126 genomes of cyanobacteria. Genome mining suggested a complex evolutionary history defined by horizontal gene transfer and rapid diversification of precursor genes. Extensive chemical analyses demonstrated that some cyanobacteria produce short linear cyanobactins with a chain length ranging from three to five amino acids. The linear peptides were N-prenylated and O-methylated on the N and C termini, respectively, and named aeruginosamide and viridisamide. These findings broaden the structural diversity of the cyanobactin family to include highly modified linear peptides with rare posttranslational modifications.

Address: Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, PO Box 56, Viikki Biocenter, Viikinkaari 9, FIN-00014, University of Helsinki, Finland

3916142e: Rev.Port.Cardiol., 647-652(2013)
Link: [ PubMED ] [ DOI ]
Title: Utility of B-type natriuretic peptide measurement in outpatients with heart failure with preserved ejection fraction.
Author: Lagoeiro Jorge,A.J., Di Calafiori Freire,M., Ribeiro,M.L., Maluhy Fernandes,L.C., Gemal Lanzieri,P., Lagoeiro Jorge,B.A., B Lage,J.G., Garcia Rosa,M.L., Tinoco Mesquita,E.
Abstract:
INTRODUCTION: Heart failure with preserved ejection fraction (HFPEF) is a highly prevalent syndrome that is difficult to diagnose in outpatients. The measurement of B-type natriuretic peptide (BNP) may be useful in the diagnosis of HFPEF, but with a different cutoff from that used in the emergency room. The aim of this study was to identify the BNP cutoff for a diagnosis of HFPEF in outpatients. METHODS AND RESULTS: This prospective, observational study enrolled 161 outpatients (aged 68.1+/-11.5 years, 72% female) with suspected HFPEF. Patients underwent ECG, tissue Doppler imaging, and plasma BNP measurement, and were classified in accordance with algorithms for the diagnosis of HFPEF. HFPEF was confirmed in 49 patients, who presented higher BNP values (mean 144.4pg/ml, median 113pg/ml, vs. mean 27.6pg/ml, median 16.7pg/ml, p<0.0001). The results showed a significant correlation between BNP levels and left atrial volume index (r=0.554, p<0.0001), age (r=0.452; p<0.0001) and E/E' ratio (r=0.345, p<0.0001). The area under the ROC curve for BNP to detect HFPEF was 0.92 (95% confidence interval: 0.87-0.96; p<0.001), and 51pg/ml was identified as the best cutoff to detect HFPEF, with sensitivity of 86%, specificity of 86% and accuracy of 86%. CONCLUSIONS: BNP levels in outpatients with HFPEF are significantly higher than in those without. A cutoff value of 51pg/ml had the best diagnostic accuracy in outpatients.

E-Mail: lagoeiro@globo.com
Address: Departamento de Medicina Clinica, Universidade Federal Fluminense, Niteroi, Rio de Janeiro, Brasil. Electronic address:

3916148e: Br.J.Pharmacol., 1027-1040(2013)
Link: [ PubMED ] [ DOI ]
Title: Effect of Mas-related gene (Mrg) receptors on hyperalgesia in rats with CFA-induced inflammation via direct and indirect mechanisms.
Author: Jiang,J., Wang,D., Zhou,X., Huo,Y., Chen,T., Hu,F., Quirion,R., Hong,Y.
Abstract:
BACKGROUND AND PURPOSE: Mas oncogene-related gene (Mrg) receptors are exclusively distributed in small-sized neurons in trigeminal and dorsal root ganglia (DRG). We investigated the effects of MrgC receptor activation on inflammatory hyperalgesia and its mechanisms. EXPERIMENTAL APPROACH: A selective MrgC receptor agonist, bovine adrenal medulla peptide 8-22 (BAM8-22) or melanocyte-stimulating hormone (MSH) or the μ-opioid receptor (MOR) antagonist CTAP was administered intrathecally (i.t.) in rats injected with complete Freund's adjuvant (CFA) in one hindpaw. Thermal and mechanical nociceptive responses were assessed. Neurochemicals were measured by immunocytochemistry, Western blot, ELISA and RT-PCR. KEY RESULTS: CFA injection increased mRNA for MrgC receptors in lumbar DRG. BAM8-22 or MSH, given i.t., generated instant short and delayed long-lasting attenuations of CFA-induced thermal hyperalgesia, but not mechanical allodynia. These effects were associated with decreased up-regulation of neuronal NOS (nNOS), CGRP and c-Fos expression in the spinal dorsal horn and/or DRG. However, i.t. administration of CTAP blocked the induction by BAM8-22 of delayed anti-hyperalgesia and inhibition of nNOS and CGRP expression in DRG. BAM8-22 also increased mRNA for MORs and pro-opiomelanocortin, along with β-endorphin content in the lumbar spinal cord and/or DRG. MrgC receptors and nNOS were co-localized in DRG neurons. CONCLUSIONS AND IMPLICATIONS: Activation of MrgC receptors suppressed up-regulation of pronociceptive mediators and consequently inhibited inflammatory pain, because of the activation of up-regulated MrgC receptors and subsequent endogenous activity at MORs. The uniquely distributed MrgC receptors could be a novel target for relieving inflammatory pain.

Address: College of Life Sciences and Provincial Key Laboratory of Developmental Biology and Neuroscience, Fujian Normal University, Fuzhou, China

3916149e: Osaka City Med.J., 53-60(2013)
Link:
Title: Reaction of plasma adiponectin level in non-small cell lung cancer patients treated with EGFR-TKIs.
Author: Umekawa,K., Kimura,T., Kudoh,S., Suzumura,T., Nagata,M., Mitsuoka,S., Matsuura,K., Oka,T., Yoshimura,N., Kira,Y., Hirata,K.
Abstract:
BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are routinely used to treat advanced non-small cell lung cancer (NSCLC) patients with activated EGFR mutations, and are associated with excellent response and improvement of performance status. Adipose tissue produces and releases substances called adipokines, which include adiponectin, leptin, resistin, and hepatocyte growth factor (HGF), etc. Previously, we reported that high levels of plasma HGF at diagnosis indicated intrinsic resistance to EGFR-TKIs. EGFR-TKIs have been hypothesized to affect these adipokines. METHODS: This prospective study, to evaluate the correlation between plasma adiponectin and insulin levels and non-hematological adverse effects in advanced NSCLC following EGFR-TKIs administration, was conducted at the Osaka City University Hospital. Plasma adiponectin and insulin levels were determined at diagnosis and on treatment day 30. RESULTS: Overall 33 patients were enrolled. We obtained plasma samples for analyses from all patients at diagnosis and from 26 patients on day 30. Increased adiponectin (13.69 to 14.42 μg/mL, p = 0.0092), and decreased insulin (404.0 to 351.2 pg/mL, p = 0.022) were observed after EGFR-TKI treatments. High levels of adiponectin at diagnosis were associated with severities of skin rash (p = 0.035). CONCLUSIONS: The adiponectin was affected by EGFR-TKI treatments for NSCLC. Besides, the adverse events by EGFR-TKIs were influenced by the plasma adipokines at diagnosis. Our study may provide useful information regarding patient outcomes to EGFR-TKI treatments. A prospective large clinical trial is warranted to clarify these results.

E-Mail: m2043009@med.osaka-cu.ac.jp
Address: Department of Respiratory Medicine, Osaka City University, Graduate School of Medicine, Japan

3916150e: Clin.Med.(London), 407-410(2013)
Link: [ PubMED ] [ DOI ]
Title: Nephrogenic diabetes insipidus partially responsive to oral desmopressin in a subject with lithium-induced multiple endocrinopathy.
Author: Kamath,C., Govindan,J., Premawardhana,A.D., Wood,S.J., Adlan,M.A., Premawardhana,L.D.
Abstract:
Lithium (Li) may cause multiple endocrinopathies, including hypercalcaemia, thyroid dysfunction and nephrogenic diabetes insipidus (NDI), but rarely in the same patient. The management of NDI remains a challenge. We report on a patient on long-term Li who had simultaneous NDI (paired serum and urine samples had abnormal osmolalities, typical of NDI, and treatment with parenteral desmopressin failed to affect urinary volume and serum osmolality), 'destructive' thyroiditis (hyperthyroidism, absent radioiodine uptake and absent thyrotrophin receptor antibodies) and primary hyperparathyroidism (compatible biochemistry, urine calcium excluding 'set point' anomalies and hypocalciuric hypercalcaemia, and normal parathyroid imaging). The thyroiditis resolved spontaneously and hypercalcaemia responded to reduction of Li dose. The NDI was unresponsive to amiloride, thiazides and ibuprofen in combination. However, urine output was reduced by 50% when a high dose of oral desmopressin was given. We conclude that Li-induced multiple endocrinopathy remains rare and, although NDI is difficult to manage, high dose oral desmopressin should be tried when other medications fail.

Address: Section of Diabetes and Endocrinology, Department of Medicine, Caerphilly Miners' and YYF Hospitals, Hengoed, Wales, UK

3916151e: Blood, 2512-2522(2013)
Link: [ PubMED ] [ DOI ]
Title: How T cells trigger the dissociation of the endothelial receptor phosphatase VE-PTP from VE-cadherin.
Author: Vockel,M., Vestweber,D.
Abstract:
The vascular endothelial (VE) receptor protein tyrosine phosphatase (VE-PTP) associates with VE-cadherin and supports endothelial cell contact integrity. This complex is rapidly dissociated by adhesion of leukocytes to endothelial cells or by vascular endothelial growth factor. We have shown recently that this dissociation is indeed required for the opening of endothelial cell contacts during leukocyte extravasation in vivo. The leukocyte receptor and signaling mechanism that stimulates VE-cadherin/VE-PTP dissociation are unknown. Here, we identify vascular cell adhesion molecule 1 as the relevant receptor for lymphocytes in this process. As signaling steps downstream of this receptor, we determined the activation of Rac1, the generation of reactive oxygen species by nicotinamide adenine dinucleotide phosphate oxidase and the activation of the redox-sensitive tyrosine kinase Pyk2 as essential for VE-cadherin/VE-PTP dissociation. These signaling steps are also required for the dissociation induced by VE growth factor. Searching for the molecular mechanism of complex dissociation, we found that a model substrate of VE-PTP represented by a tyrosine-phosphorylated peptide of Tie-2 dissociates VE-PTP from VE-cadherin when introduced with the help of a Tat peptide. We suggest that lymphocyte binding to vascular cell adhesion molecule 1 triggers a signaling process that enables a VE-PTP substrate to dissociate VE-PTP from VE-cadherin, thereby facilitating efficient transmigration.

Address: Max Planck Institute for Molecular Biomedicine, Munster, Germany

3916152e: J.Rheumatol., 1523-1528(2013)
Link: [ PubMED ] [ DOI ]
Title: Prevalence of anti-peptidylarginine deiminase type 4 antibodies in rheumatoid arthritis and unaffected first-degree relatives in indigenous North American Populations.
Author: Ferucci,E.D., Darrah,E., Smolik,I., Choromanski,T.L., Robinson,D.B., Newkirk,M.M., Fritzler,M.J., Rosen,A., El-Gabalawy,H.S.
Abstract:
OBJECTIVE: To determine whether anti-peptidylarginine deiminase type 4 (PAD4) antibodies were present in first-degree relatives (FDR) of patients with rheumatoid arthritis (RA) in 2 indigenous North American populations with high prevalence of RA. METHODS: Participants were recruited from 2 indigenous populations in Canada and the United States, including patients with RA (probands), their unaffected FDR, and healthy unrelated controls. Sera were tested for the presence of anti-PAD4 antibodies, anticyclic citrullinated peptide (anti-CCP) antibodies, and rheumatoid factor (RF). HLA-DRB1 subtyping was performed and participants were classified according to number of shared-epitope alleles present. RESULTS: Antibodies to PAD4 were detected in 24 of 82 (29.3%) probands; 2 of 147 (1.4%) relatives; and no controls (p < 0.0001). Anti-CCP was present in 39/144 (27.1%) of the relatives, and there was no overlap between positivity for anti-CCP and PAD4 in the relatives. In RA patients, anti-PAD4 antibodies were associated with disease duration (p = 0.0082) and anti-CCP antibodies (p = 0.008), but not smoking or shared-epitope alleles. CONCLUSION: Despite a significant prevalence of anti-CCP in FDR, anti-PAD4 antibodies were almost exclusively found in established RA. The prevalence of anti-PAD4 antibodies in RA is similar to the prevalence described in other populations and these autoantibodies are associated with disease duration and anti-CCP in RA.

Address: Division of Community Health Services, Alaska Native Tribal Health Consortium, Anchorage, Alaska, USA

3916153e: J.Int.Med.Res., 1612-1621(2013)
Link: [ PubMED ] [ DOI ]
Title: N-terminal prohormone B-type natriuretic peptide and weaning outcome in postoperative patients with pulmonary complications.
Author: Ma,G., Liao,W., Qiu,J., Su,Q., Fang,Y., Gu,B.
Abstract:
OBJECTIVE: To evaluate the relationship between plasma N-terminal prohormone B-type natriuretic peptide (NT-proBNP) and weaning outcomes, and the ability of NT-proBNP level to predict weaning success, in cancer patients with pulmonary complications undergoing noncardiac major surgeries. METHODS: Patients who were mechanically ventilated following postoperative respiratory failure were enrolled. NT-proBNP levels at the end of a 2-h spontaneous breathing trial were measured. Weaning was considered a success in patients who completed the trial and maintained spontaneous breathing following extubation for >48 h. RESULTS: Out of 29 patients, 22 patients weaned successfully but weaning failed in 7 patients. Plasma NT-proBNP was significantly higher in the weaning failure group than in the weaning success group. For predicting weaning success, the optimal NT-proBNP threshold value at the end of the spontaneous breathing trial was <448 ng/l (receiver operating characteristic analysis; sensitivity 68.18%, specificity 85.71%, positive predictive value 93.7% and negative predictive value 46.2%). CONCLUSION: Measuring NT-proBNP at the end of a spontaneous breathing trial may assist in predicting weaning success, as a noninvasive, quantitative and repeatable indicator of cardiac stress in patients with postsurgical respiratory failure.

Address: Department of Intensive Care Medicine, Cancer Center, Sun Yat-sen University, Guangzhou, China

3916154e: Circulation, 1085-1093(2013)
Link: [ PubMED ] [ DOI ]
Title: Temporal relationship and prognostic significance of atrial fibrillation in heart failure patients with preserved ejection fraction: a community-based study.
Author: Zakeri,R., Chamberlain,A.M., Roger,V.L., Redfield,M.M.
Abstract:
BACKGROUND: In patients with heart failure and preserved ejection fraction (HFpEF), atrial fibrillation (AF) may predate, concur with, or develop after HFpEF diagnosis. We sought to define the temporal relationship between AF and HFpEF, to identify factors associated with AF, and to determine the prognostic impact of prevalent and incident AF in HFpEF. METHOD AND RESULTS: From 1983 to 2010, 939 Olmsted County, Minnesota, residents (age, 77+/-12 years; 61% female) newly diagnosed with HFpEF (EF >/=0.50) were evaluated. Baseline rhythm classification included prior AF (>3 months before HFpEF diagnosis), concurrent AF (+/-3 months), or sinus rhythm. Incident AF (>3 months after HFpEF diagnosis) and all-cause mortality were ascertained through February 2012. Prior AF (29%) and concurrent AF (23%) were associated with older age, higher brain-type natriuretic peptide, and larger left atrial volume index at HFpEF diagnosis compared with sinus rhythm. Of HFpEF patients in sinus rhythm at diagnosis, 32% developed AF over a median follow-up of 3.7 years (interquartile range, 1.5-6.7 years; 69 events per 1000 person-years). Age and diastolic dysfunction were positively and statin use was inversely associated with incident AF. With no AF used as the referent, prior or concurrent AF (combined hazard ratio, 1.3; 95% confidence interval, 1.0-1.6; P=0.03) and incident AF, modeled as a time-dependent covariate (hazard ratio, 2.1; 95% confidence interval, 1.4-3.0; P<0.001), were independently associated with death after adjustment for pertinent covariates. CONCLUSIONS: AF occurs in two thirds of HFpEF patients at some point in the natural history and confers a poor prognosis. Further study is required to determine whether intervention for AF may improve outcomes or if statin use can prevent AF in HFpEF.

E-Mail: zakeri.rosita@mayo.edu
Address: Cardiorenal Research Laboratory and Department of Health Sciences Research, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA

3916158e: J.Nucl.Med., 1630-1636(2013)
Link: [ PubMED ] [ DOI ]
Title: Stratification of 18F-labeled PET imaging agents for the assessment of antiangiogenic therapy responses in tumors.
Author: Goggi,J.L., Bejot,R., Moonshi,S.S., Bhakoo,K.K.
Abstract:
Successful antiangiogenic therapies have been developed for the treatment of various cancers, but not all patients respond. Therefore, the early determination of therapy efficacy is essential for patient management. This study was done to evaluate the utility of various PET imaging biomarkers for early determination of the response to therapy with the antiangiogenic agent axitinib, a multiple receptor tyrosine kinase inhibitor, in tumors with diverse biologic characteristics. METHODS: Mice bearing U87-MG and MDA-MB-231 subcutaneous tumors were treated with axitinib (25 mg/kg intraperitoneally daily for 10 d), and tumor volumes were assessed with caliper measurements. The animals were concurrently imaged longitudinally with 18F-FDG, 3'-deoxy-3'-18F-fluorothymidine (18F-FLT), and 2-18F-fluoroethyl-triazolyl conjugated c(RGDyK) peptide (18F-FtRGD) to determine the optimal radiopharmaceutical for measuring the early treatment response in the 2 tumor types. RESULTS: Daily administration of axitinib successfully retarded the growth of both U87-MG and MDA-MB-231 subcutaneous tumors, with significant differences in tumor volumes being observed from day 7 after therapy on. 18F-FDG revealed a treatment efficacy response only at day 10 after treatment in both U87-MG tumor-bearing and MDA-MB-231 tumor-bearing animals. 18F-FLT afforded earlier detection of the therapy response, revealing a significant difference between drug- and vehicle-treated animals at day 3 for animals bearing U87-MG tumors and at day 7 for animals bearing the more slowly growing MDA-MB-231 tumors. 18F-FtRGD showed a rapid change in tumor retention that reached significance by day 7 in U87-MG tumor-bearing animals; in contrast, no significant difference in tumor retention was observed in MDA-MB-231 tumor-bearing animals. CONCLUSION: Longitudinal imaging with different radiopharmaceuticals displays various characteristics in different tumor types, depending on their biologic characteristics. Such studies may provide clinically important information to guide patient management and monitor the response to antiangiogenic therapy with the optiμm noninvasive imaging agent in the relevant cancer type.

E-Mail: julian_goggi@sbic.a-star.edu.sg
Address: Singapore Bioimaging Consortium (A*STAR), Helios, Singapore

3916159e: J.Clin.Pharmacol., 1168-1176(2013)
Link: [ PubMED ] [ DOI ]
Title: Safety, tolerability, and pharmacokinetics of oral and intravenous administration of GSK1322322, a peptide deformylase inhibitor.
Author: Naderer,O.J., Jones,L.S., Zhu,J., Kurtinecz,M., Dumont,E.
Abstract:
GSK1322322 is the first in a new class of antibiotics that targets peptide deformylase (PDF), an essential bacterial enzyme required for protein maturation. This randomized, double-blind, placebo-controlled, eight-cohort phase I trial enrolled 62 healthy volunteers to assess safety, tolerability, and pharmacokinetic profiles of GSK1322322. GSK1322322 was administered as a single oral or intravenous (IV) dose, escalating from 500 to 3,000 mg or repeat IV doses escalating from 500 to 1,500 mg twice daily. Upon repeat IV administration, GSK1322322 exhibits linear pharmacokinetics over time upon repeat doses as shown by time-invariant pharmacokinetics. A dose-proportional increase in area under concentration-time curve was observed after single or repeat IV dosing, whereas clearance at steady state remained generally unchanged across doses. There was minimal accumulation of GSK1322322 after repeat IV twice-daily administration. After oral tablet doses of GSK1322322 1,000 and 1,500 mg, absolute bioavailability was 69% and 56%, respectively. GSK1322322 administration at single and repeat IV doses and at supratherapeutic single IV doses of 2,000 and 3,000 mg was associated with mild-to-moderate drug-related adverse events. On the basis of the pharmacokinetics and tolerability demonstrated in this study, GSK1322322 has the potential to become the first-in-class PDF inhibitor for clinical use.

Address: GlaxoSmithKline, Research Triangle Park, NC, USA

3916160e: J.Cancer Res.Clin.Oncol., 1637-1647(2013)
Link: [ PubMED ] [ DOI ]
Title: Steroid receptor RNA activator protein (SRAP) expression as a prognostic factor in ER+ human breast tumors.
Author: Yan,Y., Penner,C.C., Skliris,G.P., Cooper,C., Nugent,Z., Blanchard,A., Hamedani,M.K., Wang,X., Myal,Y., Murphy,L.C., Leygue,E.
Abstract:
BACKGROUND: The steroid receptor RNA activator protein (SRAP) is a newly described protein modulating the activity of multiple transcription factors including the estrogen receptor (ER). We have recently reported the immunodetection by Western blot of multiple SRAP peptides in breast tissue. High expression of these peptides, assessed by tissue micro-array (TMA) analysis, was associated with poor prognosis in patients whose primary tumors were ER positive (ER+). In such studies, it is recognized that intensity as well as specificity of the signal detected directly depends upon the antibody used as well as the position of the epitope recognized. To confirm the potential relevance of SRAP as a new prognostic factor, it is critical to establish whether similar results are obtained with independent antibodies. METHODS: Two commercial anti-SRAP antibodies (742A and 743A), respectively, recognizing the N- and C-terminal extremity of the protein, were first used to analyze by Western blot SRAP expression in protein extracts from frozen breast tumor tissue sections. These antibodies were further used to investigate by immunohistochemistry (IHC) SRAP location in paraffin-embedded breast tumors. Comparative TMA analysis of 170 ER+ tumors was eventually performed in order to establish the potential associations existing between SRAP expression and clinical outcome. RESULTS: Multiple SRAP peptides were differentially detected by Western blot. Both antibodies led to similar nuclear and cytoplasmic staining in breast tissue section. A solid correlation was found (Spearman r = 0.46, P < 0.001) between 742A and 743A IHC scores. Results from both antibodies independently showed that dividing expression levels into lower 25 percentile, 26-75 percentile, and highest 25 percentile demonstrated a hazard ratio (HR) of 1.82 (P = 0.0042) for 742A antibody and 1.35 (P = 0.14) for 743A antibody. When both scores are combined, double high expressor (by 742A and 743A) was associated with a poor prognosis of breast-cancer-specific survival (Mantel-Cox: P = 0.005, HR = 2.24). CONCLUSION: Overall, our data suggest the existence in breast tumor tissue of multiple SRAP-like peptides. Assessing their expression in primary breast tumors can predict clinical outcome in ER+ breast cancer patients.

E-Mail: umyan9@cc.umanitoba.ca
Address: Manitoba Institute of Cell Biology, 675 McDermot Ave., Winnipeg, MB, R3E0V9, Canada,

3916178e: J.Clin.Gastroenterol., 834-843(2013)
Link: [ PubMED ] [ DOI ]
Title: From Diarrhea to Obesity in Prohormone Convertase 1/3 Deficiency: Age-dependent Clinical, Pathologic, and Enteroendocrine Characteristics.
Author: Bandsma,R.H., Sokollik,C., Chami,R., Cutz,E., Brubaker,P.L., Hamilton,J.K., Perlman,K., Zlotkin,S., Sigalet,D.L., Sherman,P.M., Martin,M.G., Avitzur,Y.
Abstract:
GOALS: The aim of this report is to delineate the clinical, pathologic, and enteroendocrine (EE) features of prohormone convertase 1/3 (PC1/3) deficiency in children. BACKGROUND: Prohormone convertases play a pivotal role in the activation of biologically inactive hormones. Congenital defects in the EE axis, such as PC1/3 deficiency, have been rarely reported and their pathophysiological mechanisms are largely unknown. STUDY: EE function and pathology was evaluated in 4 males (1, 2, 7, and 10 y old) from 2 families with PC1/3 deficiency at a university children's hospital. Clinical course, pathology analysis including immunohistochemistry for PC1/3, PC2, and glucagon-like peptide 1 (GLP-1) and electron microscopy, as well as EE function tests (GLP-1, GLP-2, oral glucose tolerance test) were performed. RESULTS: All (n=4) suffered from congenital severe diarrhea associated with malabsorption. The diarrhea improved during the first year of life and hyperphagia with excessive weight gain (BMI>97th percentile) became the predominant phenotype at an older age. Analysis of the enteroendocrine axis revealed high proinsulin levels (57 to 1116 pmol/L) in all patients, low serum GLP-2 levels, and impaired insulin and GLP-1 secretion after an oral glucose tolerance test at a young age, with improvement in 1 older child tested. Electron microscopy showed normal ultrastructure of enterocytes and EE cells. Immunohistochemistry revealed normal expression of chromogranin A, a marker of EE cells but markedly reduced immunostaining for PC1/3 and PC2 in all patients. CONCLUSIONS: PC1/3 deficiency is associated with an age dependent, variable clinical phenotype caused by severe abnormalities in intestinal and EE functions. Serum level of proinsulin can be used as an effective screening tool.

Address: Alberta Children's Hospital and University of Calgary, Calgary, AB, Canada paragraph signDepartment of Pediatrics, Mattel Children's Hospital, David Geffen School of Medicine at the University of California, Los Angeles, CA

3916181e: Am.J.Cardiol., 1390-1395(2013)
Link: [ PubMED ] [ DOI ]
Title: New-Onset Atrial Fibrillation After Acute Myocardial Infarction and Its Relation to Admission Biomarkers (from the TRIUMPH Registry).
Author: Parashar,S., Kella,D., Reid,K.J., Spertus,J.A., Tang,F., Langberg,J., Vaccarino,V., Kontos,M.C., Lopes,R.D., Lloyd,M.S.
Abstract:
Atrial fibrillation (AF) is an independent predictor of mortality after acute myocardial infarction (AMI). We analyzed the relation between biomarkers linked to myocardial stretch (NT-pro-brain natriuretic peptide [NT-proBNP]), myocardial damage (Troponin-T [TnT]), and inflammation (high-sensitivity C-reactive protein [hs-CRP]) and new-onset AF during AMI to identify patients at high risk for AF. In a prospective multicenter registry of AMI patients (from the Translational Research Investigating Underlying disparities in recovery from acute Myocardial infarction: Patients' Health status registry), we measured NT-proBNP, TnT, and hs-CRP in patients without a history of AF (n = 2,370). New-onset AF was defined as AF that occurred during the index hospitalization. Hierarchical multivariate logistic regression models were used to determine the association of biomarkers with new-onset AF, after adjusting for other covariates. New-onset AF was documented in 114 patients with AMI (4.8%; mean age 58 years; 32% women). For each twofold increase in NT-proBNP, there was an 18% increase in the rate of AF (odds ratio [OR] 1.18, 95% confidence interval [CI] 1.03 to 1.35; p <0.02). Similarly, for every twofold increase in hs-CRP, there was a 15% increase in the rate of AF (OR 1.15, 95% CI 1.02 to 1.30; p = 0.02). TnT was not independently associated with new-onset AF (OR 0.96, 95% CI 0.85 to 1.07; p = 0.3). NT-proBNP and hs-CRP were independently associated with new in-hospital AF after MI, in both men and women, irrespective of race. Our study suggests that markers of myocardial stretch and inflammation, but not the amount of myocardial necrosis, are important determinants of AF in the setting of AMI.

