Peptides (v.32, #10)

► Lunasin affected genes involved in cellular growth and proliferation, cellular function and maintenance, and cell to cell signaling and interaction. ► We confirmed the pro-apoptotic activity of lunasin which might be attributed to its RGD motif. ► Our findings support the potential chemopreventive and chemotherapeutic use of this novel peptide.Lunasin is a novel peptide from soybean with demonstrated chemopreventive property. We compared the effect of lunasin on gene expression of RAW 264.7 macrophages with and without lipopolysaccharide (LPS)-stimulation. Our hypothesis was that lunasin will have a differential effect in RAW 264.7 gene expression in a normal and challenged state. Analysis of the microarray data using False Discovery Rate (FDR) method resulted in the identification of 340 up-regulated and 162 down-regulated genes (FDR p-value <0.05) associated with simultaneous treatment of lunasin and LPS for 24 h. Treatment of lunasin with no LPS for 24 h resulted in the up-regulation of 855 genes and down-regulation of 397 genes. Pre-treatment of lunasin for 24 h resulted in the up-regulation of 35 genes and down-regulation of 65 genes in LPS-stimulated RAW 264.7 macrophages. GeneVenn analysis of these three sets of genes showed that there are 66 genes common among the three groups which are mostly associated with regulation of cell death, ion binding and transcription as datamined by DAVID. Analysis of the 838 genes unique to lunasin alone by functional annotation clustering tool showed that lunasin mostly affected genes associated with RNA processing, apoptosis and protein kinase activity. Further datamining of these genes by ingenuity pathway analysis (IPA) showed that lunasin affected genes involved in cellular growth and proliferation, cellular function and maintenance, and cell to cell signaling and interaction. These findings support the potential chemopreventive and chemotherapeutic use of lunasin against cancer.
Keywords: Lunasin; Microarray; Apoptosis; Datamining; Soybean;

Properties of the bubble protein, a defensin and an abundant component of a fungal exudate by Marcus Seibold; Peter Wolschann; Sabrina Bodevin; Ole Olsen (1989-1995).
► New defensin characterized (bubble protein) from Penicillium brevicompactum. ► High similarity of that protein to other defensins. ► Structure prediction for other defensins. ► Association of clinically important mycophenolic acid with the bubble protein. ► Penicillium chrysogenum contains the bubble protein in addition to defensin PAF.Colonies of the ascomycete fungus Penicillium brevicompactum Dierckx produce bright yellow-green fluorescent exudate bubbles on its surface when grown on standard plant cell culture medium. According to SDS-PAGE analysis, the exudate is enriched in one protein, named bubble protein (BP). Detailed characteristics of BP are described, and also its corresponding genomic promoter and terminator sequences that flank sequences encoding signal peptide and a precursor sequence upstream of that of the mature protein. Following on previous work, the protein is now biochemically characterized. BP, the structure of which mainly consists of beta sheets, has four very stable disulfide bridges that resist standard procedures for reduction. With such traits, BP can now be categorized as a new member of the ever growing class of defensins. Indeed, the protein revealed anti-fungal effects as it inhibits growth of the yeast Saccharomyces cerevisiae in a dose-dependent manner. Structural classification places BP into the group of proteins with a knottin fold, founding the BP superfamily. Based on genomic alignments that revealed very high homology to four proteins of related fungi, a 3D structure prediction of the corresponding proteins was made. In addition, it was discovered that the closely related fungus Penicillium chrysogenum encodes a BP homolog – in addition to its PAF protein, which also is similar to BP – further suggesting that fungi may possess more than one defensin.
Keywords: Bubble protein; Disulfide bridges; Knottins; Defensins; Antifungal protein; Penicillium brevicompactum; Host defense protein;

Antifungal action of human cathelicidin fragment (LL13–37) on Candida albicans by Jack Ho Wong; Tzi Bun Ng; Anna Legowska; Krzysztof Rolka; Mamie Hui; Chi Hin Cho (1996-2002).
► Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. ► LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form and hyphal form of Candida albicans since the nuclear stain SYTOX Green was localized in both forms. ► The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death but it might lead to the uptake of the peptides and that the peptides might have some intracellular targets.Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.
Keywords: Cathelicidin; Candida albicans; Antifungal;

