International Journal of Peptides

Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila

Mon, 29 Apr 2013 08:59:34 +0000

Tetrahymena thermophila are free-living, ciliated eukaryotes. Their behavioral response to stimuli is well characterized and easily observable, since cells swim toward chemoattractants and avoid chemorepellents. Chemoattractant responses involve increased swim speed or a decreased change in swim direction, while chemorepellent signaling involves ciliary reversal, which causes the organism to jerk back and forth, swim in small circles, or spin in an attempt to get away from the repellent. Many food sources, such as proteins, are chemoattractants for these organisms, while a variety of compounds are repellents. Repellents in nature are thought to come from the secretions of predators or from ruptured organisms, which may serve as “danger” signals. Interestingly, several peptides involved in vertebrate pain signaling are chemorepellents in Tetrahymena, including substances P, ACTH, PACAP, VIP, and nociceptin. Here, we characterize the response of Tetrahymena thermophila to three different isoforms of nociceptin. We find that G-protein inhibitors and tyrosine kinase inhibitors do not affect nociceptin avoidance. However, the calcium chelator, EGTA, and the SERCA calcium ATPase inhibitor, thapsigargin, both inhibit nociceptin avoidance, implicating calcium in avoidance. This result is confirmed by electrophysiology studies which show that 50 M nociceptin-NH2 causes a sustained depolarization of approximately 40 mV, which is eliminated by the addition of extracellular EGTA.

Interaction between Pirenzepine and Ninjinto, a Traditional Japanese Herbal Medicine, on the Plasma Gut-Regulated Peptide Levels in Humans

Yuhki Sato, Itoh Hiroki, Yosuke Suzuki, Ryosuke Tatsuta, and Masaharu Takeyama — Wed, 27 Mar 2013 14:56:00 +0000

The Japanese herbal medicine (Kampo) Ninjinto has been used for the treatment of gastroenteritis, esogastritis, gastric atony, gastrectasis, vomiting, and anorexia. The pharmacological effects of Ninjinto on the gastrointestine are due to changes in the levels of gut-regulated peptide, such as motilin, somatostatin, calcitonin gene-related peptide (CGRP), substance P, and vasoactive intestinal polypeptide (VIP). The release of these peptides is controlled by acetylcholine (ACh) from the preganglionic fibers of the parasympathetic nerve. Thus, we examined the effects of the selective M1 muscarinic receptor antagonist pirenzepine on the elevation of Ninjinto-induced plasma the area under the plasma gut-regulated peptide concentration-time curve from 0 to 240 min () in humans. Oral pretreatment with pirenzepine significantly reduced the Ninjinto-induced elevation of plasma motilin and substance P release (). Combined treatment with Ninjinto and pirenzepine significantly increased the release of plasma somatostatin () compared with administration of Ninjinto alone or placebo. Ninjinto appeared to induce the release of substance P and motilin into plasma mainly through the activation of M1 muscarinic receptors, and pirenzepine may affect the pharmacologic action of Ninjinto by the elevation of plasma substance P, motilin, and somatostatin.

Significant Increase in Salivary Substance P Level after a Single Oral Dose of Cevimeline in Humans

Sun, 24 Mar 2013 11:51:57 +0000

Cevimeline is a novel muscarinic acetylcholine receptor agonist currently being developed as a therapeutic agent for xerostomia. We examined the effects of cevimeline on salivary and plasma levels of substance-P- (SP-), calcitonin-gene-related-peptide- (CGRP-), and vasoactive-intestinal-polypeptide- (VIP-) like immunoreactive substances (ISs) in humans. An open-labeled crossover study was conducted on seven healthy volunteers. Saliva volume was measured, and saliva and venous blood samples were collected before and 30–240 min after a single oral dose of cevimeline or placebo. Salivary and plasma levels of SP-, CGRP-, and VIP-IS were measured using a highly sensitive enzyme immunoassay. A single oral dose of cevimeline resulted in significant increases in salivary but not plasma SP-IS level compared to placebo. Cevimeline administration did not alter the salivary or plasma levels of CGRP-IS or VIP-IS compared to placebo. Significant increases in salivary volume were observed after cevimeline administration compared to placebo. A significant correlation was observed between the total release of SP-IS and that of salivary volume. These findings suggest an association of SP with the enhancement of salivary secretion by cevimeline.