E-Mail: Susmita.parashar@emory.edu
Address: Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia. Electronic address:

3916182e: J.Neuroinflamm., 126-126(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Glatiramer acetate treatment effects on gene expression in monocytes of multiple sclerosis patients.
Author: Thamilarasan,M., Hecker,M., Goertsches,R.H., Paap,B.K., Schroder,I., Koczan,D., Thiesen,H.J., Zettl,U.K.
Abstract:
BACKGROUND: Glatiramer acetate (GA) is a mixture of synthetic peptides used in the treatment of patients with relapsing-remitting multiple sclerosis (RRMS). The aim of this study was to investigate the effects of GA therapy on the gene expression of monocytes. METHODS: Monocytes were isolated from the peripheral blood of eight RRMS patients. The blood was obtained longitudinally before the start of GA therapy as well as after one day, one week, one month and two months. Gene expression was measured at the mRNA level by microarrays. RESULTS: More than 400 genes were identified as up-regulated or down-regulated in the course of therapy, and we analyzed their biological functions and regulatory interactions. Many of those genes are known to regulate lymphocyte activation and proliferation, but only a subset of genes was repeatedly differentially expressed at different time points during treatment. CONCLUSIONS: Overall, the observed gene regulatory effects of GA on monocytes were modest and not stable over time. However, our study revealed several genes that are worthy of investigation in future studies on the molecular mechanisms of GA therapy.

3916183e: Cardiovasc.Diabetol., 152-152(2013)
Link: [ PubMED ] [ DOI ]
Title: Diabetes mellitus activates fetal gene program and intensifies cardiac remodeling and oxidative stress in aged spontaneously hypertensive rats.
Author: Rosa,C.M., Xavier,N.P., Henrique Campos,D., Fernandes,A.A., Cezar,M.D., Martinez,P.F., Cicogna,A.C., Gimenes,C., Gimenes,R., Okoshi,M.P., Okoshi,K.
Abstract:
BACKGROUND: The combination of systemic arterial hypertension and diabetes mellitus (DM) induces greater cardiac remodeling than either condition alone. However, this association has been poorly addressed in senescent rats. Therefore, this study aimed to analyze the influence of streptozotocin-induced DM on ventricular remodeling and oxidative stress in aged spontaneously hypertensive rats (SHR). METHODS: Fifty 18 month old male SHR were divided into two groups: control (SHR, n = 25) and diabetic (SHR-DM, n = 25). DM was induced by streptozotocin (40 mg/kg, i.p.). After nine weeks, the rats underwent echocardiography and myocardial functional study in left ventricular (LV) isolated papillary muscle preparations. LV samples were obtained to measure myocyte diameters, interstitial collagen fraction, and hydroxyproline concentration. Gene expression of atrial natriuretic peptide (ANP) and α- and β-myosin heavy chain (MyHC) isoforms was evaluated by RT-PCR. Serum oxidative stress was assessed by measuring lipid hydroperoxide concentration and superoxide dismutase and glutathione peroxidase activities. Statistics: Student's t test or Mann-Whitney test, p < 0.05. RESULTS: SHR-DM presented higher blood glucose (487 +/- 29 vs. 89.1 +/- 21.1 mg/dL) and lower body weight (277 +/- 26 vs. 339 +/- 38 g). Systolic blood pressure did not differ between groups. Echocardiography showed LV and left atrial dilation, LV diastolic and relative wall thickness decrease, and LV systolic and diastolic function impairment in SHR-DM. Papillary muscle study showed decreased myocardial contractility and contractile reserve in SHR-DM. Myocyte diameters and myocardial interstitial collagen fraction and hydroxyproline concentration did not differ between groups. Increased serum pro-oxidant activity and gene expression of ANP and β/α-MyHC ratio were observed in DM. CONCLUSION: Diabetes mellitus induces cardiac dilation and functional impairment, increases oxidative stress and activates fetal gene program in aged spontaneously hypertensive rats.

3916185e: Clin.Lab.(Heidelberg), 831-835(2013)
Link: [ PubMED ] [ DOI ]
Title: Measurement Nt-proBNP circulating concentrations in heart failure patients with a new point-of-care assay.
Author: Lepoutre,T., Rousseau,M.F., Ahn,S.A., Gruson,D.
Abstract:
BACKGROUND: BNP (Brain Natriuretic Peptide) and Nt-proBNP (N-terminal-pro-Brain Natriuretic Peptide) are valuable markers for the diagnosis and prognosis of heart failure (HF). The AQT90 FLEX is a newly released random access analyzer for point-of-care (POCT) measurement. The aim of our study was to determine Nt-pro-BNP concentrations in HF patients with the POCT assay. METHODS: Nt-proBNP levels were measured in seventy seven HF patients and in thirty seven healthy volunteers. The results were compared with a central laboratory assay. RESULTS: Nt-proBNP levels measured with the AQT90 FLEX were significantly correlated with the comparison Nt-proBNP assay and were related to HF severity. CONCLUSIONS: Nt-proBNP testing with the AQT 90 FLEX analyzer is comparable to the central lab assay and may offer the advantages of POCT testing for the diagnosis and prognosis of heart failure.

Address: Department of Laboratory Medicine, Cliniques Universitaires Saint Luc, Brussels, Belgium

3916191e: Front.Cell.Neurosci., 176-176(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: MARCH5 inactivation supports mitochondrial function during neurodegenerative stress.
Author: Fang,L., Li,J., Flammer,J., Neutzner,A.
Abstract:
Neuronal cell death is accompanied by mitochondrial dysfunction with mitochondrial maintenance critical to neuronal survival. The mitochondrial ubiquitin ligase MARCH5 has dual roles in the upkeep of mitochondrial function. MARCH5 is involved in targeted degradation of proteins harmful to mitochondria and impacts mitochondrial morphology upstream of the fission protein Drp1. In a neuronal cell model, dominant-negative MARCH5 prevents mitochondrial fragmentation during neurodegenerative stress induced by the neuron-specific reactive oxygen generator 6-hydroxydopamine, the complex I inhibitor rotenone or Alzheimer's-related amyloid β peptide. In addition, preservation of mitochondrial function in terms of membrane potential and lower reactive oxygen generation was observed following inactivation of MARCH5. Our findings connect MARCH5 to neuronal stress responses and further emphasize the link between mitochondrial dynamics and function.

Address: Department of Biomedicine, University of Basel Basel, Switzerland

3916197e: Circulation Suppl., S42-S49(2013)
Link: [ PubMED ] [ DOI ]
Title: Comparison of transendocardial and intracoronary CD34+ cell transplantation in patients with nonischemic dilated cardiomyopathy.
Author: Vrtovec,B., Poglajen,G., Lezaic,L., Sever,M., Socan,A., Domanovic,D., Cernelc,P., Torre-Amione,G., Haddad,F., Wu,J.C.
Abstract:
BACKGROUND: In an open-label blinded study, we compared intracoronary and transendocardial CD34+ cell transplantation in patients with nonischemic dilated cardiomyopathy. METHODS AND RESULTS: Of the 40 patients with dilated cardiomyopathy, 20 were randomized to receive intracoronary injection and 20 received transendocardial CD34+ cell delivery. In both groups, CD34+ cells were mobilized by filgrastim, collected via apheresis, and labeled with technetium-99m radioisotope for single-photon emission computed tomographic imaging. In the intracoronary group, cells were injected intracoronarily in the artery supplying segments of greater perfusion defect on myocardial perfusion scintigraphy. In the transendocardial group, electroanatomic mapping was used to identify viable but dysfunctional myocardium, and transendocardial cell injections were performed. Nuclear single-photon emission computed tomographic imaging for quantification of myocardial retention was performed 18 hours thereafter. At baseline, groups did not differ in age, sex, left ventricular ejection fraction, or N-terminal pro-brain natriuretic peptide levels. The number of CD34+ cells was also comparable (105 +/- 31 x 10(6) in the transendocardial group versus 103 +/- 27 x 10(6) in the intracoronary group, P=0.62). At 18 hours after procedure, myocardial retention was higher in the transendocardial group (19.2 +/- 4.8%) than in the intracoronary group (4.4 +/- 1.2%, P<0.01). At 6 months, left ventricular ejection fraction improved more in the transendocardial group (+8.1 +/- 4.3%) than in the intracoronary group (+4.2 +/- 2.3%, P=0.03). The same pattern was observed for the 6-minute walk test distance (+125 +/- 33 m in the transendocardial group versus +86 +/- 13 m in the intracoronary group, P=0.03) and N-terminal pro-brain natriuretic peptide (-628 +/- 211 versus -315 +/- 133 pg/mL, P=0.04). CONCLUSIONS: In patients with dilated cardiomyopathy, transendocardial CD34+ cell transplantation is associated with higher myocardial retention rates and greater improvement in ventricular function, N-terminal pro-brain natriuretic peptide, and exercise capacity compared with intracoronary route. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01350310.

Address: Advanced Heart Failure and Transplantation Center (B.V., G.P.), Department of Nuclear Medicine (L.L., A.S.), and Department of Hematology (M.S., P.C.), University Medical Center Ljubljana, Ljubljana, Slovenia

3916199e: Bull.Exp.Biol.Med., 175-178(2013)
Link: [ PubMED ] [ DOI ]
Title: Effect of anorexigenic peptide obestatin on platelet aggregation and osmotic resistance of erythrocytes.
Author: Khirazova,E.E., Golubeva,M.G., Maslova,M.V., Graf,A.V., Maklakova,A.S., Baizhumanov,A.A., Trofimova,L.K., Sokolova,N.A., Kamenskii,A.A.
Abstract:
We studied the effects of the anorexigenic peptide obestatin on the coagulation system and blood rheology (by the parameters of platelet aggregation and osmotic resistance of erythrocytes) in vitro and in vivo. Obestatin inhibited in vitro platelet aggregation in the entire dose range and reduced osmotic resistance of erythrocytes in all doses except 300 nmol/kg (obestatin in a dose of 300 nmol/kg had no effect on this parameter). Similar to the results of in vitro experiments, intranasal, intraperitoneal, and subcutaneous administration of obestatin in a dose of 300 nmol/kg inhibited platelet aggregation and had no effect on the osmotic resistance of erythrocytes.

E-Mail: ekhirazova@gmail.com
Address: Department of Human and Animal Physiology, Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, Russia

3916201e: PLoS Negl.Trop.Dis., e2486-e2486(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Functional and Genetic Characterization of Neuropeptide Y-Like Receptors in Aedes aegypti.
Author: Liesch,J., Bellani,L.L., Vosshall,L.B.
Abstract:
BACKGROUND: Female Aedes aegypti mosquitoes are the principal vector for dengue fever, causing 50-100 million infections per year, transmitted between human and mosquito by blood feeding. Ae. aegypti host-seeking behavior is known to be inhibited for three days following a blood meal by a hemolymph-borne humoral factor. Head Peptide-I is a candidate peptide mediating this suppression, but the mechanism by which this peptide alters mosquito behavior and the receptor through which it signals are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Head Peptide-I shows sequence similarity to short Neuropeptide-F peptides (sNPFs) that have been implicated in feeding behaviors and are known to signal through Neuropeptide Y (NPY)-Like Receptors (NPYLRs). We identified eight NPYLRs in the Ae. aegypti genome and screened each in a cell-based calcium imaging assay for sensitivity against a panel of peptides. Four of the Ae. aegypti NPYLRs responded to one or more peptide ligands, but only NYPLR1 responded to Head Peptide-I as well as sNPFs. Two NPYLR1 homologues identified in the genome of the Lyme disease vector, Ixodes scapularis, were also sensitive to Head Peptide-I. Injection of synthetic Head Peptide-I and sNPF-3 inhibited host-seeking behavior in non-blood-fed female mosquitoes, whereas control injections of buffer or inactive Head Peptide-I [Cys10] had no effect. To ask if NPYLR1 is necessary for blood-feeding-induced host-seeking inhibition, we used zinc-finger nucleases to generate five independent npylr1 null mutant strains and tested them for behavioral abnormalities. npylr1 mutants displayed normal behavior in locomotion, egg laying, sugar feeding, blood feeding, host seeking, and inhibition of host seeking after a blood meal. CONCLUSIONS: In this work we deorphanized four Ae. aegypti NPYLRs and identified NPYLR1 as a candidate sNPF receptor that is also sensitive to Head Peptide-I. Yet npylr1 alone is not required for host-seeking inhibition and we conclude that other receptors, additional peptides, or both, regulate this important behavior.

Address: Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, New York, United States of America

3916204e: PLoS One, e77176-e77176(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Rapid responses to reverse t3 hormone in immature rat sertoli cells: calcium uptake and exocytosis mediated by integrin.
Author: Zanatta,A.P., Zanatta,L., Goncalves,R., Zamoner,A., Silva,F.R.
Abstract:
There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T3 (rT3) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT3 are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT3 was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT3-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT3 may be mediated by integrin αvβ3. In addition, it was demonstrated that calcium uptake stimulated by rT3 is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT3 also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT3 modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.

Address: Departamento de Bioquimica, Centro de Ciencias Biologicas, Universidade Federal de Santa Catarina, Florianopolis-Santa Catarina, Brazil

3916207e: PLoS One, e76899-e76899(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Targeting androgen receptor/src complex impairs the aggressive phenotype of human fibrosarcoma cells.
Author: Castoria,G., Giovannelli,P., Di Donato,M., Hayashi,R., Arra,C., Appella,E., Auricchio,F., Migliaccio,A.
Abstract:
BACKGROUND: Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR). EXPERIMENTAL: Findings: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR. CONCLUSION: This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer cells and identifies new potential targets in the therapeutic approach to human cancers.

Address: Department of Biochemistry, Biophysics, and General Pathology, 2nd University of Naples, Naples, Italy

3916214e: Front.Endocrinol., 140-140(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Developmental profile and sexually dimorphic expression of kiss1 and kiss1r in the fetal mouse brain.
Author: Knoll,J.G., Clay,C.M., Bouma,G.J., Henion,T.R., Schwarting,G.A., Millar,R.P., Tobet,S.A.
Abstract:
The hypothalamic-pituitary-gonadal axis (HPG) is a complex neuroendocrine circuit involving multiple levels of regulation. Kisspeptin neurons play essential roles in controlling the HPG axis from the perspectives of puberty onset, oscillations of gonadotropin releasing hormone (GnRH) neuron activity, and the pre-ovulatory LH surge. The current studies focus on the expression of kisspeptin during murine fetal development using in situ hybridization (ISH), quantitative reverse transcription real-time PCR (QPCR), and immunocytochemistry. Expression of mRNA coding for kisspeptin (KISS1) and its receptor KISS1R was observed at embryonic (E) day 13 by ISH. At E13 and other later ages examined, Kiss1 signal in individual cells within the arcuate nucleus (ARC) appeared stronger in females than males. ISH examination of agonadal steroidogenic factor-1 (Sf1) knockout mice revealed that E17 XY knockouts (KO) resembled wild-type (WT) XX females. These findings raise the possibility that gonadal hormones modulate the expression of Kiss1 in the ARC prior to birth. The sex and genotype differences were tested quantitatively by QPCR experiments in dissected hypothalami from mice at E17 and adulthood. Females had significantly more Kiss1 than males at both ages, even though the number of cells detected by ISH was similar. In addition, QPCR revealed a significant difference in the amount of Kiss1 mRNA in Sf1 mice with WT XY mice expressing less than XY KO and XX mice of both genotypes. The detection of immunoreactive KISS1 in perikarya of the ARC at E17 indicates that early mRNA is translated to peptide. The functional significance of this early expression of Kiss1 awaits elucidation.

Address: Oregon Health and Science University , Portland, OR , USA

3916235e: Proc.Natl.Acad.Sci.U.S.A., 17790-17795(2013)
Link: [ PubMED ] [ DOI ]
Title: Putative megaenzyme DWA1 plays essential roles in drought resistance by regulating stress-induced wax deposition in rice.
Author: Zhu,X., Xiong,L.
Abstract:
Drought stress is a major limiting factor for crop production. Cuticular wax plays an important role in preventing water loss from drought stress. However, the genetic control of cuticular wax deposition under drought stress conditions has not been characterized. Here, we identified a rice gene Drought-Induced Wax Accumulation 1 (DWA1) encoding a very large protein (2,391 aa in length) containing multiple enzymatic structures, including an oxidoreductase-like domain; a prokaryotic nonribosomal peptide synthetase-like module, including an AMP-binding domain; and an allene oxide synthase-like domain. This previously unreported putative megaenzyme is conserved in vascular plants. A dwa1 KO mutant was highly sensitive to drought stress relative to the WT. DWA1 was preferentially expressed in vascular tissues and epidermal layers and strongly induced by drought stress. The dwa1 mutant was impaired in cuticular wax accumulation under drought stress, which significantly altered the cuticular wax composition of the plant, resulting in increased drought sensitivity. The mutant had reduced levels of very-long-chain fatty acids, and plants overexpressing DWA1 showed elevated levels of very-long-chain fatty acids relative to the WT. The expression of many wax-related genes was significantly suppressed in dwa1 under drought conditions. The AMP-binding domain exhibited in vitro enzymatic activity in activating long-chain fatty acids to form acyl-CoA. Our results suggest that DWA1 controls drought resistance by regulating drought-induced cuticular wax deposition in rice. This finding may have significant implications for improving the drought resistance of crop varieties.

Address: National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China

3916236e: J.Exp.Med., 2403-2414(2013)
Link: [ PubMED ] [ DOI ]
Title: Pathogenic CD4+ T cells recognizing an unstable peptide of insulin are directly recruited into islets bypassing local lymph nodes.
Author: Mohan,J.F., Calderon,B., Anderson,M.S., Unanue,E.R.
Abstract:
In the nonobese diabetic mouse, a predominant component of the autoreactive CD4+ T cell repertoire is directed against the B:9-23 segment of the insulin B chain. Previous studies established that the majority of insulin-reactive T cells specifically recognize a weak peptide-MHC binding register within the B:9-23 segment, that to the 12-20 register. These T cells are uniquely stimulated when the B:9-23 peptide, but not the insulin protein, is offered to antigen presenting cells (APCs). Here, we report on a T cell receptor (TCR) transgenic mouse (8F10) that offers important new insights into the biology of these unconventional T cells. Many of the 8F10 CD4+ T cells escaped negative selection and were highly pathogenic. The T cells were directly recruited into islets of Langerhans, where they established contact with resident intra-islet APCs. Immunogenic insulin had to be presented in order for the T cells to localize and cause disease. These T cells bypassed an initial priming stage in the pancreatic lymph node thought to precede islet T cell entry. 8F10 T cells induced the production of antiinsulin antibodies and islets contained immunoglobulin (IgG) deposited on β cells and along the vessel walls.

Address: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110

3916239e: Cardiovasc.Diabetol., 148-148(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Novel fusion of GLP-1 with a domain antibody to serum albumin prolongs protection against myocardial ischemia/reperfusion injury in the rat.
Author: Bao,W., Holt,L.J., Prince,R.D., Jones,G.X., Aravindhan,K., Szapacs,M., Barbour,A.M., Jolivette,L.J., Lepore,J.J., Willette,R.N., Deangelis,E., Jucker,B.M.
Abstract:
BACKGROUND: Glucagon-like peptide-1 (GLP-1) and its mimetics reduce infarct size in the setting of acute myocardial ischemia/reperfusion (I/R) injury. However, the short serum half-life of GLP-1 and its mimetics may limit their therapeutic use in acute myocardial ischemia. Domain antibodies to serum albumin (AlbudAbs) have been developed to extend the serum half-life of short lived therapeutic proteins, peptides and small molecules. In this study, we compared the effect of a long acting GLP-1 agonist, DPP-IV resistant GLP-1 (7--36, A8G) fused to an AlbudAb (GAlbudAb), with the effect of the GLP-1 mimetic, exendin-4 (short half-life GLP-1 agonist) on infarct size following acute myocardial I/R injury. METHODS: Male Sprague--Dawley rats (8-week-old) were treated with vehicle, GAlbudAb or exendin-4. Myocardial ischemia was induced 2 h following the final dose for GAlbudAb and 30 min post the final dose for exendin-4. In a subgroup of animals, the final dose of exendin-4 was administered (1 μg/kg, SC, bid for 2 days) 6 h prior to myocardial ischemia when plasma exendin-4 was at its miniμm concentration (Cmin). Myocardial infarct size, area at risk and cardiac function were determined 24 h after myocardial I/R injury. RESULTS: GAlbudAb and exendin-4 significantly reduced myocardial infarct size by 28% and 23% respectively, compared to vehicle (both p < 0.01 vs. vehicle) after I/R injury. Moreover, both GAlbudAb and exendin-4 markedly improved post-ischemic cardiac contractile function. Body weight loss and reduced food intake consistent with the activation of GLP-1 receptors was observed in all treatment groups. However, exendin-4 failed to reduce infarct size when administered 6 h prior to myocardial ischemia, suggesting continuous activation of the GLP-1 receptors is needed for cardioprotection. CONCLUSIONS: Cardioprotection provided by GAlbudAb, a long acting GLP-1 mimetic, following myocardial I/R injury was comparable in magnitude, but more sustained in duration than that produced by short-acting exendin-4. Very low plasma concentrations of exendin-4 failed to protect the heart from myocardial I/R injury, suggesting that sustained GLP-1 receptor activation plays an important role in providing cardioprotection in the setting of acute myocardial I/R injury. Long-acting GLP-1 agonists such as GAlbudAb may warrant additional evaluation as novel therapeutic agents to reduce myocardial I/R injury during acute coronary syndrome.

3916240e: J.Card.Fail., 678-684(2013)
Link: [ PubMED ] [ DOI ]
Title: Gremlin-1 identifies fibrosis and predicts adverse outcome in patients with heart failure undergoing endomyocardial biopsy.
Author: Mueller,K.A., Tavlaki,E., Schneider,M., Jorbenadze,R., Geisler,T., Kandolf,R., Gawaz,M., Mueller,I.I., Zuern,C.S.
Abstract:
BACKGROUND: Gremlin-1 (Grem1), an antagonist of bone morphogenetic proteins, is involved in fibrotic tissue formation in kidney and lung. The impact of myocardial Grem1 expression is unknown. We investigated the prognostic value of Grem1 expression in 214 consecutive patients with nonischemic heart failure (HF) undergoing endomyocardial biopsy. METHODS: In all patients, the following risk factors were assessed: Grem1 expression (semiquantitative score scheme ranging from 1 to 4), presence of inflammatory markers, detection of viral genome, left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), New York Heart Association functional class (NYHA), troponin I, and B-type natriuretic peptide. Degree of myocardial fibrosis was defined as an index. Study end point was a combination of all-cause death and HF-related rehospitalization within 3 years of follow-up. RESULTS: Grem1 expression significantly correlated with the degree of myocardial fibrosis (correlation coefficient r = 0.619; P < .0001). Patients with the highest Grem1 expression (score 4) showed the most severely impaired LVEF and highest LVEDD (P < .0001 and P = .030, respectively, for comparison of semiquantitative scores). During follow-up, 33 patients (15.4%) reached the study end point. Grem1 expression and NYHA >/=II were independent predictors of the end point (Grem1: hazard ratio [HR] 7.5, 95% confidence interval [CI] 1.8-32.2; P = .006; NYHA >/=II: HR 2.0, 95% CI 1.0-4.1; P = .048). CONCLUSIONS: Grem1 correlates with the degree of myocardial fibrosis and left ventricular dysfunction and is an independent predictor of adverse outcome in patients with nonischemic HF.

Address: Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Universitatsklinikum Tubingen, Tubingen, Germany

3916244e: PLoS One, e78178-e78178(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Ghrelin decreases firing activity of gonadotropin-releasing hormone (GnRH) neurons in an estrous cycle and endocannabinoid signaling dependent manner.
Author: Farkas,I., Vastagh,C., Sarvari,M., Liposits,Z.
Abstract:
The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R) in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca2+-imaging revealed a ghrelin-triggered increase of the Ca2+-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10μM) suggesting direct action of ghrelin. Estradiol (1nM) eliminated the ghrelin-evoked rise of Ca2+-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40nM-4μM) administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1) antagonist AM251 (1μM) and the intracellularly applied DAG-lipase inhibitor THL (10μM), indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner.

Address: Laboratory of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

3916245e: PLoS One, e76682-e76682(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: The Nociceptin/Orphanin FQ System Is Modulated in Patients Admitted to ICU with Sepsis and after Cardiopulmonary Bypass.
Author: Thompson,J.P., Serrano-Gomez,A., McDonald,J., Ladak,N., Bowrey,S., Lambert,D.G.
Abstract:
BACKGROUND AND OBJECTIVES: Nociceptin/Orphanin FQ (N/OFQ) is a non-classical endogenous opioid peptide that modulates immune function in vitro. Its importance in inflammation and human sepsis is unknown. The objectives of this study were to determine the relationship between N/OFQ, transcripts for its precursor (pre-pro-N/OFQ [ppNOC]) and receptor (NOP), inflammatory markers and clinical outcomes in patients undergoing cardiopulmonary bypass and with sepsis. METHODS: A prospective observational cohort study of 82 patients admitted to Intensive Care (ICU) with sepsis and 40 patients undergoing cardiac surgery under cardiopulmonary bypass (as a model of systemic inflammation). Sixty three healthy volunteers, matched by age and sex to the patients with sepsis were also studied. Clinical and laboratory details were recorded. Polymorph ppNOC and NOP receptor mRNA were determined using quantitative PCR. Plasma N/OFQ was determined using ELISA and cytokines (TNF- α, IL-8, IL-10) measured using radioimmunoassay. Data from patients undergoing cardiac surgery were recorded before, 3 and 24 hours after cardiopulmonary bypass. ICU patients with sepsis were assessed on Days 1 and 2 of ICU admission, and after clinical recovery. MAIN RESULTS: Plasma N/OFQ concentrations increased (p<0.0001) on Days 1 and 2 of ICU admission with sepsis compared to matched recovery samples. Polymorph ppNOC (p= 0.019) and NOP mRNA (p<0.0001) decreased compared to healthy volunteers. TNF-α, IL-8 and IL-10 concentrations increased on Day 1 compared to matched recovery samples and volunteers (p<0.0001). Similar changes (increased plasma N/OFQ, [p=0.0058], decreased ppNOC [p<0.0001], increased IL-8 and IL-10 concentrations [both p<0.0001]) occurred after cardiac surgery but these were comparatively lower and of shorter duration. CONCLUSIONS: The N/OFQ system is modulated in ICU patients with sepsis with similar but reduced changes after cardiac surgery under cardiopulmonary bypass. Further studies are required to clarify the role of the N/OFQ system in inflammation and sepsis, and the mechanisms involved.