► We first found the direct anti-E. coli activity of histone H3. ► The modes of antimicrobial action of Arg-rich (H3 and H4) and Lys-rich (H2B) histones are different. ► We found that the Arg-rich histones disrupt the cell membrane of ompT-expressing E. coli strains by forming blebs. ► The Lys-rich histone translocates the membrane without causing membrane lysis. ► Bacterial OmpT is involved in these modes of action through digestion of histones.There is growing evidence of the antimicrobial properties of histones and histone-derived peptides; however, most of them are specific to lysine (Lys)-rich histones (H1, H2A, and H2B). In the present study, we focused on arginine (Arg)-rich histones (H3 and H4) and investigated their antimicrobial properties in comparison with those of histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against the bacterial outer membrane protease T (OmpT) gene-expressing Escherichia coli strain JCM5491 with calculated 50% growth inhibitory concentrations of 3.8, 10, and 12.7 μM, respectively. A lysate prepared from the JCM5491 cells was capable of strongly, moderately, and slightly fragmenting histones H2B, H3, and H4, respectively. While the lysate prepared from the cells of the ompT-deleted E. coli strain BL21(DE3) did not digest these histones, the ompT-transformed BL21(DE3), termed BL21/OmpT+, cell lysate digested the histones more strongly than the JCM5491 cell lysate. Laser confocal and scanning electron microscopic analyses demonstrated that while histone H2B penetrated the cell membrane of JCM5491 or BL21/OmpT+ cells, histones H3 and H4 remained on the cell surface and subsequently disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. The BL21(DE3) cells treated with each histone showed no bleb formation, but cell integrity was affected and the cell surface was corrugated. Consequently, it is suggested that OmpT is involved in the antimicrobial properties of Arg- and Lys-rich histones and that the modes of antimicrobial action of these histones are different.
Keywords: Antimicrobial peptide; Histone H2B; Histone H3; Histone H4; Bleb formation; OmpT;

Display Omitted► The peptide BTM-P1, derived from Cry11Bb protoxins, was more active than retro-BTM-1. ► Biphasic “contraction-swelling” response of red blood cells to the peptide BTM-P1. ► The cell suicide mechanism increases the efficacy of polycationic peptides. ► The dipole moment of BTM-P1 as a possible factor of its high permeabilizing activity.Mitochondrial and plasma membrane permeabilization by polycationic peptides BTM-P1 and retro-BTM-P1 were studied. BTM-P1 was more active than its retro-analog. In the sucrose medium, the capacity of BTM-P1 to permeabilize mitochondria was lower than in salt media. In contrast, retro-BTM-P1 showed the lowest activity in the KCl medium. The efficacy of both peptides to permeabilize red blood cells was higher in the sucrose medium and depended on the nature of salt in high ionic strength media. BTM-P1, but not retro-BTM-P1, induced biphasic change in light dispersion of red blood cells with artificially generated high transmembrane potential: the initial phase of fast cell shrinkage preceded the subsequent phase of cell swelling. The shrunken red blood cells demonstrated increased sensitivity to BTM-P1 that might be explained by the cell suicide mechanism via phosphatidylserine exposure at the cell surface. As a working hypothesis, we assume that some peptide topology characteristics, such as the orientation and values of the total and local electrical dipole moments, interacting with the membrane dipole potential, as well as the asymmetric distribution of polar and non-polar side chains are important factors affecting the membrane-permeabilizing activity of polycationic peptides.
Keywords: Polycationic peptides; Membrane permeabilization; Mitochondria; Red blood cells; Cell shrinkage; Cell suicide;