Antimicrobial Lactoferrin Peptides: The Hidden Players in the Protective Function of a Multifunctional Protein

Mau Sinha, Sanket Kaushik, Punit Kaur, Sujata Sharma, and Tej P. Singh — Sun, 10 Mar 2013 13:40:07 +0000

Lactoferrin is a multifunctional, iron-binding glycoprotein which displays a wide array of modes of action to execute its primary antimicrobial function. It contains various antimicrobial peptides which are released upon its hydrolysis by proteases. These peptides display a similarity with the antimicrobial cationic peptides found in nature. In the current scenario of increasing resistance to antibiotics, there is a need for the discovery of novel antimicrobial drugs. In this context, the structural and functional perspectives on some of the antimicrobial peptides found in N-lobe of lactoferrin have been reviewed. This paper provides the comparison of lactoferrin peptides with other antimicrobial peptides found in nature as well as interspecies comparison of the structural properties of these peptides within the native lactoferrin.

Systemic Ghrelin Administration Alters Serum Biomarkers of Angiogenesis in Diet-Induced Obese Mice

M. Khazaei and Z. Tahergorabi — Thu, 28 Feb 2013 09:38:18 +0000

Introduction. Ghrelin is a gastrointestinal endocrine peptide that was initially identified as the endogenous ligand of growth hormone secretagogue receptor; however, recently, the cardiovascular effect of this peptide has been indicated. In this study, we investigated the effect of ghrelin administration on serum biomarkers of angiogenesis including leptin, nitric oxide (NO), vascular endothelial growth factor (VEGF), and its soluble receptor (VEGF receptor 1 or sFlt-1) in control- and diet-induced obese mice. Methods. Male C57BL/6 mice were randomly divided into four groups, normal diet (ND) or control, ND + ghrelin, high-fat-diet (HFD) or obese and HFD + ghrelin (/group). Obese and control groups received either HFD or ND for 15 weeks. Then, the ghrelin was injected subcutaneously 100 µg/kg twice daily for 10 days. At the end of experiment, blood samples were collected for blood glucose, serum insulin, VEGF, sFlt-1, NO, and leptin measurements. Results. The obese animals had higher serum NO and leptin concentrations without changes in serum VEGF and sFlt-1 levels compared to control. Administration of ghrelin significantly increased serum VEGF and decreased serum leptin and NO concentrations in HFD group. Conclusion. Since ghrelin changes serum biomarkers of angiogenesis, it seems that it gets involved during states with abnormal angiogenesis.

α-RgIB: A Novel Antagonist Peptide of Neuronal Acetylcholine Receptor Isolated from Conus regius Venom

Wed, 27 Feb 2013 17:54:19 +0000

Conus venoms are rich sources of biologically active peptides that act specifically on ionic channels and metabotropic receptors present at the neuromuscular junction, efficiently paralyzing the prey. Each species of Conus may have 50 to 200 uncharacterized bioactive peptides with pharmacological interest. Conus regius is a vermivorous species that inhabits Northeastern Brazilian tropical waters. In this work, we characterized one peptide with activity on neuronal acetylcholine receptor (nAChR). Crude venom was purified by reverse-phase HPLC and selected fractions were screened and sequenced by mass spectrometry, MALDI-ToF, and ESI-Q-ToF, respectively. A new peptide was identified, bearing two disulfide bridges. The novel 2,701 Da peptide belongs to the cysteine framework I, corresponding to the cysteine pattern CC-C-C. The biological activity of the purified peptide was tested by intracranial injection in mice, and it was observed that high concentrations induced hyperactivity in the animals, whereas lower doses caused breathing difficulty. The activity of this peptide was assayed in patch-clamp experiments, on nAChR-rich cells, in whole-cell configuration. The peptide blocked slow rise-time neuronal receptors, probably α3β4 and/or α3β4α5 subtype. According to the nomenclature, the new peptide was designated as α-RgIB.

Phage Display Screening for Tumor Necrosis Factor-α-Binding Peptides: Detection of Inflammation in a Mouse Model of Hepatitis

Tue, 26 Feb 2013 15:50:41 +0000

TNF-α is one of the most abundant cytokines produced in many inflammatory and autoimmune conditions such as multiple sclerosis, chronic hepatitis C, or neurodegenerative diseases. These pathologies remain difficult to diagnose and consequently difficult to treat. The aim of this work is to offer a new diagnostic tool by seeking new molecular probes for medical imaging. The target-specific part of the probe consists here of heptameric peptides selected by the phage display technology for their affinity for TNF-α. Several affinity tests allowed isolating 2 peptides that showed the best binding capacity to TNF-α. Finally, the best peptide was synthesized in both linear and cyclic forms and tested on the histological sections of concanavalin-A-(ConA-)treated mice liver. In this well-known hepatitis mouse model, the best results were obtained with the cyclic form of peptide 2, which allowed for the staining of inflamed areas in the liver. The cyclic form of peptide 2 (2C) was, thus, covalently linked to iron oxide nanoparticles (magnetic resonance imaging (MRI) contrast agent) and tested in the ConA-induced hepatitis mouse model. The vectorized nanoparticles allowed for the detection of inflammation as well as of the free peptide. These ex vivo results suggest that phage display-selected peptides can direct imaging contrast agents to inflammatory areas.