Address: Division of Anaesthesia, Critical Care and Pain Management, Department of Cardiovascular Sciences, University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom

3916247e: PLoS One, e75784-e75784(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Porphyromonas gingivalis Regulates TREM-1 in Human Polymorphonuclear Neutrophils via Its Gingipains.
Author: Bostanci,N., Thurnheer,T., Aduse-Opoku,J., Curtis,M.A., Zinkernagel,A.S., Belibasakis,G.N.
Abstract:
The Triggering Receptor Expressed on Myeloid cells 1 (TREM-1) is a cell surface receptor of the immunoglobulin superfamily, with the capacity to amplify pro-inflammatory cytokine production and regulate apoptosis. Polymorphonuclear neutrophils (PMNs) are the first line of defence against infection, and a major source of TREM-1. Porphyromonas gingivalis is a Gram-negative anaerobe highly implicated in the inflammatory processes governing periodontal disease, which is characterized by the destruction of the tooth-supporting tissues. It expresses a number of virulence factors, including the cysteine proteinases (or gingipains). The aim of this in vitro study was to investigate the effect of P. gingivalis on TREM-1 expression and production by primary human PMNs, and to evaluate the role of its gingipains in this process. After 4 h of challenge, P. gingivalis enhanced TREM-1 expression as identified by quantitative real-time PCR. This was followed by an increase in soluble (s)TREM-1 secretion over a period of 18 h, as determined by ELISA. At this time-point, the P. gingivalis-challenged PMNs exhibited diminished TREM-1 cell-membrane staining, as identified by flow cytometry and confocal laser scanning microscopy. Furthermore engagement of TREM-1, by means of anti-TREM-1 antibodies, enhanced the capacity of P. gingivalis to stimulate interleukin (IL)-8 production. Conversely, antagonism of TREM-1 using a synthetic peptide resulted in reduction of IL-8 secretion. Using isogenic P. gingivalis mutant strains, we identified the Arg-gingipain to be responsible for shedding of sTREM-1 from the PMN surface, whereas the Lys-gingipain had the capacity to degrade TREM-1. In conclusion, the differential regulation of TREM-1 by the P. gingivalis gingipains may present a novel mechanism by which P. gingivalis manipulates the host innate immune response helping to establish chronic periodontal inflammation.

Address: Oral Translational Research, Institute of Oral Biology, Center of Dental Medicine, University of Zurich, Zurich, Switzerland

3916255e: Dig.Dis.Sci., 3029-3036(2013)
Link: [ PubMED ] [ DOI ]
Title: Frequency and severity of cirrhotic cardiomyopathy and its possible relationship with bacterial endotoxemia.
Author: Karagiannakis,D.S., Vlachogiannakos,J., Anastasiadis,G., Vafiadis-Zouboulis,I., Ladas,S.D.
Abstract:
BACKGROUND: The cardiac dysfunction presented in cirrhotic patients is already known as cirrhotic cardiomyopathy. The pathogenesis of this entity is not fully understood. AIMS: The aim of this study was to evaluate the frequency and characteristics of cirrhotic cardiomyopathy and to investigate the possible role of bacterial endotoxemia on its aggravation. METHODS: Forty-five cirrhotics were studied by a tissue Doppler imaging echocardiography at rest and after stress. The diagnosis of left ventricular diastolic dysfunction was based on the latest guidelines of the American Society of Echocardiography, whereas its severity was defined by the E/e'av ratio. Endotoxemia was estimated by measuring the serum levels of lipopolysaccharide-binding protein (LBP) and cytokines. RESULTS: None of the patients had systolic dysfunction, but 17/45 (37.8 %) had a diastolic one. Patients with grade II diastolic dysfunction had significantly longer QTc (p = 0.049), larger left atrium volume (p = 0.013), higher Brain Natriuretic Peptide levels (p = 0.007) and higher LBP levels (p = 0.02), compared to those with normal cardiac function, without differences in the systemic hemodynamics and the cytokines' levels. Moreover, the severity of diastolic dysfunction as reflected by the E/e'av. was significantly correlated with the LBP levels (p = 0.002). On the multivariate analysis, the LBP was independently associated with the presence of diastolic dysfunction. CONCLUSIONS: Cirrhosis is commonly complicated by cardiac dysfunction. Patients with severe cirrhotic cardiomyopathy have higher LBP levels, which are significantly correlated with the degree of diastolic dysfunction. Our findings support a potential role of bacterial endotoxemia on the aggravation of cardiomyopathy in cirrhotic patients.

E-Mail: d_karagiannakis@hotmail.com
Address: Hepatogastroenterology Unit, 1st Department of Medicine Propaedeutic, Medical School of Athens, Laikon General Hospital, 17 Agiou Thoma Street, 11527, Athens, Greece,

3916258e: Biomed.Pharmacother., 659-663(2013)
Link: [ PubMED ] [ DOI ]
Title: Iatrogenic dry eye disease: An eledoisin/carnitine and osmolyte drops study.
Author: Nebbioso,M., Evangelista,M., Librando,A., Plateroti,A.M., Pescosolido,N.
Abstract:
BACKGROUND: To evaluate the effects of an eye drop containing eledoisin and carnitine in patients suffering from primary open-angle glaucoma (POAG) and ocular discomfort syndrome secondary to a chronically treated with eye drops containing benzalkonium chloride (BAK) as preservative. The dry eye disease was defined as a multifactorial drop disease concerning tears and ocular surface, which brings to discomfort symptoms and visual disorders with potential damage to the ocular surface. Several studies underlined the beneficial effects of secretagogues drugs, such as eledoisin. It is a neuro-peptide extracted from the salivary glands of some shellfishes. Recently, it has been also showed the protective role of carnitine in respect of the ocular surface exposed to the tear film hyperosmolarity. MATERIALS AND METHODS: This randomized double-blind pilot study has been evaluated by lubricant eye drop solutions containing eledoisin and carnitine in 40 patients with ocular discomfort syndrome secondary to POAG, since that the patients were chronically treated with eye drops which decrease eye pressure and contain BAK as preservative. The subjects filled out a questions form concerning the severity of the symptoms and their impact on daily activities. Subsequently Fluorescein Breakup Time (FBUT), Schirmer Test 1 (ST), and Ocular Protection Index (OPI) were measured at baseline and after 15days of treatment. RESULTS: At the end of therapy it was possible to match the beneficial effects of eye drops with carnitin, taurine, sodium hyaluronate and eledoisin. In fact, after 15days of treatment, patients of group 1 showed a decrease of approximately 50% concerning the severity of symptoms and a significant improvement of the tests valued. CONCLUSION: In summary, lubricant eye drops that restore physiological hosmolarity and stimulate tear production represent a promising strategy for dry eye syndrome.

E-Mail: marcella.nebbioso@uniroma1.it
Address: Department of Sense Organs, Centre of Ocular Electrophysiology, viale del policlinico 155, 00161 Rome, Italy. Electronic address:

3916264e: Folia Med.(Plovdiv), 25-32(2013)
Link: [ PubMED ] [ DOI ]
Title: Adipose tissue hormones and appetite and body weight regulators in insulin resistance.
Author: Koleva,D.I., Orbetzova,M.M., Atanassova,P.K.
Abstract:
Impaired sensitivity to insulin (the so called insulin resistance, IR) occurs in a number of genetic and acquired conditions, including obesity, non-insulin dependent diabetes mellitus, polycystic ovary syndrome (PCOS) and metabolic syndrome (MS). In this review we discuss the correlation between IR, the adipose tissue hormones and appetite and body weight regulators. Leptin acts as a major adipostat: it suppresses food intake and activates catabolic pathways associated with increased energy production. It improves the peripheral insulin sensitivity and affects β-cell function. Adiponectin is the only adipocytokine discovered so far that has anti-atherogenic properties. There is a reverse correlation between the serum adiponectin levels and the degree of obesity, IR, impaired glucose tolerance, dyslipidemia and atherosclerosis. Ghrelin stimulates food intake; of all circulating orexigenic hormones ghrelin is the most thoroughly studied. Ghrelin levels are decreased in MS and PCOS patients as this hormone is negatively correlated with body mass. Resistin is a hormone secreted by adipose tissues; a growing body of evidence suggests that it might be implicated in the link between obesity and diabetes. It has been found that the hormone's levels are significantly higher in obese people than those in normal body mass people. The recently discovered adipose tissue hormones, vaspin, visfatin, omentin-1 and their effect on IR development, have been increasingly researched.

Address: Clinic of Endocrinology and Metabolic Diseases, St George University Hospital, Plovdiv, Bulgaria

3916265e: J.Neurosci., 12705-12717(2013)
Link: [ PubMED ] [ DOI ]
Title: Neuropeptide y gates a stress-induced, long-lasting plasticity in the sympathetic nervous system.
Author: Wang,Q., Wang,M., Whim,M.D.
Abstract:
Acute stress evokes the fight-or-flight reflex, which via release of the catecholamine hormones affects the function of every major organ. Although the reflex is transient, it has lasting consequences that produce an exaggerated response when stress is reexperienced. How this change is encoded is not known. We investigated whether the reflex affects the adrenal component of the sympathetic nervous system, a major branch of the stress response. Mice were briefly exposed to the cold-water forced swim test (FST) which evoked an increase in circulating catecholamines. Although this hormonal response was transient, the FST led to a long-lasting increase in the catecholamine secretory capacity measured amperometrically from chromaffin cells and in the expression of tyrosine hydroxylase. A variety of approaches indicate that these changes are regulated postsynaptically by neuropeptide Y (NPY), an adrenal cotransmitter. Using immunohistochemistry, RT-PCR, and NPY(GFP) BAC mice, we find that NPY is synthesized by all chromaffin cells. Stress failed to increase secretory capacity in NPY knock-out mice. Genetic or pharmacological interference with NPY and Y1 (but not Y2 or Y5) receptor signaling attenuated the stress-induced change in tyrosine hydroxylase expression. These results indicate that, under basal conditions, adrenal signaling is tonically inhibited by NPY, but stress overrides this autocrine negative feedback loop. Because acute stress leads to a lasting increase in secretory capacity in vivo but does not alter sympathetic tone, these postsynaptic changes appear to be an adaptive response. We conclude that the sympathetic limb of the stress response exhibits an activity-dependent form of long-lasting plasticity.

Address: Department of Biology, Pennsylvania State University, State College, Pennsylvania 16802, USA

3916266e: J.Neurosci., 12689-12697(2013)
Link: [ PubMED ] [ DOI ]
Title: GnRH neurons elaborate a long-range projection with shared axonal and dendritic functions.
Author: Herde,M.K., Iremonger,K.J., Constantin,S., Herbison,A.E.
Abstract:
Information processing by neurons has been traditionally envisioned to occur in discrete neuronal compartments. Specifically, dendrites receive and integrate synaptic inputs while axons initiate and conduct spikes to distal neuronal targets. We report here in mice, using morphological reconstructions and electrophysiology, that the gonadotropin-releasing hormone (GnRH) neurons that control mammalian fertility do not conform to this stereotype and instead possess a single projection structure that functions simultaneously as an axon and dendrite. Specifically, we show that the GnRH neuron projection to the median eminence to control pituitary hormone secretion possesses a spike initiation site and conducts action potentials while also exhibiting spines and synaptic appositions along its entire length. Classical axonal or dendritic markers are not detectable in the projection process. Activation of ionotropic glutamate and/or GABA receptors along the GnRH neuron projection is capable of depolarizing the membrane potential and initiating action potentials. In addition, focal glutamate application to the projection is able to regulate the width of propagating spikes. These data demonstrate that GnRH neurons elaborate a previously uncharacterized neuronal projection that functions simultaneously as an axon and dendrite. This structure, termed a "dendron," greatly expands the dynamic control of GnRH secretion into the pituitary portal system to regulate fertility.

Address: Centre for Neuroendocrinology, Department of Physiology, University of Otago School of Medical Sciences, Dunedin 9054, New Zealand

3916267e: J.Neurosci., 12557-12568(2013)
Link: [ PubMED ] [ DOI ]
Title: Nitric oxide mediates selective degeneration of hypothalamic orexin neurons through dysfunction of protein disulfide isomerase.
Author: Obukuro,K., Nobunaga,M., Takigawa,M., Morioka,H., Hisatsune,A., Isohama,Y., Shimokawa,H., Tsutsui,M., Katsuki,H.
Abstract:
We addressed the role of nitric oxide (NO) in orexin neuron degeneration that has been observed under various pathological conditions. Administration of an NO donor NOC18 (50 nmol) into the third ventricle of mice resulted in a significant decrease of orexin-immunoreactive (-IR) neurons, in contrast to a modest change in melanin-concentrating hormone-IR neurons. In addition, NOC18 promoted formation of orexin-A-IR aggregates within orexin neurons. An endoplasmic reticulum stress inducer tunicamycin replicated the effect of NOC18 with regard to decrease of orexin-IR neurons and formation of aggregates. We also found that NOC18 caused an increase in S-nitrosation of protein disulfide isomerase (PDI) and a decrease in PDI activity in hypothalamic tissues. Moreover, PDI inhibitors, such as cystamine and securinine, caused a selective decrease of orexin neurons and promoted formation of orexin-A-IR aggregates. Aggregate formation in orexin-IR neurons was also induced by local injection of small interfering RNA targeting PDI. Interestingly, sleep deprivation for 7 consecutive days induced a selective decrease of orexin-IR neurons, which was preceded by aggregate formation in orexin-IR neurons and an increase in S-nitrosated PDI in the hypothalamus. Activity of neuronal NO synthase (nNOS)-positive neurons in the lateral hypothalamus as assessed by c-Fos expression was elevated in response to sleep deprivation. Finally, sleep deprivation-induced decrease of orexin-IR neurons, formation of aggregates, and S-nitrosation of PDI were not observed in nNOS knock-out mice. These results indicate that nNOS-derived NO may mediate specific pathological events in orexin neurons, including neuropeptide misfolding via S-nitrosation and inactivation of PDI.

Address: Department of Chemico-Pharmacological Sciences and Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan

3916274e: Diabetes, 3282-3291(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of HKDC1 and BACE2 as genes influencing glycemic traits during pregnancy through genome-wide association studies.
Author: Hayes,M.G., Urbanek,M., Hivert,M.F., Armstrong,L.L., Morrison,J., Guo,C., Lowe,L.P., Scheftner,D.A., Pluzhnikov,A., Levine,D.M., McHugh,C.P., Ackerman,C.M., Bouchard,L., Brisson,D., Layden,B.T., Mirel,D., Doheny,K.F., Leya,M.V., Lown-Hecht,R.N., Dyer,A.R., Metzger,B.E., Reddy,T.E., Cox,N.J., Lowe,W.L.Jr.
Abstract:
Maternal metabolism during pregnancy impacts the developing fetus, affecting offspring birth weight and adiposity. This has important implications for metabolic health later in life (e.g., offspring of mothers with pre-existing or gestational diabetes mellitus have an increased risk of metabolic disorders in childhood). To identify genetic loci associated with measures of maternal metabolism obtained during an oral glucose tolerance test at approximately 28 weeks' gestation, we performed a genome-wide association study of 4,437 pregnant mothers of European (n = 1,367), Thai (n = 1,178), Afro-Caribbean (n = 1,075), and Hispanic (n = 817) ancestry, along with replication of top signals in three additional European ancestry cohorts. In addition to identifying associations with genes previously implicated with measures of glucose metabolism in nonpregnant populations, we identified two novel genome-wide significant associations: 2-h plasma glucose and HKDC1, and fasting C-peptide and BACE2. These results suggest that the genetic architecture underlying glucose metabolism may differ, in part, in pregnancy.

E-Mail: ghayes@northwestern.edu
Address: Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA

3916278e: New Phytol., 875-887(2013)
Link: [ PubMED ] [ DOI ]
Title: The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus.
Author: Shah,F., Rineau,F., Canback,B., Johansson,T., Tunlid,A.
Abstract:
Proteins contribute to a major part of the organic nitrogen (N) in forest soils. This N is mobilized and becomes available to trees as a result of the depolymerizing activities of symbiotic ectomycorrhizal fungi. The mechanisms by which these fungi depolymerize proteins and assimilate the released N are poorly characterized. Biochemical analysis and transcriptome profiling were performed to examine the proteolytic machinery and the uptake system of the ectomycorrhizal basidiomycete Paxillus involutus during the assimilation of organic N from various protein sources and extracts of organic matter. All substrates induced secretion of peptidase activity with an acidic pH optiμm, mostly contributed by aspartic peptidases. The peptidase activity was transiently repressed by ammonium. Transcriptional analysis revealed a large number of extracellular endo- and exopeptidases. The expression levels of these peptidases were regulated in parallel with transporters and enzymes involved in the assimilation and metabolism of the released peptides and amino acids. For the first time the molecular components of the protein degradation pathways of an ectomycorrhizal fungus are described. The data suggest that the transcripts encoding these components are regulated in response to the chemical properties and the availability of the protein substrates.

Address: Department of Biology, Microbial Ecology Group, Lund University, Ecology Building, SE-223 62, Lund, Sweden

3916279e: J.Clin.Periodontol., 907-915(2013)
Link: [ PubMED ] [ DOI ]
Title: Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anti-citrullinated peptide antibody titres.
Author: Lappin,D.F., Apatzidou,D., Quirke,A.M., Oliver-Bell,J., Butcher,J.P., Kinane,D.F., Riggio,M.P., Venables,P., McInnes,I.B., Culshaw,S.
Abstract:
BACKGROUND: Anti-citrullinated protein antibody (ACPA) responses may precede clinical onset of rheumatoid arthritis. Porphyromonas gingivalis peptidylarginine deiminase can citrullinate proteins possibly inducing autoimmunity in susceptible individuals. AIM: To determine whether periodontitis, carriage of P. gingivalis, smoking and periodontal therapy influence ACPA titres. METHODS: Serum and plaque samples were collected from 39 periodontitis patients before and after non-surgical periodontal treatment, and from 36 healthy subjects. Carriage of P. gingivalis was determined by PCR of plaque DNA. ACPA was determined by anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assay (ELISA). Anti-P. gingivalis titres were determined by ELISA. RESULTS: Untreated periodontitis patients had higher anti-CCP antibody titres than healthy controls [three patients (8%) greater than manufacturer suggested assay diagnostic threshold (5 Assay Units/AU) versus none (0%); mean +/- SEM: 1.37 +/- 0.23 versus 0.40 +/- 0.10 AU, p < 0.0001]. Periodontitis patients who smoked demonstrated lower anti-P. gingivalis (15956 +/- 4385 versus 2512 +/- 1290 Units/ml, p < 0.05), but similar anti-CCP than non-smoking periodontitis patients (smokers: 1.31 +/- 0.35; non-smokers: 1.41 +/- 0.32 AU). Healthy smokers demonstrated elevated anti-CCP titres (0.75 +/- 0.19 AU), at levels between healthy non-smokers (0.15 +/- 0.05 AU) and non-smoker periodontitis patients. Six months after periodontal treatment, there were significant reductions in anti-CCP (non-smokers p < 0.05) and anti-P. gingivalis (all participants p < 0.01). CONCLUSION: In subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.

Address: Infection and Immunity, University of Glasgow Dental School, School of Medicine, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, UK

3916280e: J.Ren.Care, 128-139(2013)
Link: [ PubMED ] [ DOI ]
Title: Clinical utility of biomarkers in chronic kidney disease and chronic heart failure.
Author: Zachariah,D., Olechowski,B., Kalra,P.R.
Abstract:
Biomarkers have an increasingly important clinical role in managing patients with heart failure as well as those with kidney disease, both common conditions with generally poor prognostic outcomes and huge impacts on healthcare economics. For patients with chronic heart failure, biomarkers have become centre place in streamlining diagnostic pathways as well as identifying those with worse prognosis. There is much interest in the role for biomarkers in identifying patients at risk of acute kidney injury, although a number of these currently remain as research tools or are in the early stages of evaluation in clinical practice. Patients with cardiorenal syndrome represent a particular challenge to the clinician, and recent studies have suggested a valuable clinical role for certain biomarkers in this setting, either on their own or in combination. This paper will focus on biomarkers with a current clinical role in patients with cardiorenal disease (natriuretic peptides and neutrophil gelatinase-associated lipocalin), although brief reference will be made to other biomarkers with potential future application.

E-Mail: donahez@hotmail.com
Address: Department of Cardiology, Queen Alexandra Hospital, Portsmouth Hospitals NHS Trust, Portsmouth, Hampshire, UK

3916282e: Ren.Fail., 1204-1209(2013)
Link: [ PubMED ] [ DOI ]
Title: Hypertension, fluid overload and micro inflammation are associated with left ventricular hypertrophy in maintenance hemodialysis patients.
Author: Xu,Y., Chen,Y., Li,D., Li,J., Liu,X., Cui,C., Yu,C.
Abstract:
This cross-sectional study aims to identify the potential risk factors of left ventricular hypertrophy (LVH) in hemodialysis (HD) patients. Echocardiography, anthropometric measurements and biochemical analyses were performed for 112 HD patients. In univariate analysis, body mass index, systolic blood pressure, diastolic blood pressure, glycosylated hemoglobin, glycated albumin, high sensitivity C-reactive protein (hs-CRP), cardiac troponin T (cTnT), amino-terminal pro-B-natriuretic peptide (NT-proBNP) and carotid artery intima-media thickness were positively correlated with left ventricular mass index (LVMI); pre-albumin, serum creatinine, left ventricular ejection fraction (LVEF) and fractional shortening were negatively correlated with LVMI. Linear regression analysis showed systolic blood pressure, NT-proBNP and LVEF were independently associated with LVMI. According to a binary logistic regression model, higher systolic blood pressure, NT-proBNP and hs-CRP levels showed independent correlation with LVH. Receiver operator characteristic curves analysis showed the associations between NT-proBNP and LVH more closely than hs-CRP and cTnT. The area under the curve for NT-proBNP, hs-CRP and cTnT was 0.762 (95% CI: 0.660-0.864, p < 0.001), 0.734 (95% CI: 0.624-0.844, p < 0.001) and 0.677 (95% CI: 0.563-0.790, p = 0.004), respectively. These data support the main conclusions: hypertension, fluid overload and micro inflammation are associated with LVH in maintenance HD patients. It demonstrates traditional and nontraditional risk factors all play important roles in the development of LVH.

Address: Department of Nephrology, Tongji Hospital, Tongji University , Shanghai , China

3916287e: J.Mol.Neurosci., 503-513(2013)
Link: [ PubMED ] [ DOI ]
Title: Antiproliferative Effects of PACAP and VIP in Serum-Starved Glioma Cells.
Author: D'Amico,A.G., Scuderi,S., Saccone,S., Castorina,A., Drago,F., D'Agata,V.
Abstract:
Emerging evidence have suggested that calorie restriction (CR) is a reliable method to decrease cancer development since it produces changes in tumor microenvironment that interfere with cell proliferation, tissue invasion, and formation of metastases. Studies on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in cancer cells indicate that their influence on cell growth is either cell type specific or dependent on culture conditions. Evidence showing the effect of PACAP and VIP in glioma cells grown under conditions mimicking CR are currently unavailable. Therefore, we explored the effects of both PACAP and VIP in C6 glioma cells either grown in a normal growth medium or exposed to serum starvation, to resemble an acute condition of CR. Cell viability, expression of proteins related to cell proliferation (cyclin D1), apoptosis (Bcl2, p53, and cleaved caspase-3), and cell malignancy (GFAP and nestin) were assessed by MTT assay, immunoblot, and immunolocalization, respectively. Results demonstrated that CR significantly decreased cell proliferation, reduced levels of cyclin D1 and Bcl2, and increased the expression of p53 and cleaved caspase-3. Surprisingly, all of these CR-driven effects were further exacerbated by PACAP or VIP treatment. We also found that PACAP or VIP prevented GFAP decrease caused by CR and further reduced the expression of nestin, a prognostic marker of malignancy. In conclusion, these data demonstrate that PACAP and VIP possess antiproliferative properties against glioma cells that depend on the specific culture settings, further supporting the idea that CR might offer new avenues to improve peptide-oriented glioma cancer treatment.

Address: Section of Anatomy and Histology, Department of Bio-Medical Sciences, University of Catania, Via S.Sofia, 87, 95123, Catania, Italy

3916290e: Development(Camb.), 3486-3495(2013)
Link: [ PubMED ] [ DOI ]
Title: The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans.
Author: Masoudi,N., Fancsalszky,L., Pourkarimi,E., Vellai,T., Alexa,A., Remenyi,A., Gartner,A., Mehta,A., Takacs-Vellai,K.
Abstract:
The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis.

Address: Department of Genetics, Eotvos Lorand University, Pazmany Peter stny. 1/C, H-1117 Budapest, Hungary

3916292e: Am.J.Physiol.E, 751-759(2013)
Link: [ PubMED ] [ DOI ]
Title: Hindbrain GLP-1 receptor-mediated suppression of food intake requires a PI3K-dependent decrease in phosphorylation of membrane-bound Akt.
Author: Rupprecht,L.E., Mietlicki-Baase,E.G., Zimmer,D.J., McGrath,L.E., Olivos,D.R., Hayes,M.R.
Abstract:
Glucagon-like peptide-1 (GLP-1) receptors (GLP-1R) expressed in the nucleus tractus solitarius (NTS) are physiologically required for the control of feeding. Recently, NTS GLP-1R-mediated suppression of feeding was shown to occur via a rapid PKA-induced suppression of AMPK and activation of MAPK signaling. Unknown are the additional intracellular signaling pathways that account for the long-term hypophagic effects of GLP-1R activation. Because cAMP/PKA activity can promote PI3K/PIP3-dependent translocation of Akt to the plasma membrane, we hypothesize that hindbrain GLP-1R-mediated control of feeding involves a PI3K-Akt-dependent pathway. Importantly, the novel evidence presented here challenges the dogmatic view that PI3K phosphorylation results in an obligatory activation of Akt and instead supports a growing body of literature showing that activation of cAMP/PKA can inhibit Akt phosphorylation at the plasma membrane. Behavioral data show that inhibition of hindbrain PI3K activity by a fourth icv administration of LY-294002 (3.07 μg) attenuated the food intake- and body weight-suppressive effects of a fourth icv administration of the GLP-1R agonist exendin-4 (0.3 μg) in rats. Hindbrain administration of triciribine (10 μg), an inhibitor of PIP3-dependent translocation of Akt to the cell membrane, also attenuated the intake-suppressive effects of a fourth icv injection of exendin-4. Immunoblot analyses of ex vivo NTS tissue lysates and in vitro GLP-1R-expressing neurons (GT1-7) support the behavioral findings and show that GLP-1R activation decreases phosphorylation of Akt in a time-dependent fashion. Current data reveal the requirement of PI3K activation, PIP3-dependent translocation of Akt to the plasma membrane, and suppression in phosphorylation of membrane-bound Akt to mediate the food intake-suppressive effects of hindbrain GLP-1R activation.