An in vitro synergetic evaluation of the use of nisin and sodium fluoride or chlorhexidine against Streptococcus mutans by Zhongchun Tong; Lin Zhou; Wenkai Jiang; Rong Kuang; Jie Li; Rui Tao; Longxing Ni (2021-2026).
► Nisin and NaF combination had a synergetic effect against S. mutans, but no interaction between nisin and CHX. ► S. mutans survival showed a significant decline after treatment with nisin and NaF in combination. ► Nisin in combination with NaF possesses strong bactericidal effects on S. mutans in biofilm. ► S. mutans with the treatment of nisin and NaF in combination was severely damaged.The objective of this study is to investigate the synergetic action between nisin and sodium fluoride or chlorhexidine against Streptococcus mutans, a primary cariogenic pathogen. In the antibacterial assay, a synergetic effect on S. mutans was found between nisin and sodium fluoride, but there was no interaction between nisin and chlorhexidine by the checkerboard, the fractional inhibitory concentration (FIC) and the fractional bactericidal concentration (FBC) tests. S. mutans survival rates showed a significant decline after treatment with a combination of nisin and sodium fluoride in a time-kill study. Scanning electron microscopy showed that the damage to S. mutans with the combined nisin and sodium fluoride treatment was the most severe among all of the different single and combined antimicrobial treatments. Furthermore, in the antibiofilm test, nisin in combination with sodium fluoride produced a stronger bactericidal effect on a S. mutans biofilm for 4 h and 16 h compared with sodium fluoride alone by confocal laser scanning microscopy. Nisin in combination with sodium fluoride exerted a high bactericidal effect on S. mutans and thereby has the potential to be used as an effective drug combination to prevent dental caries.
Keywords: Streptococcus mutans; Nisin; Sodium fluoride; Chlorhexidine; Synergy;

Structural and biological characterization of mastoparans in the venom of Vespa species in Taiwan by Chun-Hsien Lin; Jason T.C. Tzen; Ching-Lin Shyu; Mars J. Yang; Wu-Chun Tu (2027-2036).
► Five cDNAs of precursor polypeptide of MPs were first identified. ► MPs adopt α-helical conformation in the presence of 40% TFE or 8 mM SDS. ► All MPs exhibit multifunctional activity, i.e., mast cell degranulation activity; antimicrobial activity against Gram-positive and Gram-negative bacteria; hemolytic activity on chicken, human, and sheep erythrocytes; membrane permeabilization on E. coli BL21.Mastoparans, a family of small peptides, are isolated from the wasp venom. In this study, six mastoparans were identified in the venom of six Vespa species in Taiwan. The precursors of these mastoparans are composed of N-terminal signal sequence, prosequence, mature mastoparan, and appendix glycine at C-terminus. These mature mastoparans all have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bond. Therefore, these peptides could be predicted to adopt an amphipathic α-helical secondary structure. In fact, the CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 8 mM SDS or 40% 2,2,2-trifluoroethanol (TFE). All mastoparans exhibit mast cell degranulation activity, antimicrobial activity against both Gram-positive and -negative bacteria tested, various degree of hemolytic activity on chicken, human, and sheep erythrocytes as well as membrane permeabilization on Escherichia coli BL21. Our results also show that the hemolytic activity of mastoparans is correlated to mean hydrophobicity and mean hydrophobic moment.
Keywords: Mastoparans; Vespa species; Mast cell degranulation; Antimicrobial activity; Hemolytic activity; Membrane permeabilization;

Evaluating antioxidative activities of amino acid substitutions on mastoparan-B by Mars J. Yang; Wen-Yuh Lin; Kuang-Hui Lu; Wu-Chun Tu (2037-2043).
► Mastoparan-B possesses good antioxidative enzyme activities resembled to SOD and GPx. ► Envenomation risks of Mastoparan-B are minor in reasonable dosage. ► Proper substitution of Mastoparan-B extends antioxidative activity.Mastoparan-B is a peptide toxin isolated from the venom of Vespa basalis, the most dangerous hornet found in Taiwan. This study is aimed to evaluate the antioxidative activities of several amino acid substitutions on MP-B, and examined the influences of mast cell degranulation and hemolytic activities in parallel with antioxidative activities. The correlations between the biological function and amino acid sequence were assessed. Our study shows original MP-B is a valuable antioxidant at low concentration in competing with nitric-oxide for oxygen molecules and possesses good antioxidative enzyme activities resembled to superoxidase dismutase and glutathione peroxidase. And there are no predominant rates of mast cell degranulation and hemolytic effects in such condition. With proper substitutions, the reducing power, DPPH scavenging activity and glutathione reductase-like enzyme activity of MP-B can increase clearly. The results demonstrate that MP-B analogs are very potential to be applicable antioxidants for other antioxidative usages.
Keywords: Antioxidative activity; Mastoparan-B; Structure–activity relationship; Substitution;