Sequence Determination of a Novel Tripeptide Isolated from the Young Leaves of Azadirachta indica A. Juss

M. Rajeswari Prabha and B. Ramachandramurty — Wed, 20 Feb 2013 10:10:47 +0000

The neem tree has long been recognized for its unique properties, both against insects and in improving human health. Every part of the tree has been used as a traditional medicine for household remedy against various human ailments, from antiquity. Although the occurrence of various phytochemicals in neem has been studied, we have identified the presence of a novel tripeptide in the young leaves of neem using a simple and inexpensive paper chromatographic method, detected by Cu(II)-ninhydrin reagent. The peptide nature of the isolated compound is confirmed by spectral studies. The sequence of the peptide is determined using de novo sequencing by tandem MS after purification.

Peptide Receptor Targeting in Cancer: The Somatostatin Paradigm

Thu, 07 Feb 2013 12:00:29 +0000

Peptide receptors involved in pathophysiological processes represent promising therapeutic targets. Neuropeptide somatostatin (SST) is produced by specialized cells in a large number of human organs and tissues. SST primarily acts as inhibitor of endocrine and exocrine secretion via the activation of five G-protein-coupled receptors, named sst1–5, while in central nervous system, SST acts as a neurotransmitter/neuromodulator, regulating locomotory and cognitive functions. Critical points of SST/SST receptor biology, such as signaling pathways of individual receptor subtypes, homo- and heterodimerization, trafficking, and cross-talk with growth factor receptors, have been extensively studied, although functions associated with several pathological conditions, including cancer, are still not completely unraveled. Importantly, SST exerts antiproliferative and antiangiogenic effects on cancer cells in vitro, and on experimental tumors in vivo. Moreover, SST agonists are clinically effective as antitumor agents for pituitary adenomas and gastro-pancreatic neuroendocrine tumors. However, SST receptors being expressed by tumor cells of various tumor histotypes, their pharmacological use is potentially extendible to other cancer types, although to date no significant results have been obtained. In this paper the most recent findings on the expression and functional roles of SST and SST receptors in tumor cells are discussed.

Identification and Characterization of a Novel Nontranslated Sequence Variant of the Human Intestinal Di-/Tripeptide Transporter, hPEPT1

Helle Bach Søndergaard, Carsten Uhd Nielsen, and Birger Brodin — Sun, 30 Dec 2012 08:47:01 +0000

The human H+-coupled di-/tripeptide transporter (hPEPT1) mediates intestinal absorption of dietary di- and tripeptides, as well as several peptidomimetic drug compounds. The aim of the present study was to investigate the possible role of the hPEPT1 variant hPEPT1-RF in hPEPT1 regulation. However, the proposed hPEPT1-RF mRNA sequence could not be detected in Caco-2 cells or in human intestinal samples. Instead, a new sequence variant, hPEPT1-RFI, was found, which is almost identical to the proposed hPEPT1-RF, except for two nucleotide insertions and one deletion that resulted in a changed open reading frame as compared to hPEPT1-RF. In vitro translation analysis showed that hPEPT1-RFI was not translated. In conclusion, the existence of hPEPT1-RF could not be confirmed; furthermore, the identified sequence variant, hPEPT1-RFI, does not appear to be translated and is therefore unlikely to have a regulatory effect on hPEPT1 transport activity.