Address: Translational Neuroscience Program, Department of Psychiatry, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania

3916293e: Am.J.Physiol.E, 736-744(2013)
Link: [ PubMED ] [ DOI ]
Title: Protein kinase C-induced activin A switches adrenocortical steroidogenesis to aldosterone by suppressing CYP17A1 expression.
Author: Hofland,J., Steenbergen,J., Hofland,L.J., Van Koetsveld,P.M., Eijken,M., Van Nederveen,F.H., Kazemier,G., De Herder,W.W., Feelders,R.A., De Jong,F.H.
Abstract:
Functional zonation of the adrenal cortex is a consequence of the zone-specific expression of P450c17 (CYP17A1) and its cofactors. Activin and inhibin peptides are differentially produced within the zones of the adrenal cortex and have been implicated in steroidogenic control. In this study, we investigated whether activin and inhibin can function as intermediates in functional zonation of the human adrenal cortex. Activin A suppressed CYP17A1 expression and P450c17 function in adrenocortical cell lines as well as in primary adrenal cell cultures. Inhibin βA-subunit mRNA and activin A protein levels were found to be increased up to 1,900-fold and 49-fold, respectively, after protein kinase C (PKC) stimulation through PMA or angiotensin II in H295R adrenocortical carcinoma cells. This was confirmed in HAC15 cells and for PMA in primary adrenal cell cultures. Both PMA and Ang II decreased CYP17A1 expression in the adrenocortical cell lines, whereas PMA concurrently suppressed CYP17A1 levels in the primary cultures. Inhibition of activin signaling during PKC stimulation through silencing of the inhibin βA-subunit or blocking of the activin type I receptor opposed the PMA-induced downregulation of CYP17A1 expression and P450c17 function. In contrast, PKA stimulation through adrenocorticotrophin or forskolin increased expression of the inhibin α-subunit and βglycan, both of which are antagonists of activin action. These data indicate that activin A acts as a PKC-induced paracrine factor involved in the suppression of CYP17A1 in the zona glomerulosa and can thereby contribute to functional adrenocortical zonation.

Address: Department of Internal Medicine

3916294e: Horm.Behav., 511-518(2013)
Link: [ PubMED ] [ DOI ]
Title: VPAC receptor signaling modulates grouping behavior and social responses to contextual novelty in a gregarious finch: A role for a putative prefrontal cortex homologue.
Author: Kingsbury,M.A., Miller,K.M., Goodson,J.L.
Abstract:
In both mammals and birds, vasoactive intestinal polypeptide (VIP) neurons and fibers are present in virtually every brain area that is important for social behavior. VIP influences aggression in birds, social recognition in rodents, and prolactin secretion in both taxa, but other possible functions in social modulation remain little explored. VIP effects are mediated by VPAC receptors, which bind both VIP and pituitary adenylate cyclase activating peptide. Within the lateral septum and medial bed nucleus of the stria terminalis, VPAC receptors are found at higher densities in gregarious finch species relative to territorial species, suggesting that VPAC receptor activation promotes social contact and/or preference for larger groups. Here we here test this hypothesis in zebra finches (Taeniopygia guttata), and also examine the relevance of VPAC receptors to anxiety-like processes. Intraventricular infusions of the VPAC receptor antagonist, neurotensin6-11 mouseVIP7-28, strongly reduce social contact when animals are tested in a novel environment, and exert sex-specific effects on grouping behavior. Specifically, VPAC receptor antagonism reduces gregariousness in females but increases gregariousness in males. Interestingly, VPAC antagonism in the medial pallium (putative prefrontal cortex homologue) significantly reduces gregariousness in both sexes, suggesting site-specific effects of VIP signaling. However, VPAC antagonism does not modulate novel-familiar social preferences in a familiar environment or general anxiety-like behaviors. The current results suggest that endogenous activation of VPAC receptors promotes social contact under novel environmental conditions, a function that may be accentuated in gregarious species. Moreover, endogenous VIP modulates gregariousness in both males and females.

E-Mail: mk24@indiana.edu
Address: Department of Biology, Indiana University, 1001 East Third Street, Bloomington, IN 47405, USA. Electronic address:

3916295e: ACS Appl.Mater.Interfaces, 8187-8194(2013)
Link: [ PubMED ] [ DOI ]
Title: Zeta potential, contact angles, and AFM imaging of protein conformation adsorbed on hybrid nanocomposite surfaces.
Author: Pinho,A.C., Piedade,A.P.
Abstract:
The sputtering deposition of gold (Au) and poly(tetrafluoroethylene) (PTFE) was used to prepare a nanocomposite hybrid thin film suitable for protein adsorption while maintaining the native conformation of the biological material. The monolithic PTFE and the nanocomposite PTFE/Au thin films, with Au content up to 1 at %, were co-deposited by r.f. magnetron sputtering using argon as a discharge gas and deposited onto 316L stainless steel substrates, the most commonly used steel in biomaterials. The deposited thin films, before and after bovine serum albumin (BSA) adsorption, were thoroughly characterized with special emphasis on the surface properties/characteristics by atomic force microscopy (AFM), zeta potential, and static and dynamic contact angle measurements, in order to assess the relationship between structure and conformational changes. The influence of a pre-adsorbed peptide (RGD) was also evaluated. The nanotopographic and chemical changes induced by the presence of gold in the nanocomposite thin films enable RGD bonding, which is critical for the maintenance of the BSA native conformation after adsorption.

Address: CEMUC-GNM, Department of Mechanical Engineering, University of Coimbra, Rua Luis Reis Santos, 3030-788 Coimbra, Portugal

3916296e: Biochim.Biophys.Acta, 2293-2307(2013)
Link: [ PubMED ] [ DOI ]
Title: Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis.
Author: Greening,D.W., Ji,H., Kapp,E.A., Simpson,R.J.
Abstract:
Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433-451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3-30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1-3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS-MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell-cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.

Address: La Trobe Institute for Molecular Science (LIMS), La Trobe University, Bundoora, Victoria, Australia

3916298e: Acta Gastro-Enterol.Belg., 246-250(2013)
Link:
Title: Increased liver stiffness values in patients with heart failure.
Author: Alegre,F., Herrero,J.I., Inarrairaegui,M., Gavira,J.J., Pujol,C., Montero,A., D'Avola,D., Prieto,J., Sangro,B., Quiroga,J.
Abstract:
BACKGROUND: Liver stiffness has been claimed to be increased in patients with heart failure. AIMS : To determine the magnitude of this increase in liver stiffness, and to clarify whether it is related to the degree of heart failure or not. METHODS : Twenty-six patients were prospectively collected, and divided in groups CHF (those with compensated chronic heart failure) and AHF (those with acute decompensated heart failure). Patients underwent routine blood chemistries, pro-BNP determination, echocardiography and transient elastography during outpatient care (group CHF) or at hospital admission (group AHF). Blood chemistries, pro-BNP and transient elastography were repeated in patients in group AHF before being discharged. RESULTS: Correlation between liver stiffness and pro-BNP levels was statistically significant (Rho = 0.747, p = 0.001). Patients in group CHF had lower values of liver stiffness and pro-BNP when compared with patients in group AHF at admission. Median liver stiffness and pro-BNP values were 6.5 vs 14.4 kPa (p = 0.009) and 1511 vs 3535 pg/ml (p = 0.025) respectively. After clinical compensation, liver stiffness decreased in all patients in group AHF. Liver stiffness was 14.4 kPa at admission and 8.2 kPa at discharge (p = 0.008). Pro-BNP values also decreased from a median of 3535 pg/ml to a median of 1098 pg/ml (p = 0.025). CONCLUSIONS: Patients with heart failure have increased liver stiffness, that appears to be related with the severity of heart failure.

E-Mail: falegre@unav.es
Address: Unidad de Hepatologia. Departamento de Medicina Interna. Clinica Universidad de Navarra, Navarra, Spain

3916299e: Indian J.Biochem.Biophys., 202-209(2013)
Link:
Title: Increased cardiac endothelin-1 and nitric oxide in adriamycin-induced acute cardiotoxicity: protective effect of Ginkgo biloba extract.
Author: El-Boghdady,N.A.
Abstract:
Cardiotoxicity and congestive heart failure are the major factors that limit the use of anti-neoplastic drug adriamycin (ADR). There is increasing experimental evidence that endothelin-1 (ET-1) and nitric oxide (NO) are vasoactive mediators that regulate cardiac performance. The present study was undertaken to investigate the role of ET-1 and NO in ADR-induced acute cardiotoxicity and to evaluate the protective effect of Ginkgo biloba extract (EGb761) in rats. A single dose of ADR (20 mg/kg i.p.) caused a significant increase in the cardiac enzyme activities of aspartate transaminases (AST), lactate dehydrogenase (LDH) and creatine phosphokinase isoenzyme (CK-MB) in the serum of animals. This was accompanied by significant increase in cardiac malondialdehyde (MDA), total antioxidant capacity (TAC), tumor necrosis factor-α (TNF-α), ET-1 and nitrite/nitrate (NOx) levels. On the other hand, reduced glutathione (GSH) was significantly depressed. Histopathological examination of heart tissues showed hyalinization of the myocardium, with interstitial edema and inflammatory exudates. Pre-treatment of the animals with EGb761 (100 mg/kg, orally) 10 days before and 5 days after ADR treatment reversed the cardiac enzyme levels to normal value, decreased cardiac MDA, TAC, TNF-α. ET-1 and NO(x), increased GSH and reversed the histopathological damage induced by ADR. In conclusion, the cardioprotective effects of EGb761 on markers of ADR-induced acute cardiotoxicity appeared to have been mediated by the regulation of inflammatory and vasoactive mediators, as well as the inhibition of membrane lipid peroxidation. Thus, EGb761 may find use as promising adjuvant therapy to ameliorate cardiotoxicity of ADR.

E-Mail: nohaelbogday@yahoo.com
Address: Biochemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt

3916300e: Anticancer Res., 3099-3103(2013)
Link: [ PubMED ]
Title: Effects of the HIF1 inhibitor, echinomycin, on growth and NOTCH signalling in leukaemia cells.
Author: Yonekura,S., Itoh,M., Okuhashi,Y., Takahashi,Y., Ono,A., Nara,N., Tohda,S.
Abstract:
Aim: To examine the effects of echinomycin, a compound that inhibits DNA-binding activity of hypoxia-inducible factor-1 (HIF1), on leukaemia cell growth. MATERIALS AND METHODS: Three acute myeloid leukaemia cell lines and three T-lymphoblastic leukaemia cell lines were cultured with echinomycin. Cell growth, mRNA and protein expression levels were examined by WST-1 assay, reverse-transcription polymerase chain reaction and immunoblotting, respectively. RESULTS: HIF1α protein was expressed in all cell lines under normoxia. Treatment with echinomycin suppressed cell growth and induced apoptosis in association with decreased mRNA expression of HIF1 targets, glucose transporter-1 (GLUT1) and B-cell CLL/lymphoma-2 (BCL2). Echinomycin also suppressed the protein expression of NOTCH1, cleaved NOTCH1, v-myc myelocytomatosis viral oncogene homolog (MYC), v-akt murine thymoma viral oncogene homolog-1 (AKT), phosphorylated AKT, mechanistic target of rapamycin (mTOR), and phosphorylated mTOR and increased that of cleaved caspase-3 in some cell lines. CONCLUSION: Echinomycin suppresses leukaemia cell growth in association with reduced NOTCH1 expression. This is the first report to show that HIF inhibitor treatment suppresses NOTCH1 signalling. HIF inhibitors could be novel candidates for a molecular-targeted therapy against leukaemia.

Address: Department of Laboratory Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-Ku, Tokyo 113-8519, Japan

3916301e: Anticancer Res., 3015-3019(2013)
Link:
Title: Correlation of immunohistopathological expression of somatostatin receptor-2 in breast cancer and tumor detection with 68Ga-DOTATOC and 18F-FDG PET imaging in an animal model.
Author: Chereau,E., Durand,L., Frati,A., Prignon,A., Talbot,J.N., Rouzier,R.
Abstract:
BACKGROUND: Fludeoxyglucose positron emission topography (18F-FDG PET) is insufficiently sensitive at detecting small or low-grade breast tumors. The characterization of somatostatin receptors (SSTR) in tumors and the development of (68)Ga-DOTATOC PET for imaging could be of interest. The aim of this study was to validate an animal model expressing SSTR2 and to correlate the immunohistochemical (IHC) analysis with 18F-FDG and (68)Ga-DOTATOC uptake in vivo. MATERIALS AND METHODS: Ten nude mice were xenografted with the ZR-75-1 breast tumor cell line. Imaging was performed with (68)Ga-DOTATOC and 18F-FDG and correlated to IHC analysis of SSTR2. RESULTS: IHC analyses showed that the tumors expressed SSTR2. On PET imaging, the tumors were barely visible with 18F-FDG, whereas with (68)Ga-DOTATOC, specific two-fold higher uptake was observed (p<0.005). CONCLUSION: Our results suggest that (68)Ga-DOTATOC PET could be used for detection of breast tumors not detected with 18F-FDG. SSTR2 status should be assessed to allow for individual treatment.

E-Mail: elisabeth.chereau@gmail.com
Address: Department of Surgical Oncology, Institut Paoli Calmettes, 232 Boulevard de Sainte-Marguerite 13009 Marseille, France

3916303e: Plant Cell, 2699-2713(2013)
Link: [ PubMED ] [ DOI ]
Title: Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis.
Author: Hsu,F.C., Chou,M.Y., Chou,S.J., Li,Y.R., Peng,H.P., Shih,M.C.
Abstract:
Transcriptional control plays an important role in regulating submergence responses in plants. Although numerous genes are highly induced during hypoxia, their individual roles in hypoxic responses are still poorly understood. Here, we found that expression of genes that encode members of the WRKY transcription factor family was rapidly and strongly induced upon submergence in Arabidopsis thaliana, and this induction correlated with induction of a large portion of innate immunity marker genes. Furthermore, prior submergence treatment conferred higher resistance to the bacterial pathogen Pseudomonas syringae in Arabidopsis. Among the WRKY genes tested, WRKY22 had the highest level of induction during the early stages of submergence. Compared with the wild type, WRKY22 T-DNA insertion mutants wrky22-1 and wrky22-2 had lower disease resistance and lower induction of innate immunity markers, such as FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1) and WRKY53, after submergence. Furthermore, transcriptomic analyses of wrky22-2 and chromatin immunoprecipitation identified several potential targets of WRKY22, which included genes encoding a TIR domain-containing protein, a plant peptide hormone, and many OLIGO PEPTIDE TRANSPORTER genes, all of which may lead to induction of innate immunity. In conclusion, we propose that submergence triggers innate immunity in Arabidopsis via WRKY22, a response that may protect against a higher probability of pathogen infection either during or after flooding.

Address: Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan

3916306e: Am.J.Primatol., 1196-1208(2013)
Link: [ PubMED ] [ DOI ]
Title: Ecology of reproduction in Sanje mangabeys (Cercocebus sanjei): Dietary strategies and energetic condition during a high fruit period.
Author: McCabe,G.M., Fernandez,D., Ehardt,C.L.
Abstract:
The ability to increase energy storage when food is abundant for later use during late gestation and early lactation is often considered the primary benefit of the capital breeding strategy (clustering conceptions during high food periods, HFP) that promotes reproductive success among females living in unpredictable environments. Capital breeding, however, may also enable preconceptive females to increase hormone production for ovulation, which has been linked to energetic condition in capital breeders, and/or allow females entering the subsequent HFP to increase their energetic condition in order to continue nursing unweaned infants. Here, we investigate whether capital breeding provides these additional benefits in 16 female Sanje mangabeys (Cercocebus sanjei) and determine the dietary strategies used to increase energetic condition (measured by urinary C-peptide: UCP) during the HFP. Fecal estradiol (fE2 ) and UCP were negatively correlated with number of cycles before conception (r = -0.591, r = -0.646, P < 0.01) and were highest in conceptive cycles. Both peri-conceptive (preconception and early gestation) and non-peri-conceptive (lactation) females increased energetic condition over the HFP (r = 0.612, r = 0.583, P < 0.001) by increasing dietary fat (r = 0.619, r = 0.703, P < 0.001) and, for non-peri-conceptive females, protein (r = 0.437, P < 0.001). Feeding intake rate (FIR) and time spent foraging and feeding did not change over the HFP; however, non-peri-conceptive females exhibited a faster FIR compared to peri-conceptive females (t = -2.324, P < 0.05), consuming almost twice as much food per unit time. The results of this study confirm that Sanje mangabeys benefit in multiple phases of the reproductive cycle by using capital breeding, which may explain how this strategy promotes female reproductive success.

Address: Department of Anthropology, University of Calgary, Calgary, AB, Canada

3916310e: Mol.Cell.Biol., 3826-3834(2013)
Link: [ PubMED ] [ DOI ]
Title: Control of energy balance by hypothalamic gene circuitry involving two nuclear receptors, neuron-derived orphan receptor 1 and glucocorticoid receptor.
Author: Kim,S.G., Lee,B., Kim,D.H., Kim,J., Lee,S., Lee,S.K., Lee,J.W.
Abstract:
Nuclear receptors (NRs) regulate diverse physiological processes, including the central nervous system control of energy balance. However, the molecular mechanisms for the central actions of NRs in energy balance remain relatively poorly defined. Here we report a hypothalamic gene network involving two NRs, neuron-derived orphan receptor 1 (NOR1) and glucocorticoid receptor (GR), which directs the regulated expression of orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) in response to peripheral signals. Our results suggest that the anorexigenic signal leptin induces NOR1 expression likely via the transcription factor cyclic AMP response element-binding protein (CREB), while the orexigenic signal glucocorticoid mobilizes GR to inhibit NOR1 expression by antagonizing the action of CREB. Also, NOR1 suppresses glucocorticoid-dependent expression of AgRP and NPY. Consistently, relative to wild-type mice, NOR1-null mice showed significantly higher levels of AgRP and NPY and were less responsive to leptin in decreasing the expression of AgRP and NPY. These results identify mutual antagonism between NOR1 and GR to be a key rheostat for peripheral metabolic signals to centrally control energy balance.

Address: Neuroscience Section, Pape Family Pediatric Research Institute, Department of Pediatrics

3916311e: Dev.Genes Evol., 389-394(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of a tyrosinase gene potentially involved in early larval shell biogenesis of the Pacific oyster Crassostrea gigas.
Author: Huan,P., Liu,G., Wang,H., Liu,B.
Abstract:
The larval shell emerges early in embryogenesis of mollusks, but the detailed mechanisms of its biogenesis remain to be determined. In this study, we cloned a tyrosinase gene (cgi-tyr1) that potentially functioned in larval shell biogenesis from the Pacific oyster Crassostrea gigas, a worldwide bivalve species. Sequence analysis of cgi-tyr1 revealed that it had typical copper-binding domains and a signal peptide. Through whole mount in situ hybridization and an electron scanning microscopic observation, we detected the expression of cgi-tyr1 firstly in the saddle-shaped shell field in trochophores, indicating that cgi-tyr1 might participate in the biogenesis of the initial non-calcified shell of trochophores. In the following development to early D-veliger, cells in the central region of shell field exhibited no detectable cgi-tyr1 expression, and cgi-tyr1 expression was sustained only in the edge of the shell field and the hinge region, indicating that cgi-tyr1 might function fundamentally in shell growth from trochophore to early D-veliger. Unexpectedly, cgi-tyr1 expression was not detected after the D-veliger stage. This indicated that other molecules might function in later shell development. Our results suggested a role for a tyrosinase gene that specifically functioned in the initial phase of the larval shell biogenesis of C. gigas. This work would shed light on future studies on larval shell development and might be helpful to understand how the molluscan shell emerged during evolution.

Address: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, 266071, China

3916313e: BMJ Case Rep., 008815-008815(2013)
Link: [ PubMED ] [ DOI ]
Title: Upper gastrointestinal bleeding in cirrhosis: varix or no varix?
Author: Ferreira,A.O., Sousa,H.T., Brito,J., Rosa,L.
Abstract:
Upper gastrointestinal bleeding from variceal origin is a frequent complication in the cirrhotic population. Duodenal variceal haemorrhage, however, is infrequent and the endoscopic management of such lesions is not straightforward. Non-endoscopic options include vasoactive drugs, transjugular intrahepatic portosystemic shunt (TIPS), transvenous obliteration and surgery as rescue therapy. We present a patient with Child-Pugh A hepatitis C virus-cirrhosis with acute bleeding from a duodenal varix. It was managed with elastic band ligation but late rebleeding occurred after 6 weeks. Gastroduodenoscopy revealed active bleeding from the ligation eschar. Band ligation and sclerosis were attempted but unsuccessful. Terlipressin was started and the patient referred for TIPS. Surprisingly, angiography showed a normal hepatic vein pressure gradient; therefore, TIPS was not performed. Haemorrhage ceased with medical treatment alone. The patient remained stable and was discharged after 10 days, being currently under evaluation for hepatitis C therapy.

E-Mail: alex.fsof@gmail.com
Address: Department of Gastroenterology and Hepatology, Centro Hospitalar Barlavento Algarvio, Portimao, Portugal

3916314e: Am.J.Clin.Pathol., 225-230(2013)
Link: [ PubMED ] [ DOI ]
Title: HepPar-1 expression in primary lung adenocarcinoma.
Author: Yousem,S.A., Lale,S., Dacic,S.
Abstract:
OBJECTIVES: To study a subset of lung adenocarcinomas with aberrant HepPar-1 antigen expression. METHODS: We performed a clinicopathologic review of 16 cases. RESULTS: Of 241 primary lung adenocarcinomas tested for expression of the HepPar-1 antigen, 16 (6.6%) were positive. The cases occurred in older smokers with an equal sex distribution. Morphologically, these pulmonary adenocarcinomas had an acinar-predominant architecture, and prominent cribriform growth, with HepPar-1-expressing tumor cells being uniformly columnar with abundant bipolar lightly eosinophilic cytoplasm. HepPar-1 was coexpressed with thyroid transcription factor 1 in 94% of cases. No EGFR mutations were identified, while 31% of cases were positive for KRAS codon 12 mutations. CONCLUSIONS: Although aberrant HepPar-1 expression was frequently seen in tumors with visceral pleural and angiolymphatic invasion, its prognostic significance is uncertain.

E-Mail: yousemsa@upmc.edu
Address: Department of Pathology, UPMC Presbyterian Shadyside, Presbyterian Campus-A610, 200 Lothrop St, Pittsburgh, PA 15213-2582, USA

3916315e: Am.J.Clin.Pathol., 177-183(2013)
Link: [ PubMED ] [ DOI ]
Title: BRAF V600E mutation analysis simplifies the testing algorithm for Lynch syndrome.
Author: Jin,M., Hampel,H., Zhou,X., Schunemann,L., Yearsley,M., Frankel,W.L.
Abstract:
OBJECTIVES: To evaluate our experience of adding reflex BRAF mutation analysis following mismatch repair (MMR) protein staining in the test algorithm for Lynch syndrome (LS), the most common inherited predisposition to colorectal cancer (CRC). METHODS: Since January 1, 2009, BRAF V600E mutation analysis has been performed at our institution for all newly diagnosed CRCs with absent MLH1 and PMS2 proteins. RESULTS: Ninety (22%) of 412 patients with CRC had at least 1 MMR absent (65 had MLH1 and PMS2 absent and 25 had other stain(s) absent). BRAF mutation was found in 36 (55%) of 65. Fifty-four (13%) of 412 patients required follow-up after addition of BRAF analysis compared with 90 who would have required follow-up without BRAF analysis. CONCLUSIONS: The addition of reflex BRAF mutation testing in CRCs with absent MLH1 and PMS2 reduced the number of patient contacts by 40% and simplified the genetic testing for LS, leading to cost and time savings.

Address: Department of Pathology, Ohio State University Wexner Medical Center, Columbus, OH 43210, USA

3916316e: J.Physiol.(London), 5071-5081(2013)
Link: [ PubMED ] [ DOI ]
Title: Permeability and contractile responses of collecting lymphatic vessels elicited by atrial and brain natriuretic peptides.
Author: Scallan,J.P., Davis,M.J., Huxley,V.H.
Abstract:
Atrial and brain natriuretic peptides (ANP and BNP, respectively) are cardiac hormones released into the bloodstream in response to hypervolaemia or fluid shifts to the central circulation. The actions of both peptides include natriuresis and diuresis, a decrease in systemic blood pressure, and inhibition of the renin-angiotensin-aldosterone system. Further, ANP and BNP elicit increases in blood microvessel permeability sufficient to cause protein and fluid extravasation into the interstitium to reduce the vascular volume. Given the importance of the lymphatic vasculature in maintaining fluid balance, we tested the hypothesis that ANP or BNP (100 nm) would likewise elevate lymphatic permeability (Ps) to serum albumin. Using a microfluorometric technique adapted to in vivo lymphatic vessels, we determined that rat mesenteric collecting lymphatic Ps to rat serum albumin increased by 2.0 +/- 0.4-fold (P = 0.01, n = 7) and 2.7 +/- 0.8-fold (P = 0.07, n = 7) with ANP and BNP, respectively. In addition to measuring Ps responses, we observed changes in spontaneous contraction amplitude and frequency from the albumin flux tracings in vivo. Notably, ANP abolished spontaneous contraction amplitude (P = 0.005) and frequency (P = 0.006), while BNP augmented both parameters by approximately 2-fold (P < 0.01 each). These effects of ANP and BNP on contractile function were examined further by using an in vitro assay. In aggregate, these data support the theory that an increase in collecting lymphatic permeability opposes the absorptive function of the lymphatic capillaries, and aids in the retention of protein and fluid in the interstitial space to counteract volume expansion.

E-Mail: huxleyv@health.missouri.edu
Address: V. H. Huxley: Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, 1 Hospital Dr., MA415, Columbia, MO 65212, USA

3916317e: J.Histochem.Cytochem., 793-801(2013)
Link: [ PubMED ] [ DOI ]
Title: Variations in Both TG1 and TG2 Isozyme-specific In Situ Activities and Protein Expressions during Mouse Embryonic Development.
Author: Itoh,M., Tatsukawa,H., Eun-Seo,L., Yamanishi,K., Kojima,S., Hitomi,K.
Abstract:
Transglutaminase (TG) is a family of enzymes that catalyzes cross-linking reactions among proteins. Using fluorescent-labeled highly reactive substrate peptides, we recently developed a system to visualize isozyme-specific in situ enzymatic activity. In the present study, we investigated the in situ activities of TG1 (skin-type) and TG2 (tissue-type) using whole mouse sections of various embryonic developmental stages and neonates. In each case, we also successfully used immunostaining of identical whole mouse sections for protein expression after detection of enzymatic activities. In general, the enzymatic activity was correlated with TG protein expression. However, in some tissues, TG protein expression patterns, which were inconsistent with the enzymatic activities, suggested that inactive TGs were produced possibly by self cross-linking or other modifications. Our method allowed us to simultaneously observe developmental variations in both TG isozyme-specific activities and protein levels in mouse embryonic and neonate tissues.