Functional characterization of codCath, the mature cathelicidin antimicrobial peptide from Atlantic cod (Gadus morhua) by Daniela C. Broekman; Alexandra Zenz; Bjarnheidur K. Gudmundsdottir; Karl Lohner; Valerie H. Maier; Gudmundur H. Gudmundsson (2044-2051).
► The Atlantic cod mature cathelicidin antimicrobial peptide was characterized. ► It was active against a fungus and Gram-negative but not a Gram-positive bacterium. ► It demonstrated a rapidly lytic mode of action. ► It did not show cytotoxicity toward a fish cell line or mammalian membrane mimetics. ► It had no activity at physiological salinity and was cleaved by bacterial proteases.Cathelicidins are among the best characterized antimicrobial peptides and have been shown to have an important role in mammalian innate immunity. We recently isolated a novel mature cathelicidin peptide (codCath) from Atlantic cod and in the present study we functionally characterized codCath. The peptide demonstrated salt sensitivity with abrogation of activity at physiological salt concentrations. In low ionic strength medium we found activity against marine and non-marine Gram-negative bacteria with an average MIC of 10 μM, weak activity against a Gram-positive bacterium (MIC 80 μM), and pronounced antifungal activity (MIC 2.5 μM). The results suggest the kinetics and mode of action of codCath to be fast killing accompanied by pronounced cell lysis. Extracellular products (ECPs) of three marine bacteria caused breakdown of the peptide into smaller fragments and the cleaved peptide lost its antibacterial activity. Proteolysis of the peptide on the other hand was abolished by prior heat-treatment of the ECPs, suggesting a protease involvement. We observed no cytotoxicity of the peptide in fish cells up to a concentration of 40 μM and the selectivity of activity was confirmed with bacterial and mammalian membrane mimetics. We conclude that the potent broad-spectrum activity of codCath hints at a role of the peptide in cod immune defense.
Keywords: Innate immunity; Fish; Extended structure; Bacterial protease; Random coil;

► An antimicrobial peptide of the palustrin-2 family was isolated from the skin of the endangered frog Odorrana ishikawae. ► The peptide consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. ► This peptide showed broad-spectrum growth inhibition against several microorganisms. ► Some synthetic analog peptides possessed greater antimicrobial activities than the native structure.Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.
Keywords: Antimicrobial peptide; Innate immunity; Frog skin; Odorrana; Microorganism;

Radical reversal of vasoactive intestinal peptide (VIP) receptors during early lymphopoiesis by Emilie E. Vomhof-DeKrey; Ashley R. Sandy; Jarrett J. Failing; Rebecca J. Hermann; Scott A. Hoselton; Jane M. Schuh; Abby J. Weldon; Kimberly J. Payne; Glenn P. Dorsam (2058-2066).
► VPAC1 is the exclusive, full-length VIP receptor expressed in early thymocyte progenitors (ETP). ► VPAC1 and VPAC2 undergo a transitory reversal in expression during thymocyte development. ► The transcription factor, Ikaros, may play a role in VPAC1 and VPAC2 reversal. ► VPAC2 knockout mouse and wild type controls show similar DN percentages. ► VIP signaling may contribute to thymocyte movement and/or protect thymocytes from cell death during early lymphopoiesis.Successful thymocyte maturation is essential for normal, peripheral T cell function. Vasoactive intestinal peptide (VIP) is a neuropeptide which is highly expressed in the thymus that has been shown to modulate thymocyte development. VIP predominantly binds two G protein coupled receptors, termed vasoactive intestinal peptide receptor 1 (VPAC1) and VPAC2, but their expression profiles in CD4/CD8 (double negative, DN) thymocyte subsets, termed DN1–4, have yet to be identified. We hypothesized that a high VPAC1:VPAC2 ratio in the earliest thymocyte progenitors (ETP cells) would be reversed during early lymphopoiesis as observed in activated, peripheral Th2 cells, as the thymus is rich in Th2 cytokines. In support of this hypothesis, high VPAC1 mRNA levels decreased 1000-fold, accompanied with a simultaneous increase in VPAC2 mRNA expression during early thymocyte progenitor (ETP/DN1) → DN3 differentiation. Moreover, arrested DN3 cells derived from an Ikaros null mouse (JE-131 cells) failed to completely reverse the VIP receptor ratio compared to wild type DN3 thymocytes. Surprisingly, VPAC2−/− mice did not show significant changes in relative thymocyte subset numbers. These data support the notion that both VPAC1 and VPAC2 receptors are dynamically regulated by Ikaros, a master transcriptional regulator for thymocyte differentiation, during early thymic development. Moreover, high VPAC1 mRNA is a novel marker for the ETP population making it enticing to speculate that the chemotactic VIP/VPAC1 signaling axis may play a role in thymocyte movement. Also, despite the results that VPAC2 deficiency did not affect thymic subset numbers, future studies are necessary to determine whether downstream T cell phenotypic changes manifest themselves, such as a propensity for a Th1 versus Th2 polarization.
Keywords: Vasoactive intestinal peptide (VIP); Receptor 1; Neuropeptide; Vasoactive intestinal peptide (VIP) receptor 2; Thymocyte maturation; Hematopoietic stem cell; Ikaros;