Erratum to “Characterization of Selective Antibacterial Peptides by Polarity Index”

Mon, 24 Dec 2012 09:16:47 +0000

Peptidomic Analysis of the Brain and Corpora Cardiaca-Corpora Allata Complex in the Bombyx mori

Mon, 17 Dec 2012 13:07:17 +0000

The silkworm, Bombyx mori, is an important economic insect for silk production. However, many of the mature peptides relevant to its various life stages remain unknown. Using RP-HPLC, MALDI-TOF MS, and previously identified peptides from B. mori and other insects in the transcriptome database, we created peptide profiles showing a total of 6 ion masses that could be assigned to peptides in eggs, including one previously unidentified peptide. A further 49 peptides were assigned to larval brains. 17 new mature peptides were identified in isolated masses. 39 peptides were found in pupal brains with 8 unidentified peptides. 48 were found in adult brains with 12 unidentified peptides. These new unidentified peptides showed highly significant matches in all MS analysis. These matches were then searched against the National Center for Biotechnology Information (NCBI) database to provide new annotations for these mature peptides. In total, 59 mature peptides in 19 categories were found in the brains of silkworms at the larval, pupal, and adult stages. These results demonstrate that peptidomic variation across different developmental stages can be dramatic. Moreover, the corpora cardiaca-corpora allata (CC-CA) complex was examined during the fifth larval instar. A total of 41 ion masses were assigned to peptides.

Peptide-Modulated Activity Enhancement of Acidic Protease Cathepsin E at Neutral pH

Masayuki Komatsu, Madhu Biyani, Sunita Ghimire Gautam, and Koichi Nishigaki — Mon, 17 Dec 2012 09:48:51 +0000

Enzymes are regulated by their activation and inhibition. Enzyme activators can often be effective tools for scientific and medical purposes, although they are more difficult to obtain than inhibitors. Here, using the paired peptide method, we report on protease-cathepsin-E-activating peptides that are obtained at neutral pH. These selected peptides also underwent molecular evolution, after which their cathepsin E activation capability improved. Thus, the activators we obtained could enhance cathepsin-E-induced cancer cell apoptosis, which indicated their potential as cancer drug precursors.

Synthesis of Hemopressin Peptides by Classical Solution Phase Fragment Condensation

P. Anantha Reddy, Sean T. Jones, Anita H. Lewin, and F. Ivy Carroll — Tue, 27 Nov 2012 11:11:51 +0000

A fragment condensation solution phase assembly of the naturally occurring CB1 inverse agonist nonapeptides, Pro-Val-Asn-Phe-Lys-Phe/Leu-Leu-Ser-His-OH (hemopressins), and two other homologues: N-terminal 2-amino acid (dipeptide) extended undecapeptide, Val-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH, and three-amino acid (tripeptide) extended dodecapeptide, Arg-Val-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH, both CB1 agonists, is reported.

Diet-Induced Obesity in Mice Overexpressing Neuropeptide Y in Noradrenergic Neurons

Suvi T. Ruohonen, Laura H. Vähätalo, and Eriika Savontaus — Thu, 18 Oct 2012 16:42:13 +0000

Neuropeptide Y (NPY) is a neurotransmitter associated with feeding and obesity. We have constructed an NPY transgenic mouse model (OE- mouse), where targeted overexpression leads to increased levels of NPY in noradrenergic and adrenergic neurons. We previously showed that these mice become obese on a normal chow. Now we aimed to study the effect of a Western-type diet in OE- and wildtype (WT) mice, and to compare the genotype differences in the development of obesity, insulin resistance, and diabetes. Weight gain, glucose, and insulin tolerance tests, fasted plasma insulin, and cholesterol levels were assayed. We found that female OE- mice gained significantly more weight without hyperphagia or decreased activity, and showed larger white and brown fat depots with no difference in UCP-1 levels. They also displayed impaired glucose tolerance and decreased insulin sensitivity. OE- and WT males gained weight robustly, but no difference in the degree of adiposity was observed. However, 40% of but none of the WT males developed hyperglycaemia while on the diet. The present study shows that female OE- mice were not protected from the obesogenic effect of the diet suggesting that increased NPY release may predispose females to a greater risk of weight gain under high caloric conditions.

Large Scale Solid Phase Synthesis of Peptide Drugs: Use of Commercial Anion Exchange Resin as Quenching Agent for Removal of Iodine during Disulphide Bond Formation

Mon, 15 Oct 2012 17:43:06 +0000

The S-acetamidomethyl (Acm) or trityl (Trt) protecting groups are widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report here a simple method for simultaneous quenching and removal of iodine and isolation of disulphide bridge peptides. The use of excess inexpensive anion exchange resin to the oxidized peptide from the aqueous acetic acid/methanol solution affords quantitative removal of iodine and other color impurities. This improves the resin life time of expensive chromatography media that is used in preparative HPLC column during the purification of peptide using preparative HPLC. Further, it is very useful for the conversion of TFA salt to acetate in situ. It was successfully applied commercially, to the large scale synthesis of various peptides including Desmopressin, Oxytocin, and Octreotide. This new approach offers significant advantages such as more simple utility, minimal side reactions, large scale synthesis of peptide drugs, and greater cost effectiveness.