Address: Graduate School of Bioagricultural Sciences (MI), Nagoya University, Nagoya, Japan

I. Immunology

3915453i: Immunogenetics, 811-821(2013)
Link: [ PubMED ] [ DOI ]
Title: Dimorphisms of the proteasome subunit β type 8 gene (PSMB8) of ectothermic tetrapods originated in multiple independent evolutionary events.
Author: Huang,C.H., Tanaka,Y., Fujito,N.T., Nonaka,M.
Abstract:
The proteasome subunit β type 8 gene (PSMB8) encodes one of the β subunits of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex class I molecules. Dimorphic alleles of the PSMB8 gene, termed A and F types, based on the deduced 31st amino acid residue of the mature protein have been reported from various vertebrates. Phylogenetic analysis revealed the presence of dichotomous ancient lineages, one comprising the F-type PSMB8 of basal ray-finned fishes, and the other comprising the A-type PSMB8 of these animals and both the F- and A-type PSMB8 of Xenopus and acanthopterygians, indicating that evolutionary history of the PSMB8 dimorphism was not straightforward. We analyzed the PSMB8 gene of five reptile and one amphibian species and found both the A and F types from all six. Phylogenetic analysis indicated that the PSMB8 F type was apparently regenerated from the PSMB8 A type at least five times independently during tetrapod evolution. Genomic typing of wild individuals of geckos and newts indicated that the frequencies of the A- and F-type alleles are not highly biased in these species. Phylogenetic analysis of each exon of the reptile PSMB8 gene suggested interallelic sequence homogenization as a possible evolutionary mechanism for the apparent recurrent regeneration of PSMB8 dimorphism in tetrapods. An extremely strong balancing selection acting on PSMB8 dimorphism was implicated in an unprecedented pattern of allele evolution.

Address: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

3915468i: J.Immunol., 3939-3947(2013)
Link: [ PubMED ] [ DOI ]
Title: Tapasin Facilitation of Natural HLA-A and -B Allomorphs Is Strongly Influenced by Peptide Length, Depends on Stability, and Separates Closely Related Allomorphs.
Author: Geironson,L., Thuring,C., Harndahl,M., Rasmussen,M., Buus,S., Roder,G., Paulsson,K.M.
Abstract:
Despite an abundance of peptides inside a cell, only a small fraction is ultimately presented by HLA-I on the cell surface. The presented peptides have HLA-I allomorph-specific motifs and are restricted in length. So far, detailed length studies have been limited to few allomorphs. Peptide-HLA-I (pHLA-I) complexes of different allomorphs are qualitatively and quantitatively influenced by tapasin to different degrees, but again, its effect has only been investigated for a small number of HLA-I allomorphs. Although both peptide length and tapasin dependence are known to be important for HLA-I peptide presentation, the relationship between them has never been studied. In this study, we used random peptide libraries from 7- to 13-mers and studied binding in the presence and absence of a recombinant truncated form of tapasin. The data show that HLA-I allomorphs are differentially affected by tapasin, different lengths of peptides generated different amounts of pHLA-I complexes, and HLA-A allomorphs are generally less restricted than HLA-B allomorphs to peptides of the classical length of 8-10 aa. We also demonstrate that tapasin facilitation varies for different peptide lengths, and that the correlation between high degree of tapasin facilitation and low stability is valid for different random peptide mixes of specific lengths. In conclusion, these data show that tapasin has specificity for the combination of peptide length and HLA-I allomorph, and suggest that tapasin promotes formation of pHLA-I complexes with high on and off rates, an important intermediary step in the HLA-I maturation process.

Address: Department of Experimental Medical Science, Immunology Section, Lund University, 221 84 Lund, Sweden

3915475i: Infect.Immun., 4121-4127(2013)
Link: [ PubMED ] [ DOI ]
Title: Characterization of sil in Invasive Group A and G Streptococci: Antibodies against Bacterial Pheromone Peptide SilCR Result in Severe Infection.
Author: Michael-Gayego,A., Dan-Goor,M., Jaffe,J., Hidalgo-Grass,C., Moses,A.E.
Abstract:
Group G β-hemolytic streptococcus (GGS) strains cause severe invasive infections, mostly in patients with comorbidities. GGS is known to possess virulence factors similar to those of its more virulent counterpart group A streptococcus (GAS). A streptococcal invasion locus, sil, was identified in GAS. sil encodes a competence-stimulating peptide named SilCR that activates bacterial quorum sensing and has the ability to attenuate virulence in GAS infections. We found that sil is present in most GGS strains (82%) but in only 25% of GAS strains, with a similar gene arrangement. GGS strains that contained sil expressed the SilCR peptide and secreted it into the growth medium. In a modified murine model of GGS soft tissue infection, GGS grown in the presence of SilCR caused a milder disease than GGS grown in the absence of SilCR. To further study the role of the peptide in bacterial virulence attenuation, we vaccinated mice with SilCR to produce specific anti-SilCR antibodies. Vaccinated mice developed a significantly more severe illness than nonvaccinated mice. Our results indicate that the sil locus is much more prevalent among the less virulent GGS strains than among GAS strains. GGS strains express and secrete SilCR, which has a role in attenuation of virulence in a murine model. We show that the SilCR peptide can protect mice from infection caused by GGS. Furthermore, vaccinated mice that produce specific anti-SilCR antibodies develop a significantly more severe infection. To our knowledge, this is a novel report demonstrating that specific antibodies against a bacterial component cause more severe infection by those bacteria.

Address: Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

3915476i: Infect.Immun., 4139-4148(2013)
Link: [ PubMED ] [ DOI ]
Title: Cellular Response to Trypanosoma cruzi Infection Induces Secretion of Defensin α-1, Which Damages the Flagellum, Neutralizes Trypanosome Motility, and Inhibits Infection.
Author: Johnson,C.A., Rachakonda,G., Kleshchenko,Y.Y., Nde,P.N., Madison,M.N., Pratap,S., Cardenas,T.C., Taylor,C., Lima,M.F., Villalta,F.
Abstract:
Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that colonic epithelial model HCT116 cells respond to Trypanosoma cruzi infection by secreting defensin α-1, which reduces infection. We also report the early effects of defensin α-1 on invasive trypomastigotes that involve damage of the flagellar structure to inhibit parasite motility and reduce cellular infection. Short exposure of defensin α-1 to trypomastigotes shows that defensin α-1 binds to the flagellum, resulting in flagellar membrane and axoneme alterations, followed by breaking of the flagellar membrane connected to the trypanosome body, leading to detachment and release of the parasite flagellum. In addition, defensin α-1 induces a significant reduction in parasite motility in a peptide concentration-dependent manner, which is abrogated by anti-defensin α-1 IgG. Preincubation of trypomastigotes with a concentration of defensin α-1 that inhibits 50% trypanosome motility significantly reduced cellular infection by 80%. Thus, human defensin α-1 is an innate immune molecule that is secreted by HCT116 cells in response to T. cruzi infection, inhibits T. cruzi motility, and plays an important role in reducing cellular infection. This is the first report showing a novel cellular innate immune response to a human parasite by secretion of defensin α-1, which neutralizes the motility of a human parasite to reduce cellular infection. The mode of activity of human defensin α-1 against T. cruzi and its function may provide insights for the development of new antiparasitic strategies.

Address: Department of Microbiology and Immunology, School of Medicine, Meharry Medical College, Nashville, Tennessee, USA

3915477i: Infect.Immun., 4100-4111(2013)
Link: [ PubMED ] [ DOI ]
Title: Relative Contributions of Lipooligosaccharide Inner and Outer Core Modifications to Nontypeable Haemophilus influenzae Pathogenesis.
Author: Morey,P., Viadas,C., Euba,B., Hood,D.W., Barberan,M., Gil,C., Grillo,M.J., Bengoechea,J.A., Garmendia,J.
Abstract:
Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.

Address: Laboratory Microbial Pathogenesis, Fundacion Investigacion Sanitaria Illes Balears (FISIB), CSIC-Govern de les Illes Balears, Bunyola, Spain

3915493i: Fish Shellfish Immunol., 1455-1465(2013)
Link: [ PubMED ] [ DOI ]
Title: A teleostean counterpart of ferritin M subunit from rock bream (Oplegnathus fasciatus): An active constituent in iron chelation and DNA protection against oxidative damage, with a modulated expression upon pathogen stress.
Author: Elvitigala,D.A., Premachandra,H.K., Whang,I., Oh,M.J., Jung,S.J., Park,C.J., Lee,J.
Abstract:
Ferritins are biological iron chelators that can sequestrate excess iron to maintain iron homeostasis in the body. Ferritins basically consist of 2 types of subunits, designated as H and L. However, another new subunit, ferritin "M" which possesses characteristic features of both the H and L subunits, was recently identified in lower vertebrates, mostly in fish. In this study, a ferritin M-like subunit from rock bream (Oplegnathus fasciatus) (RbFerM) was characterized at the molecular level, and its transcriptional profile was analyzed in healthy fish, as well as in pathogen- and mitogen-stimulated fish. Furthermore, its functional properties were evaluated using the recombinant protein. The complete coding sequence of RbFerM was 528 bp in length, encoding a 176-amino acid peptide with a calculated molecular mass of 20 kDa. In silico analysis of RbFerM revealed that it has features similar to both the mammalian ferritin subunits, H and L. Phylogenetic analysis depicted the higher evolutionary proximity of RbFerM with its fish counterparts. Quantitative real time polymerase chain reaction (PCR) analysis detected a ubiquitous transcriptional profile of RbFerM in selected tissues of rock bream, in which more pronounced expression was observed in blood and liver tissues. Significant transcriptional inductions of RbFerM were detected in liver tissues upon lipopolysaccharides (LPS), Edwardsiella tarda, Streptococcus iniae, and rock bream irido virus (RBIV) exposures in time-course immune-challenge experiments. The purified recombinant protein of RbFerM demonstrated detectable iron chelating activity that varied with the temperature. Moreover, the recombinant RbFerM rendered a detectable protection effect against iron (II) and H2O2-mediated DNA damage.

Address: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea

3915631i: Fish Shellfish Immunol., 1369-1379(2013)
Link: [ PubMED ] [ DOI ]
Title: Evidences for the involvement of an invertebrate goose-type lysozyme in disk abalone immunity: Cloning, expression analysis and antimicrobial activity.
Author: Bathige,S.D., Umasuthan,N., Whang,I., Lim,B.S., Jung,H.B., Lee,J.
Abstract:
Lysozymes are ubiquitously distributed enzymes with hydrolytic activity against bacterial peptidoglycan and function to protect organisms from microbial pathogens. In this study, an invertebrate goose-type lysozyme, designated as abLysG, was identified in the disk abalone, Haliotis discus discus. The full-length cDNA of abLysG was 894 bp in length with an open reading frame of 789 bp encoding a polypeptide of 263 amino acids containing a signal peptide and a characteristic soluble lytic transglycosylase domain. Six cysteine residues and two catalytic residues (Glu(142) and Asp(168)) conserved among molluscs were also identified. The 3D homology structural models of abLysG and hen egg white lysozyme had similar conformations of the active sites involved in the binding of substrate. BAC sequence data revealed that the genomic structure of disk abalone g-type lysozyme comprises 7 exons with 6 intervening introns. The deduced amino acid sequence of abLysG shared 45.2-61.6% similarity with those of other molluscs and vertebrates. The TFSEARCH server predicted a variety of transcription factor-binding sites in the 5'-flanking region of the abLysG gene, some of which are involved in transcriptional regulation of the lysozyme gene. abLysG expression was detected in multiple tissues with the highest expression in mantle. Moreover, qPCR analysis of abLysG mRNA expression demonstrated significant up-regulation in gill in response to infection by live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia) and bacterial mimics (LPS and PGN). Expression of the recombinant disk abalone g-type lysozyme in Escherichia coli BL21, demonstrated its bacteriolytic activity against several Gram-negative and Gram-positive bacterial species. Collectively these data suggest that abLysG is an antimicrobial enzyme with a potential role in the disk abalone innate immune system to protect it from bacterial and viral infections.

Address: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea

3915636i: Vaccine, 4736-4743(2013)
Link: [ PubMED ] [ DOI ]
Title: Improving influenza virus backbones by including terminal regions of MDCK-adapted strains on hemagglutinin and neuraminidase gene segments.
Author: Gomila,R.C., Suphaphiphat,P., Judge,C., Spencer,T., Ferrari,A., Wen,Y., Palladino,G., Dormitzer,P.R., Mason,P.W.
Abstract:
Reverse genetics approaches can simplify and accelerate the process of vaccine manufacturing by combining the desired genome segments encoding the surface glycoproteins from influenza strains with genome segments (backbone segments) encoding internal and non-structural proteins from high-growth strains. We have developed three optimized high-growth backbones for use in producing vaccine seed viruses for group A influenza strains. Here we show that we can further enhance the productivity of our three optimized backbones by using chimeric hemagglutinin (HA) and neuraminidase (NA) genome segments containing terminal regions (non-coding regions (NCRs) and coding regions for the signal peptide (SP), transmembrane domain (TMD), and cytoplasmic tail (CT)) from two MDCK-adapted high growth strains (PR8x and Hes) and the sequences encoding the ectodomains of the A/Brisbane/10/2010 (H1N1) HA and NA proteins. Viruses in which both the HA and NA genome segments had the high-growth terminal regions produced higher HA yields than viruses that contained one WT and one chimeric HA or NA genome segment. Studies on our best-performing backbone indicated that the increases in HA yield were also reflected in an increase in HA content in partially purified preparations. Our results show that the use of chimeric HA and NA segments with high-growth backbones is a viable strategy that could improve influenza vaccine manufacturing. Possible mechanisms for the enhancement of HA yield are discussed.

E-Mail: rcgomila@gmail.com
Address: Novartis Vaccines and Diagnostics, 45 Sidney Street, Cambridge, MA 02139, United States. Electronic address:

3915682i: Dev.Comp.Immunol., 694-702(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular cloning and expression of TLR in the Eisenia andrei earthworm.
Author: Skanta,F., Roubalova,R., Dvorak,J., Prochazkova,P., Bilej,M.
Abstract:
Toll-like receptors (TLRs) play an important role in defense responses to pathogens in invertebrates. Here we characterize the first TLR isolated from an oligochaete annelid, namely, Eisenia andrei (EaTLR) and show its expression pattern. The full-length EaTLR cDNA consists of 2615bp encoding a putative protein of 675 amino acids. The predicted amino acid sequence comprises of an extracellular domain containing 31 amino acid signal peptide and seven leucine-rich repeats (LRR), capped with cysteine-rich N- and C-terminal LRRs followed by a transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). TIR domains of twenty individual earthworms were sequenced and the variability suggesting the presence of a high number of TLR genes in the genome of E. andrei was observed. Phylogenetic analysis revealed the highest similarity of EaTLR with polychaete annelid, Capitella teleta and TLRs of mollusks and echinoderms. Finally, the highest constitutive expression of EaTLR was observed in the digestive tract. Gene expression was significantly increased in coelomocytes of E. andrei after the challenge with Gram-positive bacteria.

Address: Institute of Microbiology of the Academy of Sciences of the Czech Republic, v. v. i., Videnska 1083, 142 20 Prague 4, Czech Republic

3915687i: Fish Shellfish Immunol., 1406-1415(2013)
Link: [ PubMED ] [ DOI ]
Title: Genomic characterization, expression analysis, and antimicrobial function of a glyrichin homologue from rock bream, Oplegnathus fasciatus.
Author: Kasthuri,S.R., Wan,Q., Umasuthan,N., Bathige,S.D., Lim,B.S., Jung,H.B., Lee,J., Whang,I.
Abstract:
Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at (22)GFMMGFAVGMAAGAMFGTFSCLR(44). Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rock bream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, E. tarda, and S. iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream.

Address: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea

3915823i: J.Clin.Cell.Immunol., 141-141(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Immunoproteasome Activation During Early Antiviral Response in Mouse Pancreatic β-cells: New Insights into Auto-antigen Generation in Type I Diabetes?
Author: Freudenburg,W., Gautam,M., Chakraborty,P., James,J., Richards,J., Salvatori,A.S., Baldwin,A., Schriewer,J., Buller,R.M., Corbett,J.A., Skowyra,D.
Abstract:
Type 1 diabetes results from autoimmune destruction of the insulin producing pancreatic β-cells. The immunoproteasome, a version of the proteasome that collaborates with the 11S/PA28 activator to generate immunogenic peptides for presentation by MHC class I molecules, has long been implicated in the onset of the disease, but little is known about immunoproteasome function and regulation in pancreatic β-cells. Interesting insight into these issues comes from a recent analysis of the immunoproteasome expressed in pancreatic β-cells during early antiviral defenses mediated by interferon β (IFNβ), a type I IFN implicated in the induction of the diabetic state in human and animal models. Using mouse islets and the MIN6 insulinoma cell line, Freudenburg et al. found that IFNβ stimulates expression of the immunoproteasome and the 11S/PA28 activator in a manner fundamentally similar to the classic immuno-inducer IFNγ, with similar timing of mRNA accumulation and decline; similar transcriptional activation mediated primarily by the IRF1 and similar mRNA and protein levels. Furthermore, neither IFNβ nor IFNγ altered the expression of regular proteolytic subunits or prevented their incorporation into proteolytic cores. As a result, immunoproteasomes had stochastic combinations of immune and regular proteolytic sites, an arrangement that would likely increase the probability with which unique immunogenic peptides are produced. However, immunoproteasomes were activated by the 11S/PA28 only under conditions of ATP depletion. A mechanism that prevents the activation of immunoproteasome at high ATP levels has not been reported before and could have a major regulatory significance, as it could suppress the generation of immunogenic peptides as cell accumulate immunoproteasome and 11S/PA28, and activate antigen processing only when ATP levels drop. We discuss implications of these new findings on the link between early antiviral response and the onset of type 1 diabetes.

Address: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America

3915829i: J.Immunol., 3297-3307(2013)
Link: [ PubMED ] [ DOI ]
Title: Persistence of the bacterial pathogen Granulibacter bethesdensis in chronic granulomatous disease monocytes and macrophages lacking a functional NADPH oxidase.
Author: Chu,J., Song,H.H., Zarember,K.A., Mills,T.A., Gallin,J.I.
Abstract:
Granulibacter bethesdensis is a Gram-negative pathogen in patients with chronic granulomatous disease (CGD), a deficiency in the phagocyte NADPH oxidase. Repeated isolation of genetically identical strains from the same patient over years, and prolonged waxing and waning seropositivity in some subjects, raises the possibility of long-term persistence. G. bethesdensis resists killing by serum, CGD polymorphonuclear leukocytes (PMN), and antimicrobial peptides, indicating resistance to nonoxidative killing mechanisms. Although G. bethesdensis extends the survival of PMN, persistent intracellular bacterial survival might rely on longer-lived macrophages and their precursor monocytes. Therefore, we examined phagocytic killing by primary human monocytes and monocyte-derived macrophages (MDM). Cells from both normal and CGD subjects internalized G. bethesdensis similarly. G. bethesdensis stimulated superoxide production in normal monocytes, but to a lesser degree than in normal PMN. Normal but not CGD monocytes and MDM killed G. bethesdensis and required in vitro treatment with IFN-γ to maintain this killing effect. Although in vitro IFN-γ did not enhance G. bethesdensis killing in CGD monocytes, it restricted growth in proportion to CGD PMN residual superoxide production, providing a potential method to identify patients responsive to IFN-γ therapy. In IFN-γ-treated CGD MDM, G. bethesdensis persisted for the duration of the study (7 d) without decreasing viability of the host cells. These results indicate that G. bethesdensis is highly resistant to oxygen-independent microbicides of myeloid cells, requires an intact NADPH oxidase for clearance, and can persist long-term in CGD mononuclear phagocytes, most likely relating to the persistence of this microorganism in infected CGD patients.

Address: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

3915852i: Vaccine, 4923-4930(2013)
Link: [ PubMED ] [ DOI ]
Title: Antigenicity and immunogenicity of a novel chimeric peptide antigen based on the P. vivax circumsporozoite protein.
Author: Cespedes,N., Arevalo-Herrera,M., Felger,I., Reed,S., Kajava,A.V., Corradin,G., Herrera,S.
Abstract:
BACKGROUND: Plasmodium vivax circumsporozoite (PvCS) protein is a major sporozoite surface antigen involved in parasite invasion of hepatocytes and is currently being considered as vaccine candidate. PvCS contains a dimorphic central repetitive fragment flanked by conserved regions that contain functional domains. METHODS: We have developed a chimeric 137-mer synthetic polypeptide (PvCS-NRC) that includes the conserved region I and region II-plus and the two natural repeat variants known as VK210 and VK247. The antigenicity of PvCS-NRC was tested using human sera from PNG and Colombia endemic areas and its immunogenicity was confirmed in mice with different genetic backgrounds, the polypeptide formulated either in Alum or GLA-SE adjuvants was assessed in inbred C3H, CB6F1 and outbred ICR mice, whereas a formulation in Montanide ISA51 was tested in C3H mice. RESULTS: Antigenicity studies indicated that the chimeric peptide is recognized by a high proportion (60-70%) of residents of malaria-endemic areas. Peptides formulated with either GLA-SE or Montanide ISA51 adjuvants induced stronger antibody responses as compared with the Alum formulation. Sera from immunized mice as well as antigen-specific affinity purified human IgG antibodies reacted with sporozoite preparations in immunofluorescence and Western blot assays, and displayed strong in vitro inhibition of sporozoite invasion (ISI) into hepatoma cells. CONCLUSIONS: The polypeptide was recognized at high prevalence when tested against naturally induced human antibodies and was able to induce significant immunogenicity in mice. Additionally, specific antibodies were able to recognize sporozoites and were able to block sporozoite invasion in vitro. Further evaluation of this chimeric protein construct in preclinical phase e.g. in Aotus monkeys in order to assess the humoral and cellular immune responses as well as protective efficacy against parasite challenge of the vaccine candidate must be conducted.

Address: Malaria Vaccine and Drug Development Center (MVDC), Carrera 37 2Bis No. 5E-08, A.A. 25574 Cali, Colombia

3915872i: Clin.Exp.Immunol., 414-423(2013)
Link: [ PubMED ] [ DOI ]
Title: Dimorphic HLA-B signal peptides differentially influence HLA-E- and natural killer cell-mediated cytolysis of HIV-1-infected target cells.
Author: Merino,A.M., Sabbaj,S., Easlick,J., Goepfert,P., Kaslow,R.A., Tang,J.
Abstract:
As a mechanism of self-protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA-E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK-mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2-methionine (P2-M) having higher HLA-E binding affinity than P2-threonine (P2-T). All HLA-A and HLA-C molecules carry P2-M, whereas HLA-B products have either P2-M or P2-T. Epidemiological evidence suggests that P2-M is unfavourable in the context of HIV-1 infection, being associated with accelerated acquisition of HIV-1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK-mediated killing of CD4+ T cells and monocyte-derived macrophages infected with two laboratory-adapted HIV-1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA-B P2 genotypes (M/M, M/T and T/T), NK-mediated cytolysis was elevated consistently for P2-T in a dose-dependent manner for all cell and virus combinations tested (P = 0.008-0.03). Treatment of target cells with an anti-HLA-E monoclonal antibody restored NK-mediated cytolysis of cells expressing P2-M. Observations on cell lysis were also substantiated by measurements of HIV-1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti-HIV-1 function mediated by NK cells is compromised by P2-M, corroborating the association of HLA-B genotype encoding P2-M with accelerated HIV-1 acquisition.

Address: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

3915922i: Vaccine, 4802-4811(2013)
Link: [ PubMED ] [ DOI ]
Title: Vaccination with Flt3L-induced CD8α+ dendritic cells prevents CD4+ T helper cell-mediated experimental autoimmune myocarditis.
Author: Valaperti,A., Nishii,M., Germano,D., Liu,P.P., Eriksson,U.
Abstract:
Experimental autoimmune myocarditis (EAM) represents a CD4+ T helper (Th) cell-mediated mouse model of inflammatory heart disease. Interferon (IFN)-γ, typically produced by Th1 cells, reduces EAM severity in myosin heavy-chain-(MyHC)-α peptide/Complete Freund adjuvant-immunized mice. Thus, developing a vaccination strategy that promotes differentiation of Th1 cells may be beneficial in EAM. FMS-like tyrosine kinase 3 ligand (Flt3L)-induced splenic CD8α+ dendritic cells (DC), which produce interleukin (IL)-12p35, were identified to selectively induce biased differentiation towards Th1. Mice vaccinated with MyHC-α-loaded Flt3L-induced splenic CD8α+ DC were protected from EAM. In contrast, when Flt3L-induced splenic CD8α+ DC were pre-stimulated and over-activated with LPS and αCD40 antibodies or loaded with unspecific OVA(323-339) peptide instead of MyHC-α peptide, mice developed similar disease scores as non-vaccinated controls. Vaccination efficacy depended on IFN-γ, since CD8α+-vaccinated IFN-γR(-/-) mice were not protected. Importantly, splenic CD8α+ vaccination was independent of regulatory T cells. Taken together, Flt3L-induced dendritic cell-based antigen-specific vaccination limits expansion of auto-reactive Th cells and protects mice from autoimmune heart inflammation.

Address: Division of Cardioimmunology, Cardiovascular Research, Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Switzerland

3915952i: J.Immunol., 3025-3036(2013)
Link: [ PubMED ] [ DOI ]
Title: CD8+ T cells produce the chemokine CXCL10 in response to CD27/CD70 costimulation to promote generation of the CD8+ effector T cell pool.
Author: Peperzak,V., Veraar,E.A., Xiao,Y., Babala,N., Thiadens,K., Brugmans,M., Borst,J.
Abstract:
Various cell types can produce the chemokine CXCL10 in response to IFN-γ stimulation. CXCL10 is generally viewed as a proinflammatory chemokine that promotes recruitment of CD8+ and Th1-type CD4+ effector T cells to infected or inflamed nonlymphoid tissues. We show that CXCL10 plays a role during CD8+ T cell priming in the mouse. Genome-wide expression profiling revealed the Cxcl10 gene as a target of CD27/CD70 costimulation in newly activated CD8+ T cells. CD27/CD70 costimulation is known to promote activated T cell survival, but CXCL10 did not affect survival or proliferation of primed CD8+ T cells in vitro. Accordingly, CXCL10 could not fully rescue CD27 deficiency in mice infected with influenza virus. Rather, CXCL10 acted as chemoattractant for other activated CD8+ T cells. It signaled downstream of CD27 in a paracrine fashion to promote generation of the CD8+ effector T cell pool in the Ag-draining lymph nodes. Consistently, CD8+ T cells required expression of the CXCL10 receptor CXCR3 for their clonal expansion in a CD27/CD70-dependent peptide-immunization model. Our findings indicate that CXCL10, produced by primed CD8+ T cells in response to CD27/CD70 costimulation, signals to other primed CD8+ T cells in the lymph node microenvironment to facilitate their participation in the CD8+ effector T cell pool.