Vasoactive intestinal peptide reduces oxidative stress in pancreatic acinar cells through the inhibition of NADPH oxidase by Nao Fujimori; Takamasa Oono; Hisato Igarashi; Tetsuhide Ito; Taichi Nakamura; Masahiko Uchida; David H. Coy; Robert T. Jensen; Ryoichi Takayanagi (2067-2076).
► Oxidative stress by H2O2 increased reactive oxygen species (ROS) in acini. ► ROS increased dose-dependently in damaged acini, thereby reducing the viability. ► Vasoactive intestinal peptide (VIP) reduced ROS and ameliorated cell viability. ► VIP up-regulated superoxide dismutase (SOD) 2 and inhibited NADPH oxidase activity. ► VIP affects the mechanism of ROS production through induction of cAMP/PKA pathway.Vasoactive intestinal peptide (VIP) attenuates experimental acute pancreatitis (AP) by inhibition of cytokine production from inflammatory cells. It has been suggested that reactive oxygen species (ROS) as well as cytokines play pivotal roles in the early pathophysiology of AP. This study aimed to clarify the effect of VIP on the oxidative condition in pancreas, especially pancreatic acinar cells (acini). Hydrogen peroxide (H2O2)-induced intracellular ROS, assessed with CM-H2DCFDA, increased time- and dose-dependently in acini isolated from rats. Cell viability due to ROS-induced cellular damage, evaluated by MTS assay, was decreased with ≥100 μmol/L H2O2. VIP significantly inhibited ROS production from acini and increased cell viability in a dose-dependent manner. Expression of antioxidants including catalase, glutathione reductase, superoxide dismutase (SOD) 1 and glutathione peroxidase was not altered by VIP except for SOD2. Furthermore, Nox1 and Nox2, major components of NADPH oxidase, were expressed in pancreatic acini, and significantly increased after H2O2 treatment. Also, NADPH oxidase activity was provoked by H2O2. VIP decreased NADPH oxidase activity, which was abolished by PKA inhibitor H89. These results suggested that VIP affected the mechanism of ROS production including NADPH oxidase through induction of a cAMP/PKA pathway. In conclusion, VIP reduces oxidative stress in acini through the inhibition of NADPH oxidase. These results combined with findings of our previous study suggest that VIP exerts its protective effect in pancreatic damage, not only through an inhibition of cytokine production, but also through a reduction of the injury caused by oxidative stress.
Keywords: Vasoactive intestinal peptide; Reactive oxygen species; Antioxidant; NADPH oxidase;