Natural Peptides with Potential Applications in Drug Development, Diagnosis, and/or Biotechnology

Mirian A. F. Hayashi, Frédéric Ducancel, and Katsuhiro Konno — Thu, 09 Aug 2012 09:53:25 +0000

A Meta-Analysis of the Therapeutic Effects of Glucagon-Like Peptide-1 Agonist in Heart Failure

Mohammed Munaf, Pierpaolo Pellicori, Victoria Allgar, and Kenneth Wong — Sun, 01 Jul 2012 13:59:36 +0000

We conducted a meta-analysis of the existing literature of the therapeutic effects of using GLP-1 agonists to improve the metabolism of the failing heart. Animal studies showed significant improvement in markers of cardiac function, such as left ventricular ejection fraction (LVEF), with regular GLP-1 agonist infusions. In clinical trials, the potential effects of GLP-1 agonists in improving cardiac function were modest: LVEF improved by 4.4% compared to placebo (95% C.I 1.36–7.44, 𝑃=0.005). However, BNP levels were not significantly altered by GLP-1 agonists in heart failure. In two trials, a modest increase in heart rate by up to 7 beats per minute was noted, but meta-analysis demonstrated this was not significant statistically. The small number of studies plus variation in the concentration and length of the regime between the trials would limit our conclusions, even though statistically, heterogeneity chi-squared tests did not reveal any significant heterogeneity in the endpoints tested. Moreover, studies in non-diabetics with heart failure yielded conflicting results. In conclusion, the use of GLP-1 agonists has at best a modest effect on ejection fraction improvement in heart failure, but there was no significant improvement in BNP levels in the meta-analysis.

Effects of Endurance and Resistance Training on Calcitonin Gene-Related Peptide and Acetylcholine Receptor at Slow and Fast Twitch Skeletal Muscles and Sciatic Nerve in Male Wistar Rats

Mon, 18 Jun 2012 10:19:58 +0000

The aim of this study was to investigate effects of endurance and resistance training (ET and RT) on CGRP and AChRs at slow and fast twitch muscles and sciatic nerve in rats. Twenty-five male rats were randomly assigned into three groups including sedentary (SED), endurance training (ET), and resistance training (RT). Animals of ET exercised for 12 weeks, five times/week, and 60 min/day at 30 m/min. Animals of RT were housed in metal cage with 2 m high wire-mesh tower, with water bottles set at the top. 48 h after the last session of training protocol, animals were anaesthetized. The right sciatic nerves were removed; then, Soleus (SOL) and Tibialis anterior (TA) muscles were excised and immediately snap frozen in liquid nitrogen. All frozen tissues were stored at −80°C. Results showed that, after both ET and RT, CGRP content as well as AChR content of SOL and TA muscles significantly increased. But there was no significant difference among groups at sciatic nerve’ CGRP content. In conclusion, data demonstrate that ET and RT lead to changes of CGRP and AChR content of ST and FT muscles. The changes indicate to the importance of neuromuscular activity.

Pan-PPAR Agonist, Bezafibrate, Restores Angiogenesis in Hindlimb Ischemia in Normal and Diabetic Rats

M. Khazaei, E. Salehi, and B. Rashidi — Sun, 03 Jun 2012 08:20:38 +0000

Introduction. The aim of this study was to investigate the effect of bezafibrate as a pan-PPAR agonist on angiogenesis and serum nitrite, the main metabolite of nitric oxide (NO), vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) concentrations in hindlimb ischemia model of normal and type I diabetic rats. Methods. 28 male Wistar rats were divided into control and diabetic groups. Then, all rats underwent unilateral hindlimb ischemia. After recovery, they were randomly assigned to one of the following experimental groups: (1) control; (2) control + bezafibrate (400 mg/kg/day); (3) diabetic; (4) diabetic + beztafibrate. After three weeks, blood samples were taken and capillary density was evaluated in the gasterocnemius muscle of ischemic limb. Results. Bezafibrate increased capillary density and capillary/fiber ratio in ischemic leg of diabetic and control rats (𝑃<0.05). Serum VEGF and VEGFR-2 concentrations did not alter after bezafibrate administration, however, serum nitrite concentration was significantly higher in bezafibrate-treated groups than non-treated groups (𝑃<0.05). Discussion. It seems that bezafibrate, as a pan PPAR agonist, restores angiogenesis in hindlimb ischemic diabetic animals and is useful for prevention and/or treatment of peripheral artery disease in diabetic subjects.