Address: Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

3915955i: Infect.Immun., 3975-3983(2013)
Link: [ PubMED ] [ DOI ]
Title: Augmentation of epithelial resistance to invading bacteria by using mRNA transfections.
Author: Zou,X., Sorenson,B.S., Ross,K.F., Herzberg,M.C.
Abstract:
To protect against invading bacteria, oral epithelial cells appear to use two effector antimicrobial peptides (AMPs): calprotectin (S100A8-S100A9 heterodimer [S100A8/A9]) in the cytosol and cathelicidin antimicrobial protein (CAMP) in endosomes. We sought to learn whether innate immunity might be augmented benignly to increase resistance against invasive bacteria. Epithelial cells were transiently transfected with mRNA constructs containing either the CAMP, S100A8, and S100A9 open reading frames, A8-IRES-A9 (fusion sequence), or A8-nIRES-A9 (fusion with native internal ribosome entry site [IRES] sequence). CAMP, S100A8, and S100A9 protein levels generally peaked between 16 and 44 h after mRNA transfection, depending on the construct; CAMP was processed to LL-37 over time. Following transfection with the respective mRNAs, CAMP and S100A8/A9 each independently increased resistance of epithelial cells to invasion by Listeria and Salmonella for up to 48 h; tandem S100A8/A9 constructs were also effective. Cotransfection to express S100A8/A9 and CAMP together augmented resistance, but synergy was not seen. Independent of the new proteins produced, transfection reduced cell viability after 48 h by 20%, with only 2% attributable to apoptosis. Taken together, these results suggest that epithelial cell resistance to invasive pathogens can be augmented by transient transfection of antimicrobial mRNAs into epithelial cells.

Address: Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, Minnesota, USA

3915993i: Vaccine, 4647-4654(2013)
Link: [ PubMED ] [ DOI ]
Title: A universal virus-like particle-based vaccine for human papillomavirus: longevity of protection and role of endogenous and exogenous adjuvants.
Author: Tumban,E., Peabody,J., Peabody,D.S., Chackerian,B.
Abstract:
Antibodies targeting epitopes within the amino terminus of the minor capsid protein L2 of human papillomavirus (HPV) are broadly neutralizing against diverse HPV isolates. We have constructed bacteriophage virus-like particle (VLP)-based vaccines that display short L2 peptides and elicit high-titer and broadly protective antibody responses. Here, we further characterize two additional features of these VLP-based vaccines; the longevity of protection and the role of endogenous and exogenous adjuvants on the magnitude and characteristics of the antibody response. We show that vaccinated mice have long-lived antibody responses against L2, persisting over 18 months after vaccination. Vaccinated mice were strongly protected against infection by diverse HPV pseudoviruses over a year after immunization. We also show that exogenous and endogenous adjuvants (LPS and encapsidated single-stranded RNA) have minor effects on antibody titers. Immunization with VLPs containing encapsidated ssRNA predominantly shifts the response to a Th1, rather than a Th2-like response. Importantly, immunization with L2-VLPs (without endogenous and exogenous adjuvants) in the presence of alum hydroxide elicited a robust antibody response.

E-Mail: etumban@salud.unm.edu
Address: Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. Electronic address:

3915994i: Vaccine, 4834-4840(2013)
Link: [ PubMED ] [ DOI ]
Title: An unbiased peptide-wide discovery approach to select Mycobacterium tuberculosis antigens that target CD8+ T cell response during infection.
Author: Cayabyab,M.J., Qin,L., Kashino,S.S., Izzo,A., Campos-Neto,A.
Abstract:
Accruing data strongly support the possible role of CD8+ T cells in immunity against tuberculosis (TB). Multivalent vaccines against Mycobacterium tuberculosis (Mtb) that incorporate CD8+ T cell antigens with those that elicit CD4+ T cells are therefore highly desirable. To screen for potential CD8+ T cell antigens that are produced by Mtb during infection, we isolated pathogen-derived peptides that bound to MHC Class I molecules expressed in adherent splenocytes obtained from Mtb-infected mice. Mass spectroscopy analysis revealed the following four nonamer peptides that had 100% homology with Mtb proteins: DGYVGAPAH (MT_0401), TTMPLFAD (MT_1164), RSGAATPVR (MT_2160.1) and LAAVVGVVL (MT_0078). The gene MT_0401 codes the protein 5'-phosphoribosylglycinamide transformylase 2 and the other three genes code for hypothetical proteins with unknown function. The NCBI/Blast analysis showed that among the four peptides DGYVGAPAH had the highest maxiμm alignment score and lowest E value (number of alignments expected by chance). Therefore, we assessed whether MT_0401 expressed in two genetic vaccine formulations was capable of stimulating CD8+ T cell response that is specific to DGYVGAPAH peptide. When mice were immunized with a recombinant plasmid DNA and an E1/E3-deleted Adenovirus 5 expressing MT0401 protein, using both homologous and heterologous prime-boost protocols, they developed strong DGYVGAPAH-specific CD8+ T cell response as well as antibody and CD4+ specific T cell response to the full length MT0401 protein. Equally important was the observation that mice infected with Mtb developed DGYVGAPAH-specific CD8+ T cell responses in both spleen and lungs. These results demonstrate that Mtb antigens that are processed and presented via MHC Class I machinery can be readily identified by the described approach and may be useful candidate antigens to stimulate specific CD8+ T cell responses in vaccine development programs.

Address: Global Infectious Disease Research Center, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, USA

3915995i: Vaccine, 4508-4515(2013)
Link: [ PubMED ] [ DOI ]
Title: Peptide nanofiber hydrogel adjuvanted live virus vaccine enhances cross-protective immunity to porcine reproductive and respiratory syndrome virus.
Author: Li,X., Galliher-Beckley,A., Huang,H., Sun,X., Shi,J.
Abstract:
Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in swine farms worldwide and is a major source of economic loss and animal suffering. Rapid genetic variation of PRRSV makes it difficult for current vaccines to confer protection against newly emerging strains. We recently demonstrated that a novel peptide nanofiber hydrogel (H9e) could act as a potent adjuvant for killed H1N1 vaccines. Therefore, the objective of this study was to evaluate H9e as an adjuvant for PRRSV modified live virus (MLV) vaccines. Pigs were vaccinated with Ingelvac PRRSV MLV with or without H9e adjuvant before being challenged with the VR-2332 (parental vaccine strain) or MN184A (genetically diverse strain) PRRSV. Pigs vaccinated with MLV+H9e had higher levels of circulating vaccine virus. More importantly, pigs vaccinated with MLV+H9e had improved protection against challenge by both PRRSV strains, as demonstrated by reduced challenge-induced viremia compared with pigs vaccinated with MLV alone. Pigs vaccinated with MLV+H9e had lower frequency of T-regulatory cells and IL-10 production but higher frequency of Th/memory cells and IFN-γ secretion than that in pigs vaccinated with MLV alone. Taken together, our studies suggest that the peptide nanofiber hydrogel H9e, when combined with the PRRSV MLV vaccine, can enhance vaccine efficacy against two different PRRSV strains by modulating both host humoral and cellular immune responses.

Address: Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States

3915996i: J.Immunol.Methods, 44-55(2013)
Link: [ PubMED ] [ DOI ]
Title: Development of a biosensor-based immunogenicity assay capable of blocking soluble drug target interference.
Author: Weeraratne,D.K., Lofgren,J., Dinnogen,S., Swanson,S.J., Zhong,Z.D.
Abstract:
As with other protein therapeutics, trebananib (AMG 386), an investigational peptide Fc-fusion protein ("peptibody") that inhibits angiogenesis by neutralizing the interaction of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) with the Tie2 receptor, has the potential to trigger an immune response in cancer patients treated with the therapeutic. An electrochemiluminescence bridging anti-drug antibody (ADA) assay that was utilized to support early-phase clinical trials in the development of trebananib was found to lack adequate sensitivity and drug tolerance in later-phase clinical studies when higher doses of trebananib were administered. Therefore, we developed a surface plasmon resonance (SPR) immunoassay method utilizing a secondary confirmatory detector antibody (goat anti-human IgG F[ab']2) known to cross-react with human IgG and IgM to better assess the potential impact of immunogenicity on the pharmacokinetics, pharmacodynamics, and toxicity of trebananib. The SPR method was more sensitive than the electrochemiluminescence bridging assay because of signal amplification from the confirmatory binding of the detector antibody; drug tolerance was improved since antibody binding avidity does not affect detection on this platform. Despite the inability of the confirmatory detector antibody to bind angiopoietins in protein-free buffer, false-positive ADA results were generated from patient serum samples containing Ang1 and Ang2 through an apparently specific binding between the angiopoietins and the confirmatory detector antibody, likely mediated by the interaction of the angiopoietins with serum immunoglobulins. Addition to the sample diluent of a human antibody that specifically binds to Ang1 and Ang2 with high affinity resulted in a complete block of angiopoietin interference without affecting ADA detection. This biosensor-based assay provides a reliable method for assessing immunogenicity in phase 3 clinical trials.

E-Mail: dohanw@amgen.com
Address: Department of Clinical Immunology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. Electronic address:

3915997i: J.Immunol.Methods, 168-172(2013)
Link: [ PubMED ] [ DOI ]
Title: The abrogation of TCR-independent interactions with human serum ensures a selective capture of therapeutic virus-specific CD8+ T-cells by Multimer Technology in Adoptive Immunotherapy.
Author: Beloki,L., Ciaurriz,M., Mansilla,C., Bandres,E., Rodriguez-Calvillo,M., Ramirez,N., Olavarria,E.
Abstract:
Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8+ T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes. Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8+ T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.

Address: Oncohematology Research Group, Navarrabiomed-Fundacion Miguel Servet, Pamplona, Spain

3915998i: Fish Shellfish Immunol., 1240-1248(2013)
Link: [ PubMED ] [ DOI ]
Title: The involvement of PDGF/VEGF related factor in regulation of immune and neuroendocrine in Chinese mitten crab Eriocheir sinensis.
Author: Li,F., Xu,L., Gai,X., Zhou,Z., Wang,L., Zhang,H., Gai,Y., Song,L., Yu,J., Liang,C.
Abstract:
Members of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family have been implicated in cell proliferation, cell differentiation, and cell migration, vascular development, angiogenesis and neural development. In the present study, a novel PDGF/VEGF related factor gene was cloned and identified in Chinese mitten crab Eriocheir sinensis (designated as EsPVF1). The full-length cDNA of EsPVF1 was of 1173 bp, consisting a 5' untranslated region (UTR) of 54 bp, a 3' UTR of 1131 bp with a poly (A) tail, and an open reading frame (ORF) of 588 bp encoding 196 amino acid residues. A signal peptide of 20 amino acid residues, a PDGF/VEGF homology growth factor domain of 81 amino acids, and a typical cysteine knot motif (CXCXC) were identified in the deduced amino acid sequence of EsPVF1. By fluorescent quantitative real-time PCR, the EsPVF1 mRNA was detected ubiquitously in the select tissues of hemocytes, gonad, heart, muscle, hepatopancreas and gill, with the high abundance in hemocytes and gonad. The mRNA expression level of EsPVF1 was up-regulated and reached the highest at 24 h after Vibrio anguillarum challenge, while it was induced at 3 h, 6 h, 12 h, 24 h and 48 h compared with the untreated group after Pichia pastoris GS115 challenge. Tissue injury also induced the mRNA expression of EsPVF1 in hemocytes of crabs, and the expression level increased obviously at 8 h. The cDNA fragment encoding mature peptide of EsPVF1 was recombined and expressed in Escherichia coli BL21 (DE3) pLysS. Biogenic amine in hemolymph pre-incubated with recombinant protein of EsPVF1 (rEsPVF1) was detected by fluorimetric method. Norepinephrine and dopamine in hemolymph incubated with rEsPVF1 were higher than that in the blank group. Therefore, EsPVF1 could significantly provoke the release of norepinephrine and dopamine. The results collectively indicated that EsPVF1 was involved in regulation of the immune response and neuroendocrine system in crabs.

E-Mail: lifengmei@qust.edu.cn
Address: Qingdao University of Science and Technology, Qingdao 266042, China

3916015i: Immunobiology, 1354-1360(2013)
Link: [ PubMED ] [ DOI ]
Title: Macrophages engulfing apoptotic thymocytes produce retinoids to promote selection, differentiation, removal and replacement of double positive thymocytes.
Author: Sarang,Z., Garabuczi,E., Joos,G., Kiss,B., Toth,K., Ruhl,R., Szondy,Z.
Abstract:
The thymus provides the microenvironment in which thymocytes develop into mature T-cells, and interactions with thymic stromal cells are thought to provide the necessary signals for thymocyte maturation. Recognition of self-MHC by T-cells is a basic requirement for mature T-cell functions, and those thymocytes that do not recognize or respond too strongly to the peptide-loaded self-MHC molecules found in the thymus undergo apoptosis. As a result, 95% of the thymocytes produced will die and be subsequently cleared by macrophages. This review describes a complex crosstalk between developing thymocytes and engulfing macrophages which is mediated by retinoids produced by engulfing macrophages. The interaction results in the harmonization of the rate of cell death of dying double positive cells with their clearance and replacement, and in promotion of the differentiation of the selected cells in the thymus.

Address: Section of Dental Biochemistry, Department of Biochemistry and Molecular Biology, Research Center of Molecular Medicine, University of Debrecen, Nagyerdei krt. 98, 4012 Debrecen, Hungary

3916045i: J.Immunol., 3287-3296(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification of a phage-encoded Ig-binding protein from invasive Neisseria meningitidis.
Author: Muller,M.G., Ing,J.Y., Cheng,M.K., Flitter,B.A., Moe,G.R.
Abstract:
Ig-binding proteins are employed by a variety of organisms to evade the immune system. To our knowledge, we now report for the first time that meningococcal strains from several capsular groups exhibit Ig-binding activity that is dependent on human serum factors. A protein mediating Ig binding was identified as T and B cell-stimulating protein B (TspB) by immunoprecipitation and by mass spectroscopic analysis of tryptic peptides. Recombinant TspB and derivatives verified Ig binding, with a preference for human IgG2 Fc, and localized the IgG-binding region to a highly conserved subdomain of TspB. Antiserum produced in mice against the conserved subdomain detected the presence of TspB on the cell surface by flow cytometry when bacteria were grown in the presence of human serum. By fluorescence microscopy, we observed formation of an extracellular matrix having characteristics of a biofilm containing TspB, human IgG, DNA, and large aggregates of bacteria. TspB is encoded by gene ORF6 in prophage DNA, which others have shown is associated with invasive meningococcal strains. Knocking out ORF6 genes eliminated IgG binding and formation of large bacterial aggregates in biofilm. Reintroduction of a wild-type ORF6 gene by phage transduction restored the phenotype. The results show that TspB mediated IgG binding and aggregate/biofilm formation triggered by factors in human serum. As has been observed for other Ig-binding proteins, the activities mediated by TspB may provide protection against immune responses, which is in accordance with the association of prophage DNA carrying ORF6 with invasive meningococcal strains.

Address: Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA

3916074i: Proc.Natl.Acad.Sci.U.S.A., 13916-13920(2013)
Link: [ PubMED ] [ DOI ] [ OpenAccess]
Title: Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation.
Author: Venkatraman Girija,U., Gingras,A.R., Marshall,J.E., Panchal,R., Sheikh,M.A., Gal,P., Schwaeble,W.J., Mitchell,D.A., Moody,P.C., Wallis,R.
Abstract:
Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca2+-coordinating residues. The data explain the Ca2+-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.

Address: Department of Infection, University of Leicester, Leicester LE1 9HN, United Kingdom

3916106i: Mol.Immunol., 22-27(2014)
Link: [ PubMED ] [ DOI ]
Title: HLA-B27 and antigen presentation: At the crossroads between immune defense and autoimmunity.
Author: Sorrentino,R., Bockmann,R.A., Fiorillo,M.T.
Abstract:
The HLA-B27 is historically studied as a susceptibility factor in spondyloarthropathies and, primarily, in ankylosing spondylitis (AS). Over the recent years however, it has been rediscovered as protective factor against some severe viral infections. This is due to the high capacity of virus-specific, HLA-B27-restricted CD8+ T cells for both intrinsic (i.e. polyfunctionality, high avidity, low sensitivity to Treg cell-mediated suppression) and extrinsic (i.e. rapid and efficient antigen processing and presentation) factors. It is tempting to speculate that these two aspects are not independent and that the association of B27 molecules to autoimmunity is the downside of this superior functional efficacy which, in given genetic backgrounds and environmental conditions, can support a chronic inflammation leading to spondyloarthropathies. Still, the pathogenic role of HLA-B27 molecules in AS is elusive. Here, we focus on the biology of HLA-B27 from the genetics to the biochemistry and to the structural/dynamical properties of B27:peptide complexes as obtained from atomistic molecular dynamics simulation. Overall, the results point at the antigen presentation as the key event in the disease pathogenesis. In particular, an extensive comparison of HLA-B*2705 and B*2709 molecules, that differ in a single amino acid (Asp116 to His116) and are differentially associated with AS, indicates that position 116 is crucial for shaping the entire peptide-presenting groove.

Address: Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza, University of Rome, Italy

3916107i: Mol.Immunol., 12-21(2014)
Link: [ PubMED ] [ DOI ]
Title: ERAP1 structure, function and pathogenetic role in ankylosing spondylitis and other MHC-associated diseases.
Author: Alvarez-Navarro,C., Lopez de Castro,J.A.
Abstract:
The endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in the final processing of Major Histocompatibility Complex class I (MHC-I) ligands and with a significant influence in the stability and immunological properties of MHC-I proteins. ERAP1 polymorphism is associated with ankylosing spondylitis among HLA-B27-positive individuals and the altered enzymatic activity of natural variants has significant effects on the HLA-B27 peptidome, suggesting a critical pathogenetic role of peptides in this disease. Likewise, the association of ERAP1 with other MHC-I associated disorders and its epistasis with their susceptibility MHC alleles point out to a general role of the MHC-I peptidome in these diseases. The functional interaction between ERAP1 and HLA-B27 or other MHC-I molecules may be related to the processing of specific epitopes, or to a more general peptide-dependent influence on other biological features of the MHC-I proteins. In addition, from a consideration of the reported functions of ERAP1, including its involvement in angiogenesis and macrophage activation, a more complex and multi-level influence in the inflammatory and immune pathways operating in these diseases cannot be ruled out.

Address: Centro de Biologia Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientificas and Universidad Autonoma de Madrid), Madrid, Spain

3916110i: J.Immunol., 2072-2081(2013)
Link: [ PubMED ] [ DOI ]
Title: Simultaneous TCR and CD244 signals induce dynamic downmodulation of CD244 on human antiviral T cells.
Author: Pacheco,Y., McLean,A.P., Rohrbach,J., Porichis,F., Kaufmann,D.E., Kavanagh,D.G.
Abstract:
Various cosignaling molecules on T cells can contribute to activation, inhibition, or exhaustion, depending on context. The surface receptor signaling lymphocytic activation molecule (SLAM) family receptor CD244 (2B4/SLAMf4) has been shown to be capable of either inhibitory or enhancing effects upon engagement of its ligand CD48 (SLAMf2). We examined phenotypes of CD8 T cells from HIV+ and HIV(neg) human donors, specific for HIV and/or respiratory syncytial virus. Cultured and ex vivo CD8 T cells expressed PD-1, CD244, and TIM-3. We found that ex vivo CD8 T cells downregulated CD244 in response to superantigen. Furthermore, cognate peptide induced rapid downregulation of both CD244 and TIM-3, but not PD-1, on CD8 T cell clones. CD244 downmodulation required simultaneous signaling via both TCR and CD244 itself. Using a pH-sensitive fluorophore conjugated to avidin-Ab tetramers, we found that CD244 crosslinking in the presence of TCR signaling resulted in rapid transport of CD244 to an acidic intracellular compartment. Downregulation was not induced by PMA-ionomycin, or prevented by PI3K inhibition, implicating a TCR-proximal signaling mechanism. CD244 internalization occurred within hours of TCR stimulation and required less peptide than was required to induce IFN-γ production. The degree of CD244 internalization varied among cultured CD8 T cell lines of different specificities, and correlated with the enhancement of IFN-γ production in response to CD48 blockade in HIV+, but not HIV(neg), subjects. Our results indicate that rapid CD244 internalization is induced by a two-signal mechanism and plays a role in modulation of antiviral CD8 T cell responses by CD48-CD244 signaling.

Address: Ragon Institute of MGH, MIT and Harvard, Massachusetts General Hospital, Cambridge, MA 02139, USA

3916119i: Immunogenetics, 785-793(2013)
Link: [ PubMED ] [ DOI ]
Title: Molecular cloning, genomic structure, and tissue distribution of EW135, a novel chicken egg white protein with group B scavenger receptor cysteine-rich domains.
Author: Yoo,W., Nakamura,T., Asanuma,H., Matsushita,M.
Abstract:
Approximately 80 proteins are reported to be present in chicken egg white. The major function of egg white proteins isolated so far is to defend the egg yolk against infections. We recently isolated a novel protein termed EW135 from chicken egg white. In this paper, we have determined the complete amino acid sequence of EW135 based on cDNA cloning. EW135 consists of 970 amino acids with a putative signal peptide of 17 amino acids. It is composed exclusively of tandem repeats of nine group B scavenger receptor cysteine-rich (SRCR) domains separated by eight seven-amino acid peptides. The features of consensus sequences found in the group B SRCR domain were well conserved in EW135. The EW135 gene consists of putative 11 exons, with each SRCR domain being encoded by a single exon. Reverse transcription PCR showed that EW135 is expressed in only the oviduct among the 11 types of tissues tested. EW135 is a second soluble protein belonging to the group B SRCR domain superfamily identified in chickens. One of the important functions of proteins belonging to the group B SRCR domain superfamily is to recognize pathogens in innate immunity. It is, therefore, conceivable that EW135 could be involved in host defense in egg white.

Address: Department of Applied Biochemistry, Tokai University, Hiratsuka, 259-1292, Japan

3916139i: J.Neuroimmunol., 20-27(2013)
Link: [ PubMED ] [ DOI ]
Title: Suppression of MOG- and PLP-induced experimental autoimmune encephalomyelitis using a novel multivalent bifunctional peptide inhibitor.
Author: Badawi,A.H., Siahaan,T.J.
Abstract:
Previously, bifunctional peptide inhibitors (BPI) with a single antigenic peptide have been shown to suppress experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner. In this study, a multivalent BPI (MVBMOG/PLP) with two antigenic peptides derived from myelin oligodendrocyte glycoprotein (MOG38-50) and myelin proteolipid protein (PLP139-151) was evaluated in suppressing MOG38-50- and PLP139-151-induced EAE. MVBMOG/PLP significantly suppressed both models of EAE even when there was some evidence of epitope spreading in the MOG38-50-induced EAE model. In addition, MVBMOG/PLP was found to be more effective than PLP-BPI and MOG-BPI in suppressing MOG38-50-induced EAE. Thus, the development of MVB molecules with broader antigenic targets can lead to suppression of epitope spreading in EAE.

Address: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, United States

3916140i: Theriogenology, 597-608(2013)
Link: [ PubMED ] [ DOI ]
Title: Antibody selection for immunocytochemical characterization of the male reproductive system in Psittaciformes.
Author: Reitemeier,S., Hanse,M., Hahn,A., Schmidt,V., Steinbach-Sobiraj,K., Tatzner,S., Krautwald-Junghanns,M.E., Einspanier,A.
Abstract:
The success of breeding programs is limited by the sparse knowledge about endocrine regulation and biochemical reactions in the psittacine male tract. The immunocytochemical analysis of parrots' testicular tissues provides an insight into their reproductive system but is often hampered by the lack of reliable antibodies. In the present study, we tested a large panel of antibodies raised against steroid receptors, steroidogenic enzymes, relaxin peptides including their receptors, and proliferation markers on paraffin sections of testicular tissue from eight psittacine genera representing three continents. All investigated species displayed the tested markers in somatic and germ cells of testis and epididymis, even though cell distribution was partly heterogenous and in species-specific patterns. The 17β-hydroxysteroid-dehydrogenase-2, 3β-hydroxysteroid-dehydrogenase, and smooth muscle actin allowed the cross-species differentiation between active and nonactive gonads. The remaining steroidogenic enzymes, steroid receptors, relaxin peptides, and Ki67 proved to be suitable to define reproductive activity depending on the parrot species. Adapting immunocytochemical methods to different psittacines was successful, though various cellular expression patterns do not allow the transfer of results among different parrot species. However, the availability of a reliable repertory of sexual markers is important to examine reproductive biology of psittacine birds.

Address: Institute of Physiological Chemistry, Veterinary Faculty, University of Leipzig, Leipzig, Germany

3916238i: Int.Immunopharmacol., 880-884(2013)
Link: [ PubMED ] [ DOI ]
Title: The role of TLR2/JNK/NF-κB pathway in amyloid β peptide-induced inflammatory response in mouse NG108-15 neural cells.
Author: Lin,W., Ding,M., Xue,J., Leng,W.
Abstract:
The TLR2-mediated neuroinflammatory activation has been involved in the pathogenesis of Alzheimer's disease (AD) associated with amyloid β(Aβ) deposition. In neuronal damage, JNK and NF-κB pathways contribute to TLR2-dependent secretion of proinflammatory cytokines. However, the role of TLR2/JNK/NF-κB pathway on Aβ-induced inflammatory response in nerve cell damage remains unclear. In the present study, Aβ1-42 was used to induce mouse NG108-15 neural cell injury. The cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT). The levels of tumor necrosis factor (TNF)-α, monocyte chemoattractant protein(MCP)-1 and interleukin (IL)-10 in culture supernatant were measured by ELISA. western blot analysis was performed to detect the expressions of JNK and p-65 NF-κB proteins. Immunofluorescence assay was also performed to examine the p-JNK and p-65 NF-κB activation. As a result, Aβ1-42 incubation for 36h inhibited remarkedly the cell viability of NG108-15, and increased significantly the levels of inflammatory cytokines TNF-α, MCP-1 and IL-10, as well as enhanced the expressions of JNK and p-65 NF-κB in western blot analysis and immunofluorescence assay. However, the pre-incubation with anti-TLR2 (OPN301, 1μg/ml) or JNK inhibitor SP600125 (10μg/ml) prior to Aβ1-42 administration, these upregulation events were all reduced. These results suggested that the induction of Aβ1-42 on proinflammatory cytokine generation might be associated with TLR2-dependent JNK/NF-κB signal pathway, at least partially. Our findings indicated that blockade of TLR2/JNK/NF-κB pathway could be beneficial in the pathogenesis of AD.

Address: Department of Neurology, Jintan People's Hospital, Jiangsu, Jintan 213200, PR China

3916257i: Vaccine, 4611-4618(2013)
Link: [ PubMED ] [ DOI ]
Title: Boosters of a therapeutic HIV-1 vaccine induce divergent T cell responses related to regulatory mechanisms.
Author: Lind,A., Brekke,K., Sommerfelt,M., Holmberg,J.O., Aass,H.C., Baksaas,I., Sorensen,B., Dyrhol-Riise,A.M., Kvale,D.
Abstract:
Therapeutic human immunodeficiency virus (HIV) vaccines aim to reduce disease progression by inducing HIV-specific T cells. Vacc-4x are peptides derived from conserved domains within HIV-1 p24 Gag. Previously, Vacc-4x induced T cell responses in 90% of patients which were associated with reduced viral loads. Here we evaluate the effects of Vacc-4x boosters on T cell immunity and immune regulation seven years after primary immunization. Twenty-five patients on effective antiretroviral therapy received two Vacc-4x doses four weeks apart and were followed for 16 weeks. Vacc-4x T cell responses were measured by proliferation (CFSE), INF-γ, CD107a, Granzyme B, Delayed-Type Hypersensitivity test (DTH) and cytokines and chemokines (Luminex). Functional regulation of Vacc-4x-specific T cell proliferation was estimated in vitro using anti-IL-10 and anti-TGF-ss monoclonal antibodies. Vacc-4x-specific CD8+ T cell proliferation increased in 80% after either the first (64%) or second (16%) booster. Only 40% remained responders after two boosters with permanently increased Vacc-4x-specific proliferative responses (p=0.005) and improved CD8+ T cell degranulation, IFN-γ production and DTH. At baseline, responders had higher CD8+ T cell degranulation (p=0.05) and CD4+ INF-γ production (p=0.01), whereas non-responders had higher production of proinflammatory TNF-α, IL-1α and IL-1ss (p<0.045) and regulatory IL-10 (p=0.07). Notably, IL-10 and TGF-ss mediated downregulation of Vacc-4x-specific CD8+ T cell proliferation increased only in non-responders (p<0.001). Downregulation during the study correlated to higher PD-1 expression on Vacc-4x-specific CD8+ T cells (r=0.44, p=0.037), but was inversely correlated to changes in Vacc4x-specific CD8+ T cell proliferation (r=-0.52, p=0.012). These findings show that Vacc-4x boosters can improve T cell responses in selected patients, but also induce vaccine-specific downregulation of T cell responses in others. Broad surveillance of T cell functions during immunization may help to individualize boosting, where assessment of vaccine-related immune regulation should be further explored as a potential new parameter.