Amylin effect in extrapancreatic tissues participating in glucose homeostasis, in normal, insulin-resistant and type 2 diabetic state by P. Moreno; A. Acitores; I. Gutiérrez-Rojas; B. Nuche-Berenguer; M. El Assar; L. Rodriguez-Mañas; R. Gomis; I. Valverde; M. Visa; W.J. Malaisse; A. Novials; N. González; M.L. Villanueva-Peñacarrillo (2077-2085).
► In liver, amylin normalizes mRNA-GLUT-2 and glycogen content IR and T2D rats. ► In muscle, amylin normalizes the reduced glycogen synthase a activity in IR rats. ► In adipocytes, amylin normalizes altered glucose transport (GT) in IR and T2D rats. ► In adipocytes, amylin highly improves insulin-induced GT in N, IR and T2D rats.Amylin is co-secreted with insulin, responds to the same stimuli, is anorectic, lowers body weight by reducing fat mass, and is proposed for diabetes treatment. We examined the effect of a 3-day constant infusion of close to physiological doses of amylin in Wistar rats, on glucotransporter expression, glycogen content (G), glycogen synthase a activity (GSa) and glucose transport (GT), in liver, muscle and fat from insulin resistant (IR) and type 2 diabetic (T2D) models, compared to normal (N) animals; plasma glucose and insulin were measured. Plasma insulin in IR was higher than in N or T2D, and amylin normalized the value. In both, IR and T2D, liver G was lower than normal, accompanied by GLUT-2, mRNA and protein, higher and lower, respectively, than in N; amylin normalized G in both groups, without changes in GLUT-2, except for an mRNA increase in T2D. In IR and T2D, muscle GSa was reduced, together with respective over- and under-GLUT-4 expression; amylin induced only a trend toward GSa normalization in both groups. In isolated adipocytes, GT and GLUT-4 in IR and T2D were lower and higher, respectively, than in N; after amylin, not only GT was normalized in both groups but also the response to insulin was much more pronounced, including that in N, without major changes in GLUT-4. This suggests that the beneficial effect of amylin in states running with altered glucose homeostasis could occur by partially acting on the hexose metabolism of the liver and mainly on that of the adipose tissue.
Keywords: Amylin; Insulin-resistance; Diabetes;

Effect of different doses of GLP-2 (Teduglutide) on acute esophageal lesion due to acid-pepsin perfusion in male rats by Rohallah Moloudi; Fatemeh Nabavizadeh; Hossein Nahrevanian; Gholamreza Hassanzadeh (2086-2090).
► Acid-pepsin perfusion by pump induced esophageal lesions in rat. ► Pre-administration of GLP-2 (Teduglutide) with acid-pepsin perfusion, significantly reduced esophageal complications and lesion size. ► The GLP-2 (Teduglutide) healing effect on esophageal lesion due to NO pathway.Gastro-esophageal reflux currently is widespread disorders with dangerous complications. GLP-2 is a peptide that has trophic and anti-inflammatory effects on gastrointestinal mucosa. The aim of this study was to evaluate the protective role of GLP-2 in esophageal mucosa lesion due to perfusion acid-pepsin. Thirty-six male rats were used in this study and divided into six groups. They were control, acid-pepsin, GLP-2 20 μg, GLP-2 30 μg, GLP-2 40 μg and GLP-2 50 μg/kg groups. Esophageal blood flow, plasma NO metabolite, esophageal tissue NO metabolites and histological study of esophagus were performed as indicators of esophageal damage following acid-pepsin perfusion. Results showed that GLP-2 significantly increased plasma and tissue NO metabolites in comparison to acid-pepsin group. Also histological study showed significantly fewer lesions in the most effective dose GLP-2 30 μg in comparison to acid-pepsin group, our results show that GLP-2 could be useful for the treatment of esophageal in animal model.
Keywords: GLP-2 (Teduglutide); Gastro esophageal reflux; Nitric oxide; Blood flow; Mucous membrane;