Antimicrobial Peptides as Infection Imaging Agents: Better Than Radiolabeled Antibiotics

Thu, 17 May 2012 10:52:48 +0000

Nuclear medicine imaging techniques offer whole body imaging for localization of number and site of infective foci inspite of limitation of spatial resolution. The innate human immune system contains a large member of important elements including antimicrobial peptides to combat any form of infection. However, development of antibiotics against bacteria progressed rapidly and gained popularity over antimicrobial peptides but even powerful antimicrobials failed to reduce morbidity and mortality due to emergence of mutant strains of bacteria resulting in antimicrobial resistance. Differentiation between infection and inflammation using radiolabeled compounds with nuclear medicine techniques has always been a dilemma which is still to be resolved. Starting from nonspecific tracers to specific radiolabeled tracers, the question is still unanswered. Specific radiolabeled tracers included antibiotics and antimicrobial peptides which bind directly to the bacteria for efficient localization with advanced nuclear medicine equipments. However, there are merits and demerits attributed to each. In the current paper, radiolabeled antibiotics and radiolabeled peptides for infection localization have been discussed starting with the background of primitive nonspecific tracers. Radiolabeled antimicrobial peptides have certain merits compared with labeled antibiotics which make them superior agents for localization of infective focus.

Proline Rich Motifs as Drug Targets in Immune Mediated Disorders

Mythily Srinivasan and A. Keith Dunker — Wed, 16 May 2012 10:13:20 +0000

The current version of the human immunome network consists of nearly 1400 interactions involving approximately 600 proteins. Intermolecular interactions mediated by proline-rich motifs (PRMs) are observed in many facets of the immune response. The proline-rich regions are known to preferentially adopt a polyproline type II helical conformation, an extended structure that facilitates transient intermolecular interactions such as signal transduction, antigen recognition, cell-cell communication and cytoskeletal organization. The propensity of both the side chain and the backbone carbonyls of the polyproline type II helix to participate in the interface interaction makes it an excellent recognition motif. An advantage of such distinct chemical features is that the interactions can be discriminatory even in the absence of high affinities. Indeed, the immune response is mediated by well-orchestrated low-affinity short-duration intermolecular interactions. The proline-rich regions are predominantly localized in the solvent-exposed regions such as the loops, intrinsically disordered regions, or between domains that constitute the intermolecular interface. Peptide mimics of the PRM have been suggested as potential antagonists of intermolecular interactions. In this paper, we discuss novel PRM-mediated interactions in the human immunome that potentially serve as attractive targets for immunomodulation and drug development for inflammatory and autoimmune pathologies.

Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi

Brian Hall, Carley Squires, and Keith K. Parker — Wed, 09 May 2012 12:16:59 +0000

Peptide mimics of intracellular loop 2 (ic2) of the human 5HT1a receptor have been studied with respect to their ability to inhibit agonist binding via interference with receptor-G-protein coupling. These peptides give shallow concentration-effect relationships. Additionally, these peptides have been studied with respect to their ability to trigger the signal transduction system of this Gi-coupled receptor. Two signaling parameters have been quantified: concentration of intracellular cAMP and changes in incorporation into the G protein of a stable analog of GTP. In both cases, peptide mimics near midloop of ic2 actually show agonist activity with efficacy falling off toward both loop termini near TM 3 and TM 4. Previous results have suggested that the loop region near the TM3/ic2 interface is primarily responsible for receptor-G-protein coupling, while the current result emphasizes the mid-ic2 loop region's ability to activate the G protein following initial coupling. A limited number of peptides from the receptor's TM5/ic3 loop vicinity were also studied regarding agonist inhibition and G-protein activation. These peptides provide additional evidence that the human 5HT1a receptor, TM5/ic3 loop region, is involved in both coupling and activation actions. Overall, these results provide further information about potential pharmacological intervention and drug development with respect to the human 5HT1a receptor/G-protein system. Finally, the structural evidence generated here provides testable models pending crystallization and X-ray analysis of the receptor.