Address: Department of Infectious Diseases, Oslo University Hospital, Oslo, Norway

3916272i: J.Immunol., 2700-2707(2013)
Link: [ PubMED ] [ DOI ]
Title: NSP4 is stored in azurophil granules and released by activated neutrophils as active endoprotease with restricted specificity.
Author: Perera,N.C., Wiesmuller,K.H., Larsen,M.T., Schacher,B., Eickholz,P., Borregaard,N., Jenne,D.E.
Abstract:
Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer-based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1', P2', P3, P4, and P2 positions were tested. This enabled us to construct an α1-proteinase inhibitor variant (Ile-Lys-Pro-Arg-/-Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefevre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils.

Address: Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum Munchen, Member of the German Center for Lung Research, 81377 Munich, Germany

3916273i: J.Immunol., 2299-2307(2013)
Link: [ PubMED ] [ DOI ]
Title: Inhibition of TGF-β1 signaling promotes central memory T cell differentiation.
Author: Takai,S., Schlom,J., Tucker,J., Tsang,K.Y., Greiner,J.W.
Abstract:
This study affirmed that isolated CD8+ T cells express mRNA and produce TGF-β following cognate peptide recognition. Blockage of endogenous TGF-β with either a TGF-β-blocking Ab or a small molecule inhibitor of TGF-βRI enhances the generation of CD62L(high)/CD44(high) central memory CD8+ T cells accompanied with a robust recall response. Interestingly, the augmentation within the central memory T cell pool occurs in lieu of cellular proliferation or activation, but with the expected increase in the ratio of the Eomesoderm/T-bet transcriptional factors. Yet, the signal transduction pathway(s) seems to be noncanonical, independent of SMAD or mammalian target of rapamycin signaling. Enhancement of central memory generation by TGF-β blockade is also confirmed in human PBMCs. The findings underscore the role(s) that autocrine TGF-β plays in T cell homeostasis and, in particular, the balance of effector/memory and central/memory T cells. These results may provide a rationale to targeting TGF-β signaling to enhance Ag-specific CD8+ T cell memory against a lethal infection or cancer.

Address: Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

3916286i: Immunogenetics, 711-724(2013)
Link: [ PubMED ] [ DOI ]
Title: NetMHCIIpan-3.0, a common pan-specific MHC class II prediction method including all three human MHC class II isotypes, HLA-DR, HLA-DP and HLA-DQ.
Author: Karosiene,E., Rasmussen,M., Blicher,T., Lund,O., Buus,S., Nielsen,M.
Abstract:
Major histocompatibility complex class II (MHCII) molecules play an important role in cell-mediated immunity. They present specific peptides derived from endosomal proteins for recognition by T helper cells. The identification of peptides that bind to MHCII molecules is therefore of great importance for understanding the nature of immune responses and identifying T cell epitopes for the design of new vaccines and immunotherapies. Given the large number of MHC variants, and the costly experimental procedures needed to evaluate individual peptide-MHC interactions, computational predictions have become particularly attractive as first-line methods in epitope discovery. However, only a few so-called pan-specific prediction methods capable of predicting binding to any MHC molecule with known protein sequence are currently available, and all of them are limited to HLA-DR. Here, we present the first pan-specific method capable of predicting peptide binding to any HLA class II molecule with a defined protein sequence. The method employs a strategy common for HLA-DR, HLA-DP and HLA-DQ molecules to define the peptide-binding MHC environment in terms of a pseudo sequence. This strategy allows the inclusion of new molecules even from other species. The method was evaluated in several benchmarks and demonstrates a significant improvement over molecule-specific methods as well as the ability to predict peptide binding of previously uncharacterised MHCII molecules. To the best of our knowledge, the NetMHCIIpan-3.0 method is the first pan-specific predictor covering all HLA class II molecules with known sequences including HLA-DR, HLA-DP, and HLA-DQ. The NetMHCpan-3.0 method is available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0 .

E-Mail: edita@cbs.dtu.dk
Address: Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Building 208, Kemitorvet, 2800, Lyngby, Denmark,

3916302i: J.Immunol., 2174-2183(2013)
Link: [ PubMED ] [ DOI ]
Title: Bacterial CD1d-restricted glycolipids induce IL-10 production by human regulatory T cells upon cross-talk with invariant NKT cells.
Author: Venken,K., Decruy,T., Aspeslagh,S., Van Calenbergh,S., Lambrecht,B.N., Elewaut,D.
Abstract:
Invariant NKT (iNKT) cells and CD4+CD25+FOXP3+ regulatory T cells (Tregs) are important immune regulatory T cells with Ag reactivity to glycolipids and peptides, respectively. However, the functional interplay between these cells in humans is poorly understood. We show that Tregs suppress iNKT cell proliferation induced by CD1d-restricted glycolipids, including bacterial-derived diacylglycerols, as well as by innate-like activation. Inhibition was related to the potency of iNKT agonists, making diacylglycerol iNKT responses very prone to suppression. Cytokine production by iNKT cells was differentially modulated by Tregs because IL-4 production was reduced more profoundly compared with IFN-γ. A compelling observation was the significant production of IL-10 by Tregs after cell contact with iNKT cells, in particular in the presence of bacterial diacylglycerols. These iNKT-primed Tregs showed increased FOXP3 expression and superior suppressive function. Suppression of iNKT cell responses, but not conventional T cell responses, was IL-10 dependent, suggesting that there is a clear difference in mechanism between the Treg-mediated inhibition of these cell types. Our data highlight a physiologically relevant interaction between human iNKT and Tregs upon pathogen-derived glycolipid recognition that has a significant impact on the design of iNKT cell-based therapeutics.

Address: Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium

G. Others

3915523g: Methods Mol.Biol., 255-269(2014)
Link: [ PubMED ] [ DOI ]
Title: Fourier transform infrared spectroscopy of peptides.
Author: Bakshi,K., Liyanage,M.R., Volkin,D.B., Middaugh,C.R.
Abstract:
Fourier transform infrared (FTIR) spectroscopy provides data that are widely used for secondary structure characterization of peptides. A wide array of available sampling methods permits structural analysis of peptides in diverse environments such as aqueous solution (including optically turbid media), powders, detergent micelles, and lipid bilayers. In some cases, side chain vibrations can also be resolved and used for tertiary structure and chemical analysis. Data from several low-resolution spectroscopic techniques, including FTIR, can be combined to generate an empirical phase diagram, an overall picture of peptide structure as a function of environmental conditions that can aid in the global interpretation of large amounts of spectroscopic data.

Address: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA

3915524g: Methods Mol.Biol., 247-253(2014)
Link: [ PubMED ] [ DOI ]
Title: Circular dichroism of peptides.
Author: Bakshi,K., Liyanage,M.R., Volkin,D.B., Middaugh,C.R.
Abstract:
Circular dichroism measures the difference between the absorbance of left- and right-handed circularly polarized light, and can be used to monitor the secondary structure of peptides (far UV) and the tertiary structure of larger polypeptides (near UV). This technique is especially useful for helix-coil transitions and other aspects of structural alterations. Data from several low-resolution spectroscopic techniques, including CD, can be combined to generate an overall picture of peptide structure as a function of environmental conditions.

Address: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA

3915525g: Methods Mol.Biol., 237-246(2014)
Link: [ PubMED ] [ DOI ]
Title: Fluorescence spectroscopy of peptides.
Author: Liyanage,M.R., Bakshi,K., Volkin,D.B., Middaugh,C.R.
Abstract:
In peptides, steady-state fluorescence can be used to measure the intrinsic fluorescence of Trp, Tyr, and Phe residues, as well as associated dyes, which can change upon exposure to different environmental conditions. This technique can be thus used to detect changes in the conformational states of peptides. Time-resolved fluorescence can also be used to study fast motions of peptides and their interactions with fluorescence dyes. Data from several low-resolution spectroscopic techniques, including fluorescence, can be combined to generate an overall picture of peptide structure as a function of environmental conditions.

Address: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA

3915526g: Methods Mol.Biol., 225-236(2014)
Link: [ PubMED ] [ DOI ]
Title: Ultraviolet absorption spectroscopy of peptides.
Author: Liyanage,M.R., Bakshi,K., Volkin,D.B., Middaugh,C.R.
Abstract:
UV absorption spectroscopy is commonly used with peptides for determining concentration and enzyme activity, but high-resolution UV spectra can also provide information on peptide secondary and tertiary structure and association behavior. New developments using temperature- and cation-dependent high-resolution second derivative absorption methods can also provide information concerning peptide dynamics. Data from several low-resolution spectroscopic techniques, including UV absorption, can be combined to generate an overall picture of peptide structure as a function of environmental conditions.

Address: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA

3915527g: Methods Mol.Biol., 213-223(2014)
Link: [ PubMED ] [ DOI ]
Title: Evaluation of prenylated peptides for use in cellular imaging and biochemical analysis.
Author: Ochocki,J.D., Igbavboa,U., Wood,W.G., Arriaga,E.A., Wattenberg,E.V., Distefano,M.D.
Abstract:
Protein prenylation involves the addition of a farnesyl (C15) or geranylgeranyl (C20) isoprenoid moiety onto the C-terminus of approximately 2 % of all mammalian proteins. This hydrophobic modification serves to direct membrane association of the protein. Due to the finding that the oncogenic protein Ras is naturally prenylated, several researchers have developed inhibitors of the prenyltransferase enzymes as cancer therapeutics. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation in living cells. Using a combination of flow cytometry and confocal microscopy, we have shown that synthetic fluorescently labeled prenylated peptides enter a variety of different cell types. Additionally, using capillary electrophoresis we have shown that these peptides can be detected in minute quantities from lysates of cells treated with these peptides. This method will allow for further study of the enzymology of protein prenylation in living cells.

Address: Department of Chemistry, University of Minnesota, Minneapolis, MN, USA

3915528g: Methods Mol.Biol., 185-211(2014)
Link: [ PubMED ] [ DOI ]
Title: Peptide optimization and conjugation strategies in the development of molecularly targeted magnetic resonance imaging contrast agents.
Author: Kolodziej,A.F., Zhang,Z., Overoye-Chan,K., Jacques,V., Caravan,P.
Abstract:
Peptides are highly selective, high-affinity ligands for a diverse array of disease targets, but suitably derivatizing them for application as diagnostic or therapeutic agents often presents a significant challenge. Covalent modification with metal chelates frequently results in decreased binding affinity, so a variety of strategies must be explored to find suitable locations for modification and facile peptide conjugation chemistries that maintain or enhance binding affinity. In this chapter, we present a paradigm for systematically optimizing peptide binding and determining the favorable sites and methods for peptide conjugation. This strategy is illustrated by two case studies of peptide-based targeted gadolinium contrast agents: EP-2104R for diagnosis of thrombosis and EP-3533 for diagnosis of cardiac perfusion and fibrosis. Two different architectures for the peptide-metal complex conjugation were designed: EP-2104R contains a total of four gadolinium (Gd) chelates linked at the N- and C-termini, whereas EP-3533 is derivatized with three Gd chelates, two on the N-terminus and one on a lysine side chain. Detailed protocols are provided for two Gd chelate conjugation methods.

Address: EPIX Pharmaceuticals, Lexington, MA, USA

3915529g: Methods Mol.Biol., 171-183(2014)
Link: [ PubMED ] [ DOI ]
Title: Peptide labelling strategies for imaging agents.
Author: Huhtala,T., Weisell,J., Rytkonen,J., Narvanen,A.
Abstract:
The main goal in modern biomedicine is to develop specific diagnostic and therapeutic agents for different diseases. Especially in cancer research tumor targeted molecules are the key factor in the development of new anti-tumor drugs. In addition, the early diagnosis of the disease is an important factor for a successful therapy. Synthetic peptides have been shown to be specific targeting agents for next generation diagnostic and therapeutic agents. Noninvasive in vivo imaging using targeting molecules provides modern method for the diagnosis of the pathological alterations like cancer. To evaluate the usefulness of a synthetic peptide for in vivo diagnostic purposes the preclinical biodistribution and targeting studies are essential. Today the widely used preclinical imaging modalities for the biodistribution and tissue alteration studies in experimental animals are single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). Together with conventional histochemistry, the biodistribution and tissue/cell location can be determined. In this chapter we describe the conjugation and labelling methods of the peptides for histochemistry and for the molecular imaging with SPECT and MRI modalities.

Address: Department of Biotechnology and Molecular Medicine, University of Eastern Finland, Kuopio, Finland

3915530g: Methods Mol.Biol., 159-170(2014)
Link: [ PubMED ] [ DOI ]
Title: Assessment of antimicrobial (host defense) peptides as anti-cancer agents.
Author: Douglas,S., Hoskin,D.W., Hilchie,A.L.
Abstract:
Cationic antimicrobial (host defense) peptides (CAPs) are able to kill microorganisms and cancer cells, leading to their consideration as novel candidate therapeutic agents in human medicine. CAPs can physically associate with anionic membrane structures, such as those found on cancer cells, causing pore formation, intracellular disturbances, and leakage of cell contents. In contrast, normal cells are less negatively-charged and are typically not susceptible to CAP-mediated cell death. Because the interaction of CAPs with cells is based on charge properties rather than cell proliferation, both rapidly dividing and quiescent cancer cells, as well as multidrug-resistant cancer cells, are targeted by CAPs, making CAPS potentially valuable as anti-cancer agents.CAPs often exist as families of peptides with slightly different amino acid sequences. In addition, libraries of synthetic peptide variants based on naturally occurring CAP templates can be generated in order to improve upon their action. High-throughput screens are needed to quickly and efficiently assess the suitability of each CAP variant. Here we present the methods for assessing CAP-mediated cytotoxicity against cancer cells (suspension and adherent) and untransformed cells (measured using the tritiated thymidine-release or MTT assay), and for discriminating between cell death caused by necrosis (measured using lactate dehydrogenase- or (51)Cr-release assays), or apoptosis and necrosis (single-stranded DNA content measured by flow cytometry). In addition the clonogenic assay, which assesses the ability of single transformed cells to multiply and produce colonies, is described.

Address: Department of Microbiology & Immunology, Dalhousie University, Halifax, NS, Canada

3915531g: Methods Mol.Biol., 147-158(2014)
Link: [ PubMED ] [ DOI ]
Title: Evaluation of peptides as protease inhibitors and stimulators.
Author: Koistinen,H., Hekim,C., Wu,P., Narvanen,A., Stenman,U.H.
Abstract:
Changes in proteolytic activity are associated with several diseases, including cancer. Proteases are potential drug targets and targeting of proteases is used for treatment of various conditions/diseases, like high blood pressure and HIV. We present here detailed protocols for basic evaluation of the effects of peptides on the activity of proteases, using kallikrein-related peptidases KLK2 and KLK3 (also known as hK2 and PSA), and trypsin as examples. KLK2 and KLK3 are major prostatic proteases, and they are potential targets for prostate cancer treatment. KLK2 has trypsin-like activity and KLK3 chymotrypsin-like activity. By phage display technology, we have developed peptides that specifically stimulate KLK3-activity and other peptides that inhibit KLK2 or trypsin. The effect of the peptides on the proteolytic activity of proteases can be studied using substrates, the cleavage of which generates detectable signal, allowing rapid evaluation of protease activity. The cleavage of protein substrates can be detected by SDS-PAGE, followed by staining of the proteins. We also describe graphical analysis of the IC50-value, the effect of a peptide on Michaelis-Menten constant (K m) and the maximal reaction rate (V max).

Address: Department of Clinical Chemistry, Biomedicum, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland

3915532g: Methods Mol.Biol., 125-145(2014)
Link: [ PubMED ] [ DOI ]
Title: Engineering Peptide Therapeutics Using MIMETIBODY Technology.
Author: Picha,K., Huang,C., Bugelski,P., O'Neil,K.
Abstract:
The MIMETIBODY platform was developed to expand the opportunities for application of biotherapeutics. While the utility of antibodies as antagonists has been well demonstrated, their application as agonists has been more challenging. For steric reasons, antibodies may be less well suited to perform as agonists or as inhibitors of GPCRs. In contrast, many bioactive peptides function as agonists or by interaction with GPCRs but their development as therapeutics has been challenging due to their small size and metabolic lability. The MIMETIBODY platform has been used to develop a variety of stable, long-lived molecules with intrinsic activities similar to that of their parent peptides. This chapter describes methods for construction of expression plasmids, expression and purification strategies, and methods for characterizing the activity of these novel proteins.

Address: Centocor Research and Development, Inc., A Division of Johnson & Johnson Company, Radnor, PA, USA

3915533g: Methods Mol.Biol., 97-105(2014)
Link: [ PubMED ] [ DOI ]
Title: Identification of ideal peptides for heterovalent ligands.
Author: Shrivastava,A., Von Wronski,M., Tweedle,M.F., Nunn,A.D.
Abstract:
Recent use of hetero-dimerization to improve the affinity of peptide ligands has made peptides an attractive alternative to small molecules and proteins. Using this strategy, we have developed peptides with affinities comparable to antibodies and specificities often better than small molecules or antibodies. These peptides can be used as a delivery vehicle for drugs or diagnostics, especially in the case of tumor targeting cytotoxic drugs or targeted diagnostics. We describe here an assay to identify an ideal pair of peptides suitable for heterovalent ligands.

Address: Ernst Felder Laboratories, Bracco Research USA, Princeton, NJ, USA

3915534g: Methods Mol.Biol., 81-96(2014)
Link: [ PubMED ] [ DOI ]
Title: Bioprospecting open reading frames for peptide effectors.
Author: Xiong,L., Scott,C.
Abstract:
Recent successes in the development of small-molecule antagonists of protein-protein interactions designed based on co-crystal structures of peptides bound to their biological targets confirm that short peptides derived from interacting proteins can be high-value ligands for pharmacologic validation of targets and for identification of druggable sites. Evolved sequence space is likely to be enriched for interacting peptides, but identifying minimal peptide effectors within genomic sequence can be labor intensive. Here we describe the use of incremental truncation to diversify genetic material on the scale of open reading frames into comprehensive libraries of constituent peptides. The approach is capable of generating peptides derived from both continuous and discontinuous sequence elements, and is compatible with the expression of free linear or backbone cyclic peptides, with peptides tethered to amino- or carboxyl-terminal fusion partners or with the expression of peptides displayed within protein scaffolds (peptide aptamers). Incremental truncation affords a valuable source of molecular diversity to interrogate the druggable genome or evaluate the therapeutic potential of candidate genes.

Address: Department of Biochemistry & Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA

3915535g: Methods Mol.Biol., 67-80(2014)
Link: [ PubMED ] [ DOI ]
Title: Affinity selection using filamentous phage display.
Author: Kulseth,M.A., Fagerlund,A., Myrset,A.H.
Abstract:
Display of peptides on filamentous phage, phage display, is an in vitro selection technique well suited for identification of therapeutic peptide binders for a huge variety of protein targets. The peptides are identified in a process where phage libraries are subjected to affinity selection towards a particular protein target. A successful outcome of an affinity selection is dependent on proper surveillance of the phage life cycle, to make sure that the selection is based on affinity for the target, not on bias in phage propagation rate. In this chapter we present two approaches for protein target presentation and a protocol for phage rescue and propagation, which includes several controls to ensure that all phages initially eluted from the protein target are given equal conditions during the following amplification and selection steps.

Address: Department of Medical Genetics, Rikshospitalet, Oslo University Hospital, Oslo, Norway

3915536g: Methods Mol.Biol., 51-66(2014)
Link: [ PubMED ] [ DOI ]
Title: T7 lytic phage-displayed peptide libraries: construction and diversity characterization.
Author: Krumpe,L.R., Mori,T.
Abstract:
In this chapter, we describe the construction of T7 bacteriophage (phage)-displayed peptide libraries and the diversity analyses of random amino acid sequences obtained from the libraries. We used commercially available reagents, Novagen's T7Select system, to construct the libraries. Using a combination of biotinylated extension primer and streptavidin-coupled magnetic beads, we were able to prepare library DNA without applying gel purification, resulting in extremely high ligation efficiencies. Further, we describe the use of bioinformatics tools to characterize library diversity. Amino acid frequency and positional amino acid diversity and hydropathy are estimated using the REceptor LIgand Contacts website http://relic.bio.anl.gov . Peptide net charge analysis and peptide hydropathy analysis are conducted using the Genetics Computer Group Wisconsin Package computational tools. A comprehensive collection of the estimated number of recombinants and titers of T7 phage-displayed peptide libraries constructed in our lab is included.

Address: Molecular Targets Laboratory, SAIC-Frederick, Inc., Frederick National Laboratory, Frederick, MD, USA

3915537g: Methods Mol.Biol., 35-50(2014)
Link: [ PubMED ] [ DOI ]
Title: Engineering bioactive peptide-based therapeutic molecules.
Author: Ryu,J.S., Cho,A.Y., Seo,S.W., Min,H.
Abstract:
Peptides are increasingly emerging as human therapeutic drugs. By screening very large phage display libraries, novel bioactive peptides that bind to the target of interest with desired biological properties can be identified. Peptides that are obtained in this fashion become the basis for therapeutic molecule development. However, naked peptides are usually not sufficient to be therapeutic molecules by themselves. They need to be chemically modified or conjugated to other molecules to obtain desired physicochemical and in vivo properties. In this chapter, we describe a general methodology of identifying bioactive peptides by biopanning of peptide phage libraries. As an example of therapeutic peptide modifications, we also describe a method for fusing the peptides to the Fc portion of antibody molecule to increase in vivo stability and activity.

Address: Bio Lab, Emerging Technology Center, SAIT, Samsung Electronics, Giheung-Gu, Yongin-Si, Gyeonggi-Do, Republic of Korea

3915538g: Methods Mol.Biol., 19-33(2014)
Link: [ PubMED ] [ DOI ]
Title: Construction of a filamentous phage display peptide library.
Author: Fagerlund,A., Myrset,A.H., Kulseth,M.A.
Abstract:
The concept of phage display is based on insertion of random oligonucleotides at an appropriate location within a structural gene of a bacteriophage. The resulting phage will constitute a library of random peptides displayed on the surface of the bacteriophages, with the encoding genotype packaged within each phage particle. Using a phagemid/helper phage system, the random peptides are interspersed between wild-type coat proteins. Libraries of phage-expressed peptides may be used to search for novel peptide ligands to target proteins. The success of finding a peptide with a desired property in a given library is highly dependent on the diversity and quality of the library. The protocols in this chapter describe the construction of a high-diversity library of phagemid vector encoding fusions of the phage coat protein pVIII with random peptides, from which a phage library displaying random peptides can be prepared.

Address: Department of Pharmaceutical Biosciences, University of Oslo, Oslo, Norway

3915589g: Methods Mol.Biol., 677-685(2014)
Link: [ PubMED ] [ DOI ]
Title: Identification of thioredoxin target disulfides using isotope-coded affinity tags.
Author: Hagglund,P., Bunkenborg,J., Maeda,K., Finnie,C., Svensson,B.
Abstract:
Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with "light" ((12)C) and "heavy" (13C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems.

Address: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark

3915590g: Methods Mol.Biol., 621-632(2014)
Link: [ PubMED ] [ DOI ]
Title: Tandem metal-oxide affinity chromatography for enhanced depth of phosphoproteome analysis.
Author: Beckers,G.J., Hoehenwarter,W., Rohrig,H., Conrath,U., Weckwerth,W.
Abstract:
In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool to study protein phosphorylation because it allows unbiased localization, and site-specific quantification, of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy to identify phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite the high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. Tandem metal-oxide affinity chromatography (MOAC) represents a robust and highly selective approach for the identification and site-specific quantification of low abundant phosphoproteins that is based on the successive enrichment of phosphoproteins and -peptides. This strategy combines protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, tryptic digestion of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides. Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and, thus, allows probing of the phosphoproteome to unprecedented depth.

Address: Department of Molecular Systems Biology, University of Vienna, Vienna, Austria

3915610g: Methods Mol.Biol., 211-225(2013)
Link: [ PubMED ] [ DOI ]
Title: Engineering and utilization of reporter cell lines for cell-based assays of transmembrane receptors.
Author: Lluis,M.W., Yin,H.
Abstract:
Transmembrane receptors, a subset of integral membrane proteins, are the receivers that transduce an extracellular chemical message into an intracellular response. Accordingly, these proteins are of particular interest in the scientific community and are probably best studied as part of a cellular system. Herein, we detail the engineering of a fluorescent and bioluminescent reporter cell line for a transmembrane receptor and how to employ it in a directed evolution screen that identifies peptide regulators of receptor activity.

Address: Department of Chemistry and Biochemistry and the BioFrontiers Institute, University of Colorado at Boulder, Boulder, CO, USA

3915611g: Methods Mol.Biol., 197-210(2013)
Link: [ PubMED ] [ DOI ]
Title: Design of transmembrane peptides: coping with sticky situations.
Author: Rath,A., Deber,C.M.
Abstract:
Membrane proteins have central roles in cellular processes ranging from nutrient uptake to cell-cell communication, and are key drug targets. However, research on α-helical integral membrane proteins is in its relative infancy vs. water-soluble proteins, largely because of their water insolubility when extracted from their native membrane environment. Peptides with sequences that correspond to the membrane-spanning segments of α-helical integral membrane proteins, termed transmembrane (TM) peptides, provide valuable tools for the characterization of these molecules. Here we describe in detail protocols for the design of TM peptides from the sequences of natural α-helical integral membrane proteins and outline strategies for their synthesis and for improving their solubility properties.