Pyrazinamide Effects on Cartilage Type II Collagen Amino Acid Composition

Larysa B. Bondarenko and Valentina M. Kovalenko — Mon, 07 May 2012 15:06:05 +0000

Introduction. Current therapeutic regimens with first-line antitubercular agents are associated to a high rate of adverse effects which could cause pronounced changes in collagen's contents and structure. Investigation of these changes is very important for optimization of antitubercular therapy and minimization of treatment-caused harm. The aim of present paper was to investigate potential effect of pyrazinamide on male rats' cartilage type II collagen amino acid composition. Materials and Methods. Wistar albino male rats (160–200 g b.w.) were divided into three groups: I—received pyrazinamide per os at a dose of 1000 mg/kg b.w./day; II—at a dose of 2000 mg/kg b.w./day, in both groups it was given for 60 days; III—control. After 60 days of the experiment, rats of the experimental (groups I and II) and control groups were sacrificed and the amino acids contents of male rat cartilage type II collagens were determined using amino acid analyzer. Results and Discussion. The study of pyrazinamide effects (administered in different doses) on rat cartilage type II collagen amino acid contents demonstrated presence of dose-dependent pyrazinamide-mediated quantitative and qualitative changes in these rat extracellular matrix proteins in comparison with control.

Amyloid Beta Peptide Slows Down Sensory-Induced Hippocampal Oscillations

Fernando Peña-Ortega and Ramón Bernal-Pedraza — Sun, 29 Apr 2012 11:40:55 +0000

Alzheimer’s disease (AD) progresses with a deterioration of hippocampal function that is likely induced by amyloid beta (Aβ) oligomers. Hippocampal function is strongly dependent on theta rhythm, and disruptions in this rhythm have been related to the reduction of cognitive performance in AD. Accordingly, both AD patients and AD-transgenic mice show an increase in theta rhythm at rest but a reduction in cognitive-induced theta rhythm. We have previously found that monomers of the short sequence of Aβ (peptide 25–35) reduce sensory-induced theta oscillations. However, considering on the one hand that different Aβ sequences differentially affect hippocampal oscillations and on the other hand that Aβ oligomers seem to be responsible for the cognitive decline observed in AD, here we aimed to explore the effect of Aβ oligomers on sensory-induced theta rhythm. Our results show that intracisternal injection of Aβ1–42 oligomers, which has no significant effect on spontaneous hippocampal activity, disrupts the induction of theta rhythm upon sensory stimulation. Instead of increasing the power in the theta band, the hippocampus of Aβ-treated animals responds to sensory stimulation (tail pinch) with an increase in lower frequencies. These findings demonstrate that Aβ alters induced theta rhythm, providing an in vivo model to test for therapeutic approaches to overcome Aβ-induced hippocampal and cognitive dysfunctions.

Characterization of Selective Antibacterial Peptides by Polarity Index

Sun, 01 Apr 2012 19:11:04 +0000

In the recent decades, antibacterial peptides have occupied a strategic position for pharmaceutical drug applications and became subject of intense research activities since they are used to strengthen the immune system of all living organisms by protecting them from pathogenic bacteria. This work proposes a simple and easy statistical/computational method through a peptide polarity index measure by which an antibacterial peptide subgroup can be efficiently identified, that is, characterized by a high toxicity to bacterial membranes but presents a low toxicity to mammal cells. These peptides also have the feature not to adopt to an alpha-helicoidal structure in aqueous solution. The double-blind test carried out to the whole Antimicrobial Peptide Database (November 2011) showed an accuracy of 90% applying the polarity index method for the identification of such antibacterial peptide groups.

Identification of Histoplasma-Specific Peptides in Human Urine

Mon, 26 Mar 2012 08:42:58 +0000

Histoplasmosis is a severe dimorphic fungus infection, which is often difficult to diagnose due to similarity in symptoms to other diseases and lack of specific diagnostic tests. Urine samples from histoplasma-antigen-positive patients and appropriate controls were prepared using various sample preparation strategies including immunoenrichment, ultrafiltration, high-abundant protein depletion, deglycosylation, reverse-phase fractions, and digest using various enzymes. Samples were then analyzed by nanospray tandem mass spectrometry. Accurate mass TOF scans underwent molecular feature extraction and statistical analysis for unique disease makers, and acquired MS/MS data were searched against known human and histoplasma proteins. In human urine, some 52 peptides from 37 Histoplasma proteins were identified with high confidence. This is the first report of identification of a large number of Histoplasma-specific peptides from immunoassay-positive patient samples using tandem mass spectrometry and bioinformatics techniques. These findings may lead to novel diagnostic markers for histoplasmosis in human urine.

Angiotensin-Converting Enzyme 2 (ACE2) Is a Key Modulator of the Renin Angiotensin System in Health and Disease

Chris Tikellis and M. C. Thomas — Tue, 20 Mar 2012 14:36:57 +0000

Angiotensin-converting enzyme 2 (ACE2) shares some homology with angiotensin-converting enzyme (ACE) but is not inhibited by ACE inhibitors. The main role of ACE2 is the degradation of Ang II resulting in the formation of angiotensin 1–7 (Ang 1–7) which opposes the actions of Ang II. Increased Ang II levels are thought to upregulate ACE2 activity, and in ACE2 deficient mice Ang II levels are approximately double that of wild-type mice, whilst Ang 1–7 levels are almost undetectable. Thus, ACE2 plays a crucial role in the RAS because it opposes the actions of Ang II. Consequently, it has a beneficial role in many diseases such as hypertension, diabetes, and cardiovascular disease where its expression is decreased. Not surprisingly, current therapeutic strategies for ACE2 involve augmenting its expression using ACE2 adenoviruses, recombinant ACE2 or compounds in these diseases thereby affording some organ protection.

Effect of Rabbit Epididymal Antimicrobial Peptide, REHb𝜷P, on LPS-Induced Proinflammatory Cytokine Responses in Human Vaginal Cells In Vitro

K. V. R. Reddy, D. Sukanya, M. S. Patgaonkar, and C. Selvaakumar — Wed, 14 Mar 2012 09:05:57 +0000

Antimicrobial peptides (AMP’s) protect epithelial surfaces including epididymis against pathogens and play a key role in orchestrating various defensive responses. Recently, we have identified one such AMP, rabbit epididymal hemoglobin-β subuit (REHbβP) from the epididymal fluid of rabbit, Oryctologus cuniculus. The demonstration of a protective role of REHbβP in epididymal epithelial cells (EPEC’s) led us to investigate: (1) the identification of LPS interactive domain in REHbβP, and (2) whether the REHbβP of rabbit origin mediates vaginal cellular immune responses of another species (human). HeLa-S3, human vaginal epithelial cells (hVECs) were exposed to LPS or the LPS-stimulated cells treated with REHbβP or neutral peptide, nREHbβP. Effect of LPS and cytokines (IL-6 and IL-1𝛼) and chemokines (IL-8, MCP-1) levels was determined in the culture supernatants. In response to the LPS, hVECs synthesized these mediators and the levels were significantly higher than controls. This enhancing effect was ameliorated when the LPS-induced hVECs were treated with REHbβP. Similar results were obtained on NF-κB protein and hBD-1 mRNA expression. Confocal microscopy studies revealed that REHbβP attenuated the LPS-induced internalization of E. coli by macrophages. The chemotaxis studies performed using Boyden chamber Transwell assay, which showed elevated migration of U937 cells when the supernatants of LPS-induced hVECs were used, and the effect was inhibited by REHbβP. REHbβP was found to be localized on the acrosome of rabbit spermatozoa, suggesting its role in sperm protection beside sperm function. In conclusion, REHbβP may have the potential to develop as a therapeutic agent for reproductive tract infections (RTI’s).

Molecular Cloning and Sequence Analysis of the cDNAs Encoding Toxin-Like Peptides from the Venom Glands of Tarantula Grammostola rosea

Tadashi Kimura, Seigo Ono, and Tai Kubo — Wed, 29 Feb 2012 10:05:10 +0000

Tarantula venom glands produce a large variety of bioactive peptides. Here we present the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared from the venom glands of the Chilean common tarantula, Grammostola rosea. The cDNA sequences of about 1500 clones out of 4000 clones were analyzed after selection using several criteria. Forty-eight novel toxin-like peptides (GTx1 to GTx7, and GTx-TCTP and GTx-CRISP) were predicted from the nucleotide sequences. Among these peptides, twenty-four toxins are ICK motif peptides, eleven peptides are MIT1-like peptides, and seven are ESTX-like peptides. Peptides similar to JZTX-64, aptotoxin, CRISP, or TCTP are also obtained. GTx3 series possess a cysteine framework that is conserved among vertebrate MIT1, Bv8, prokineticins, and invertebrate astakines. GTx-CRISP is the first CRISP-like protein identified from the arthropod venom. Real-time PCR revealed that the transcripts for TCTP-like peptide are expressed in both the pereopodal muscle and the venom gland. Furthermore, a unique peptide GTx7-1, whose signal and prepro sequences are essentially identical to those of HaTx1, was obtained.