Address: Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, Toronto, ON, Canada

3915612g: Methods Mol.Biol., 159-180(2013)
Link: [ PubMED ] [ DOI ]
Title: On the role of NMR spectroscopy for characterization of antimicrobial peptides.
Author: Porcelli,F., Ramamoorthy,A., Barany,G., Veglia,G.
Abstract:
Antimicrobial peptides (AMPs) provide a primordial source of immunity, conferring upon eukaryotic cells resistance against bacteria, protozoa, and viruses. Despite a few examples of anionic peptides, AMPs are usually relatively short positively charged polypeptides, consisting of a dozen to about a hundred amino acids, and exhibiting amphipathic character. Despite significant differences in their primary and secondary structures, all AMPs discovered to date share the ability to interact with cellular membranes, thereby affecting bilayer stability, disrupting membrane organization, and/or forming well-defined pores. AMPs selectively target infectious agents without being susceptible to any of the common pathways by which these acquire resistance, thereby making AMPs prime candidates to provide therapeutic alternatives to conventional drugs. However, the mechanisms of AMP actions are still a matter of intense debate. The structure-function paradigm suggests that a better understanding of how AMPs elicit their biological functions could result from atomic resolution studies of peptide-lipid interactions. In contrast, more strict thermodynamic views preclude any roles for three-dimensional structures. Indeed, the design of selective AMPs based solely on structural parameters has been challenging. In this chapter, we will focus on selected AMPs for which studies on the corresponding AMP-lipid interactions have helped reach an understanding of how AMP effects are mediated. We will emphasize the roles of both liquid- and solid-state NMR spectroscopy for elucidating the mechanisms of action of AMPs.

Address: Department of Chemistry, University of Minnesota, Minneapolis, MN, USA

3915613g: Methods Mol.Biol., 125-142(2013)
Link: [ PubMED ] [ DOI ]
Title: Solvation models and computational prediction of orientations of peptides and proteins in membranes.
Author: Lomize,A.L., Pogozheva,I.D.
Abstract:
Membrane-associated peptides and proteins function in the highly heterogeneous environment of the lipid bilayer whose physico-chemical properties change non-monotonically along the bilayer normal. To simulate insertion of peptides and proteins into membranes and correctly reproduce the energetics of this process, an adequate solvation model and physically realistic representation of the lipid bilayer should be employed. We present a brief overview of the existing solvation models and their application for prediction of binding affinities and orientations of proteins in membranes. Particular emphasis is placed on the recently proposed PPM method, the corresponding web server, and the OPM database that were designed for positioning in membranes of integral and peripheral proteins with known three-dimensional structures.

Address: Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA

3915614g: Methods Mol.Biol., 19-36(2013)
Link: [ PubMED ] [ DOI ]
Title: Measurement of transmembrane peptide interactions in liposomes using Forster resonance energy transfer (FRET).
Author: Khadria,A., Senes,A.
Abstract:
Present day understanding of the thermodynamic properties of integral membrane proteins (IMPs) lags behind that of water-soluble proteins due to difficulties in mimicking the physiological environment of the IMPs in order to obtain a reversible folded system. Despite such challenges faced in studying these systems, significant progress has been made in the study of the oligomerization of single span transmembrane helices. One of the primary methods available to characterize these systems is based on Forster resonance energy transfer (FRET). FRET is a widely used spectroscopic tool that provides proximity data that can be fitted to obtain the energetics of a system. Here we discuss various technical aspects related to the application of FRET to study transmembrane peptide oligomerization in liposomes. The analysis is based on FRET efficiency relative to the concentration of the peptides in the bilayer (peptide:lipid ratio). Some important parameters that will be discussed include labeling efficiency, sample homogeneity, and equilibration. Furthermore, data analysis has to be performed keeping in mind random colocalization of donors and acceptors in liposome vesicles.

Address: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA

3915615g: Methods Mol.Biol., 3-18(2013)
Link: [ PubMED ] [ DOI ]
Title: Use of thiol-disulfide exchange method to study transmembrane peptide association in membrane environments.
Author: Cristian,L., Zhang,Y.
Abstract:
The development of methods for reversibly folding membrane proteins in a two-state manner remains a considerable challenge for studies of membrane protein stability. In recent years, a variety of techniques have been established and studies of membrane protein folding thermodynamics in the native bilayer environments have become feasible. Here we present the thiol-disulfide exchange method, a promising experimental approach for investigating the thermodynamics of transmembrane (TM) helix-helix association in membrane-mimicking environments. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. This experimental method provides a broadly applicable tool for thermodynamic studies of folding, oligomerization, and helix-helix interactions of membrane proteins.

Address: Influmedix, Inc., Radnor, PA, USA

3915761g: Methods Mol.Biol., 323-343(2013)
Link: [ PubMED ] [ DOI ]
Title: Structure-based prediction of Major Histocompatibility Complex (MHC) epitopes.
Author: Bordner,A.J.
Abstract:
Because of the enormous diversity of both MHC proteins and peptide epitopes, computational epitope prediction methods are needed in order to supplement limited experimental data. These prediction methods are useful for guiding experiments and have many potential biomedical applications. Unlike popular sequence-based methods, structure-based epitope prediction methods can predict epitopes for multiple MHC types with highly distinct peptide binding propensities. In this chapter, we describe in detail our previously developed structure-based epitope prediction methods for both class I and class II MHC proteins. We also discuss the relative advantages and disadvantages of sequence-based versus structure-based methods and how to evaluate prediction performance.

Address: Mayo Clinic, Scottsdale, AZ, USA

3915762g: Methods Mol.Biol., 245-264(2013)
Link: [ PubMED ] [ DOI ]
Title: Construction and screening of an antigen-derived peptide library displayed on yeast cell surface for CD4+ T cell epitope identification.
Author: Wen,F., Zhao,H.
Abstract:
Identification of T cell epitopes is a critical, but often difficult step in studying T cell function and developing peptide-based vaccines and immunotherapies. Unlike antibodies that recognize free soluble antigens, T cell receptor (TCR) recognizes its epitope bound to major histocompatibility complex (MHC) expressed on antigen presenting cells (APCs). In addition, the examination of T cell epitope activity requires the use of professional APCs, which are difficult to isolate, expand, and maintain. To address these issues, we have developed a facile, accurate, and high-throughput method for T cell epitope mapping by screening antigen-derived peptide libraries in complex with MHC protein displayed on yeast cell surface. Here, we use hemagglutinin and influenza A virus X31/A/Aichi/68 as examples to describe the key steps in identification of CD4+ T cell epitopes from a single antigenic protein and the entire genome of a pathogen, respectively. Methods for single-chain peptide-MHC complex vector design, yeast surface display, peptide library generation in Escherichia coli, and functional screening in Saccharomyces cerevisiae are discussed.

Address: Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA

3915763g: Methods Mol.Biol., 231-243(2013)
Link: [ PubMED ] [ DOI ]
Title: Identification and quantitation of MHC class II-bound peptides from mouse spleen dendritic cells by immunoprecipitation and mass spectrometry analysis.
Author: Bozzacco,L., Yu,H.
Abstract:
Advances in immunology and immune therapies require knowledge of antigenic peptide sequences that are presented on MHC class II and class I molecules of antigen presenting cells. The most specialized antigen presenting cells are dendritic cells (DCs). In the past, the small number of DCs that could be isolated from mouse spleen prevented direct analysis of the MHC II peptide repertoire presented by DCs. Here we describe a protocol that integrates immunological methods (in vivo enrichment of mouse spleen DCs by Flt3L treatment and immunoprecipitation of MHC II-peptide complexes), mass spectrometry analysis and peptide synthesis (LC-MS/MS and quantitation analysis for non tryptic peptides) to identify and quantitate the endogenous peptides that are bound to MHC II molecules on DCs. The described method produces quantitative data that are reproducible and reliable enough to cover a wide range of peptide copy numbers. We propose the application of this method in future studies to quantitatively investigate the MHC II repertoire on DCs presented during viral infections or different immunizations in vaccine development research.

Address: Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA

3915764g: Methods Mol.Biol., 211-229(2013)
Link: [ PubMED ] [ DOI ]
Title: Cell-based arrays for the identification of interacting polypeptide domains or epitopes.
Author: Maier,R.H., Maier,C.J., Onder,K.
Abstract:
The specific regions on proteins which are responsible for protein-protein interaction are called interacting domains, or epitopes in case of antigen-antibody binding. These domains are one feature to characterize proteins and are important in clinical diagnostics and research. For the mapping of such domains the use of protein/peptide arrays has become popular. Regardless of which kind of array, the major requirements are a high number of candidates arranged in the array, high quality, ease of use, and cost-effectiveness. Here, the authors describe a general protocol for mapping the interacting domains of proteins demonstrated by a high affinity protein interaction, the interaction of an antibody to an antigen. The chapter describes a stepwise protocol from library production to the verification of the domain by the use of an automated cell-based polypeptide array, which comprises the named requirements of a good array.

Address: Division of Molecular Dermatology, Department of Dermatology, Paracelsus Private Medical University, Salzburg, Austria

3915765g: Methods Mol.Biol., 181-195(2013)
Link: [ PubMED ] [ DOI ]
Title: Enrichment and characterization of glycopeptide epitopes from complex mixtures.
Author: Tessier,L., Fulton,K.M., Twine,S.M.
Abstract:
Antigen posttranslational modifications, including glycosylation, are recognized by the innate and adaptive arms of the immune system. Analytical approaches, including mass spectrometry and allied techniques, have allowed advances in the enrichment and identification of glyco-antigens, particularly T-cell epitopes. Similarly, major advances have been made in the identification, isolation, and detailed characterization of prokaryotic and eukaryotic glycoproteins and glycopeptides. In particular, peptide centric approaches are now capable of enriching low level glycopeptides from highly complex peptide mixtures. Similarly, advanced mass spectrometry methods allow identification of glycopeptides, characterization of glycans, and mapping of modification sites. Herein, we describe methods developed in our laboratory for the broad study of glycopeptides and illustrate how these approaches can be exploited to further our understanding of the identity and nature of glycopeptide epitopes in various diseases or auto immune disorders.

Address: Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, Canada

3915766g: Methods Mol.Biol., 141-153(2013)
Link: [ PubMED ] [ DOI ]
Title: An immunoproteomics approach to screen the antigenicity of the influenza virus.
Author: Downard,K.M.
Abstract:
The structure and antigenicity of protein antigens of the influenza virus are screened in a single step employing an immunoproteomics approach. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) coupled to gel electrophoresis is used both to identify viral antigens and screen their antigenicity. Earlier evidence that antigen-antibody complexes can survive on MALDI targets has allowed both the primary structure and antigenicity of viral strains to be rapidly screened with the specific localization of protein epitopes. The approach is anticipated to have a greater role in the future surveillance of the virus and should also aid in the development of immunogenic peptide constructs as alternatives to whole virus for vaccination.

Address: School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia

3915767g: Methods Mol.Biol., 79-95(2013)
Link: [ PubMED ] [ DOI ]
Title: Antigen discovery using whole-genome phage display libraries.
Author: Beghetto,E., Gargano,N.
Abstract:
In the last two decades phage display technology has been used for investigating complex biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda-display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen-Streptococcus pneumoniae-and the use of this library for the molecular dissection of the human B-cell response against bacterial infection and colonization.

Address: Sigma-Tau Industrie Farmaceutiche Riunite SpA, Rome, Italy

3915768g: Methods Mol.Biol., 21-57(2013)
Link: [ PubMED ] [ DOI ]
Title: Immunoproteomics: current technology and applications.
Author: Fulton,K.M., Twine,S.M.
Abstract:
The varied landscape of the adaptive immune response is determined by the peptides presented by immune cells, derived from viral or microbial pathogens or cancerous cells. The study of immune biomarkers or antigens is not new and classical methods such as agglutination, enzyme-linked immunosorbent assay, or Western blotting have been used for many years to study the immune response to vaccination or disease. However, in many of these traditional techniques, protein or peptide identification has often been the bottleneck. Recent advances in genomics and proteomics, has led to many of the rapid advances in proteomics approaches. Immunoproteomics describes a rapidly growing collection of approaches that have the common goal of identifying and measuring antigenic peptides or proteins. This includes gel based, array based, mass spectrometry, DNA based, or in silico approaches. Immunoproteomics is yielding an understanding of disease and disease progression, vaccine candidates, and biomarkers. This review gives an overview of immunoproteomics and closely related technologies that are used to define the full set of antigens targeted by the immune system during disease.

Address: Human Health Therapeutics, National Research Council Canada, Ottawa, ON, Canada

3916161g: Methods Mol.Biol., 527-539(2014)
Link: [ PubMED ] [ DOI ]
Title: Separation of the plant Golgi apparatus and endoplasmic reticulum by free-flow electrophoresis.
Author: Parsons,H.T., Fernandez-Nino,S.M., Heazlewood,J.L.
Abstract:
Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The plant Golgi and endoplasmic reticulum (ER) are similar in density and are therefore separated with difficulty using standard techniques such as density centrifugation. Purification of the ER and Golgi apparatus permits a biochemical and proteomic characterization which can reveal the division of processes between these compartments. Here we describe complete separation between the ER and more negatively charged Golgi compartments using ZE-FFE. We also describe techniques for assigning proteins to partially separated ER and the less negatively charged Golgi compartments.

Address: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

3916162g: Methods Mol.Biol., 379-389(2014)
Link: [ PubMED ] [ DOI ]
Title: Back to Osborne. Sequential Protein Extraction and LC-MS Analysis for the Characterization of the Holm Oak Seed Proteome.
Author: Romero-Rodriguez,M.C., Maldonado-Alconada,A.M., Valledor,L., Jorrin-Novo,J.V.
Abstract:
It is impossible to capture in just one experiment all or most of the total set of protein species that constitute the cell's proteome. Thus, according to our results, and even considering that they depend on the experimental system carried out (plant, yeast, fungi, or bacteria), the best protein extraction protocol yielded less than 20 % of the total amount of proteins, as determined by the Kjeldahl method. For this reason, protein cataloguing and the whole proteome characterization require the use of firstly, fractionation techniques at the cellular, subcellular, protein, or peptide level, and secondly, the use of complementary approaches.Within our current research on Holm oak (Quercus ilex subsp. ballota), we aim to characterize its seed proteome. For that we have optimized an experimental workflow in which the Osborne sequential protein extraction (Osborne, Science 28:417-427, 1908) is combined with downstream electrophoretic protein separation or shotgun MS analysis. In general, it can be used to study any plant seed, as well as to investigate on seed maturation and germination, genotype characterization, allergens identification, food traceability, and substantial equivalence, among others.

Address: Agroforestry and Plant Biochemistry and Proteomics Research Group, Department of Biochemistry and Molecular Biology, University of Cordoba, Cordoba, Spain

3916163g: Methods Mol.Biol., 333-346(2014)
Link: [ PubMED ] [ DOI ]
Title: Proteome analysis of orphan plant species, fact or fiction?
Author: Carpentier,S.C., America,T.
Abstract:
Biological research has focused in the past on model organisms and most of the functional genomics studies in the field of plant sciences are still performed on model species or reference species that are characterized to a great extent. However, numerous non-model plants are essential as food, feed, or energy resource. Some features and processes are unique to these plant species or families and cannot be approached via a model plant. The power of all proteomic and transcriptomic methods, i.e., high throughput identification of candidate gene products, tends to be lost in orphan species due to the lack of genomic information, the complexity of the genome (protein inference problem, polyploidy) or due to the sequence divergence to a related sequenced reference variety or to a related model organism. Nevertheless, a proteomics approach has a great potential to study orphan species. This chapter reviews concisely orphan plants from a proteomic angle and provides an outline of the problems encountered when initiating the proteome analysis of a non-model organism. We discuss briefly the problems and solutions for orphan plants associated with sample preparation and focus further on the difficulties associated with protein redundancy in polyploid species and the protein inference issue which is particularly associated with a peptide based proteomics approach.

Address: Faculty of Bioscience Engineering, Department of Biosystems, K.U. Leuven, Leuven, Belgium

3916164g: Methods Mol.Biol., 303-313(2014)
Link: [ PubMED ] [ DOI ]
Title: Medicago truncatula Proteomics for Systems Biology: Novel Rapid Shotgun LC-MS Approach for Relative Quantification Based on Full-Scan Selective Peptide Extraction (Selpex).
Author: Castillejo,M.A., Staudinger,C., Egelhofer,V., Wienkoop,S.
Abstract:
Medicago truncatula has become the focus of systems biology research for improved legume crop breeding. In plant systems biology, several comparative studies have been carried out using liquid chromatography shotgun mass spectrometry (LC-MS/MS) and database-dependent protein identification analyses in combination with the spectral count for relative quantification. In order to receive optimal protein identification rates and spectral count quantification, data-dependent tandem mass spectrometry with LC separation of more than 1 h is required. Thus LC-MS/MS analyses time is the bottleneck for high-throughput research of experiments with high sample number.We describe a novel method, called full-scan (FS) selective peptide extraction, that allows for comparative quantification of target peptides combined with a significant reduction in LC-MS analysis time. In future, it will be a useful tool to detect 15N-labeled selected peptide patterns for the targeted analysis of protein turnover and synthesis. We provide a first reference library of selected target peptides generated for M. truncatula leaf tissue. These peptides are also suitable candidates for selective reaction monitoring approaches.

Address: Department of Molecular Systems Biology, University of Vienna, Vienna, Austria

3916165g: Methods Mol.Biol., 275-288(2014)
Link: [ PubMED ] [ DOI ]
Title: Arabidopsis proteomics: a simple and standardizable workflow for quantitative proteome characterization.
Author: Rodiger,A., Agne,B., Baerenfaller,K., Baginsky,S.
Abstract:
Arabidopsis is the model plant of choice for large-scale proteome analyses, because its genome is well annotated, essentially free of sequencing errors, and relatively small with little redundancy. Furthermore, most Arabidopsis organs are susceptible to standard protein solubilization protocols making protein extraction relatively simple. Many different facets of functional plant proteomics were established with Arabidopsis such as mapping the subcellular proteomes of organelles, proteo-genomic peptide mapping, and numerous studies on the dynamic changes in protein modification and protein abundances. As most standard proteomics technologies are now routinely applied, research interest is increasingly shifting towards the reverse genetic characterization of gene function at the proteome level, i.e., by profiling the quantitative proteome of wild type in comparison with mutant plant tissue. We report here a simple, standardizable protocol for the large-scale comparative quantitative proteome characterization of different Arabidopsis organs based on normalized spectral counting and suggest a statistical framework for data interpretation. Based on existing organellar proteome maps, proteins can be assigned to organelles, thus allowing the identification of organelle-specific responses.

Address: Institut fur Biochemie, Abteilung Pflanzenbiochemie, Martin-Luther- Universitat Halle-Wittenberg, Halle (Saale), Germany

3916166g: Methods Mol.Biol., 209-222(2014)
Link: [ PubMED ] [ DOI ]
Title: Selected reaction monitoring mass spectrometry: a methodology overview.
Author: Ebhardt,H.A.
Abstract:
Moving past the discovery phase of proteomics, the term targeted proteomics combines multiple approaches investigating a certain set of proteins in more detail. One such targeted proteomics approach is the combination of liquid chromatography and selected or multiple reaction monitoring mass spectrometry (SRM, MRM). SRM-MS requires prior knowledge of the fragmentation pattern of peptides, as the presence of the analyte in a sample is determined by measuring the m/z values of predefined precursor and fragment ions. Using scheduled SRM-MS, many analytes can robustly be monitored allowing for high-throughput sample analysis of the same set of proteins over many conditions. In this chapter, fundaments of SRM-MS are explained as well as an optimized SRM pipeline from assay generation to data analyzed.

Address: Institute of Molecular Systems Biology, Eidgenossische Technische Hochschule (ETH) Zurich, Zurich, Switzerland

3916167g: Methods Mol.Biol., 199-208(2014)
Link: [ PubMED ] [ DOI ]
Title: Mass Western for absolute quantification of target proteins and considerations about the instrument of choice.
Author: Lyon,D., Weckwerth,W., Wienkoop,S.
Abstract:
The Mass Western describes the absolute quantification of proteins based on stable isotope labeled integral standard peptides and liquid chromatography coupled selective reaction monitoring triple quadrupole mass spectrometry (LC-SRM/MS). Here, we present a detailed workflow including tips and we discuss advantages and disadvantages of using different types of MS for absolute quantification.

Address: Department of Molecular Systems Biology, University of Vienna, Vienna, Austria

3916168g: Methods Mol.Biol., 185-198(2014)
Link: [ PubMED ] [ DOI ]
Title: Combining chymotrypsin/trypsin digestion to identify hydrophobic proteins from oil bodies.
Author: Vermachova,M., Purkrtova,Z., Santrucek,J., Jolivet,P., Chardot,T., Kodicek,M.
Abstract:
Oil bodies, lipid-storage organelles, are stabilized by a number of specific proteins. These proteins are very hydrophobic, which complicates their identification by "classical" proteomic protocols using trypsin digestion. Due to the lack of trypsin cleavage sites, the achievable protein coverage is limited or even insufficient for reliable protein identification. To identify such proteins and to enhance their coverage, we introduced a modified method comprising standard three-step procedure (SDS-PAGE, in-gel digestion, and LC-MS/MS analysis). In this method, chymotrypsin, single or in combination with trypsin, was used, which enabled to obtain proteolytic peptides from the hydrophobic regions and to identify new oil bodies' proteins. Our method can be easily applied to identification of other hydrophobic proteins.

Address: Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague, Czech Republic

3916169g: Methods Mol.Biol., 155-169(2014)
Link: [ PubMED ] [ DOI ]
Title: Differential Plant Proteome Analysis by Isobaric Tags for Relative and Absolute Quantitation (iTRAQ).
Author: Martinez-Esteso,M.J., Casado-Vela,J., Selles-Marchart,S., Pedreno,M.A., Bru-Martinez,R.
Abstract:
Protein relative quantitation is one of the main targets in many proteomic experiments. Among the range of techniques available for both top-down and bottom-up approaches, isobaric tags for relative and absolute quantitation (iTRAQ) have gained positions within the top-rank techniques used for this purpose in the recent years. Briefly, each iTRAQ reagent consists of three different components: a reporter group (with a variable mass in the range of 114-117 amu), a balance group, and an amino-reactive group. The isobaric nature of iTRAQ-labeled peptides adds a signal to every peptide in the sample which is detectable in both MS and MS/MS spectra, thus enhancing the sensitivity of detection. During MS/MS, the reporter groups are released as singly charged ions with m/z ratios ranking from 114 to 117 amu, visible in the low mass region of MS/MS spectra. The iTRAQ technology can be used to analyze up to four different samples using the 4-plex kit (reporter groups 114-115 amu) or can be scaled up to eight different samples using the 8-plex kit (reporter groups 113-121 amu). In this chapter, we focus on the experimental procedures typically using 4-plex labeling, including tips leading to successful application of iTRAQ technology for the analysis of plant protein mixtures.

Address: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University of Alicante, Alicante, Spain

3916170g: Methods Mol.Biol., 111-129(2014)
Link: [ PubMED ] [ DOI ]
Title: Plant proteomics methods to reach low-abundance proteins.
Author: Boschetti,E., Righetti,P.G.
Abstract:
The question of low-abundance proteins from biological tissues is still a major issue. Technologies have been devised to improve the situation and in the last few years a method based on solid-phase combinatorial peptide ligand libraries has been extensively applied to animal extracts. This method has also been extended to plant extracts taking advantage of findings from previous experience. Detailed methods are described and their pertinence highlighted according to various situations of plant sample origin, size of the sample, and analytical methods intended to be used for protein identifications.

Address: EB-Consult, Croissy, France

3916171g: Methods Mol.Biol., 15-27(2014)
Link: [ PubMED ] [ DOI ]
Title: From Proteomics to Systems Biology: MAPA, MASS WESTERN, PROMEX, and COVAIN as a User-Oriented Platform.
Author: Weckwerth,W., Wienkoop,S., Hoehenwarter,W., Egelhofer,V., Sun,X.
Abstract:
Genome sequencing and systems biology are revolutionizing life sciences. Proteomics emerged as a fundamental technique of this novel research area as it is the basis for gene function analysis and modeling of dynamic protein networks. Here a complete proteomics platform suited for functional genomics and systems biology is presented. The strategy includes MAPA (mass accuracy precursor alignment; http://www.univie.ac.at/mosys/software.html ) as a rapid exploratory analysis step; MASS WESTERN for targeted proteomics; COVAIN ( http://www.univie.ac.at/mosys/software.html ) for multivariate statistical analysis, data integration, and data mining; and PROMEX ( http://www.univie.ac.at/mosys/databases.html ) as a database module for proteogenomics and proteotypic peptides for targeted analysis. Moreover, the presented platform can also be utilized to integrate metabolomics and transcriptomics data for the analysis of metabolite-protein-transcript correlations and time course analysis using COVAIN. Examples for the integration of MAPA and MASS WESTERN data, proteogenomic and metabolic modeling approaches for functional genomics, phosphoproteomics by integration of MOAC (metal-oxide affinity chromatography) with MAPA, and the integration of metabolomics, transcriptomics, proteomics, and physiological data using this platform are presented. All software and step-by-step tutorials for data processing and data mining can be downloaded from http://www.univie.ac.at/mosys/software.html .

Address: Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, Austria

3916172g: Methods Mol.Biol., 3-13(2014)
Link: [ PubMED ] [ DOI ]
Title: Plant proteomics methods and protocols.
Author: Jorrin-Novo,J.V.
Abstract:
In this first, introductory chapter, it is intended to summarize from a methodological point of view the state of the art in plant proteomics, focusing on mass spectrometry-based strategies. Thus, this chapter is mainly directed at beginners or at those trying to get into the field, rather than at those with real experience or a long trajectory in plant proteomics research. The different alternative workflows, methods, techniques, and protocols from the experimental design to the data analysis will be briefly commented, with cross references to previous monographs and reviews, as well as to the rest of the book chapters. The difficulty of working with proteins, together with the power, limitations, and challenges of the approach will also be briefly discussed.Proteins, as molecular entities, and the cell proteome, as a whole, are much more complex than what we thought in the past and can be studied in a single experiment. Because of that, fractionation and complementary strategies are required for its study. The MS analysis of complex samples may result in up to 100,000-peptide spectra that cannot be easily analyzed with standard procedures. Therefore, proteomics, more than other -omics, needs a dry lab, time, and an effort in data mining.As main conclusion, it can be stated that proteomics is in its beginnings. It is starting to make important contributions to a proper gene annotation, identification, and characterization of gene products or protein species and to the knowledge of living organisms, having also an enormous application potential to translational research. However, and despite its great potential, and as in any other experimental approach, it is far from being a Pandora's Box. In the case of plant research, the full potential of proteomics is quite far from being totally exploited, and second-, third-, and fourth-generation proteomics techniques are still of very limited use. Most of the plant proteomics papers so far published belong to the descriptive, subcellular, and comparative proteomics subgroup, mainly using a few experimental model systems-those whose genome has been sequenced-and being from a biological point of view quite descriptive and speculative. From now on we should put more emphasis on the study of posttranslational proteomics and interactomics, and move to targeted, hypothesis-driven approaches. Furthermore, and even more important, we should move to data validation through other -omics or classical biochemical strategies, in an attempt to get a deeper, real, and more accurate view and understanding of cell biology. In the modern Systems Biology concept, proteomics must be considered as a part of a global, multidisciplinary approach. Making biological sense of a proteomics experiment requires a proper experimental design, data validation, interpretation, and publication policy.

Address: Agroforestry and Plant Biochemistry and Proteomics Research Group, Department of Biochemistry and Molecular Biology, University of Cordoba, Agrifood Campus of International Excellence, ceiA3, Cordoba, Spain