Effects of kisspeptin-10 on progesterone secretion in cultured chicken ovarian granulosa cells from preovulatory (F1–F3) follicles by Yunqi Xiao; Yingdong Ni; Yanbing Huang; Jing Wu; Roland Grossmann; Ruqian Zhao (2091-2097).
► Kisspeptin immunoreactive substance is expressed in chicken ovarian granulosa cells. ► Kisspeptin-10 (Kp-10) stimulates progesterone secretion in these cells from preovulatory (F1–F3) follicles. ► Stimulated progesterone secretion parallels upregulation of StAR, P450scc and 3β-HSD mRNA expression.The effect of kisspeptin-10 (Kp-10) on the secretion of progesterone (P4) was investigated in cultured granulosa cells from F1 to F3 follicles of hens. The results showed that granulosa cells were stained with clear signals for kisspeptin using immunocytochemistry with the specific antibody against Kp-10. Among 10, 100 and 1000 nM concentrations tested, 100 nM Kp-10 treated for 24 h significantly increased P4 secretion in granulosa cells from F1 to F3 follicles. After 24 h and 48 h of treatment, 100 nM Kp-10 showed a significant increase in P4 secretion, while after 72 h of treatment P4 secretion was markedly decreased by Kp-10 compared to the control group (P  < 0.05). F1 and F2/3 cells treated with 100 nM Kp-10 for 24 h showed significantly increased viability (P  < 0.05) and which was in parallel to a marked increase in P4 secretion (P  < 0.01). Real-time PCR results showed that the gene expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc) and the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) in F1 and F2/3 granulosa cells was significantly up-regulated by 24 h–100 nM Kp-10 treatment (P  < 0.05 versus P  < 0.01, respectively). However, there was no significant difference in StAR, P450scc and 3β-HSD protein content between control and the Kp-10 treated group (P  > 0.05). These results indicate that Kp-10 stimulates P4 secretion in cultured chicken granulosa cells, which was associated with an up-regulation in StAR, P450scc and 3β-HSD gene transcription.
Keywords: Kisspeptin-10; Progesterone; StAR; P450scc; 3β-HSD; Chicken granulosa cells;

Gastrin-releasing peptide induces itch-related responses through mast cell degranulation in mice by Tsugunobu Andoh; Takashi Kuwazono; Jung-Bum Lee; Yasushi Kuraishi (2098-2103).
► The role of gastrin-releasing peptide released from primary afferent on itch was demonstrated. ► Gastrin-releasing peptide elicits scratching, an itch-related response, in mice though BB2 bombesin receptor. ► Gastrin-releasing peptide-induced scratching is inhibited by H1 histamine receptor antagonists and PAR2 proteinase-activated receptor antagonist. ► Gastrin-releasing peptide-induced scratching may be elicited through the activation of mast cells.Gastrin-releasing peptide (GRP), secreted from the central terminals of primary afferents, is involved in the transmission of itch signals in the spinal dorsal horn. Although primary afferents containing GRP are distributed throughout the skin, the role of peripherally released GRP in the itch response is unknown. We investigated whether GRP acts on the skin to induce an itch response in mice. Intradermal injections of GRP18–27 (1–300 nmol/site) elicited scratching. GRP18–27-induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone hydrochloride, the BB2 bombesin receptor antagonist RC-3095, the H1 histamine receptor antagonists fexofenadine hydrochloride and chlorpheniramine maleate, and the PAR2 proteinase-activated receptor antagonist FSLLRY-NH2. Mast cell deficiency significantly, but not completely, reduced the GRP18–27-induced scratching. BB2 bombesin receptors are present in mast cells in the skin, and intradermal injection of GRP18–27, not only induced scratching, but also led to mast cell degranulation. GRP18–27-induced mast cell degranulation was inhibited by the BB2 bombesin receptor antagonist RC-3095. These results suggest that peripherally released GRP can induce an itch response, at least partly, through activation of BB2 receptors present in the mast cells, triggering their degradation and the release of histamine and the serine proteinase, tryptase.
Keywords: Itch, Gastrin-releasing peptide; GRP18–27; BB2 bombesin receptor; Scratching; Mast cell;

Oxytocin in the rat caudate nucleus influences pain modulation by Jun Yang; Yan-Juan Pan; Ying Zhao; Pei-Yong Qiu; Lu Lu; Peng Li; Feng Chen; Xi-Qing Yan; Da-Xin Wang (2104-2107).
► Intra-CdN microinjection of OXT receptor antagonist decreased the pain threshold. ► Intra-CdN microinjection of OXT increased the pain threshold. ► OXT receptor antagonist can attenuate the analgesic role induced intra-CdN administration of OXT. ► Pain stimulation could increase OXT concentration in CdN perfusion liquid. ► OXT in the CdN was involved in pain modulation via OXT receptors.Our previous studies have demonstrated that oxytocin (OXT) in the central nervous system plays a role in pain modulation. Many studies have found that caudate nucleus (CdN) enriches OXT and OXT receptors by the methods of historadioautograph and gene expression. The communication was designed to investigate OXT effect in the rat CdN on pain modulation. The results showed that (1) intra-CdN microinjection of OXT receptor antagonist, desGly-NH2, d(CH2)5[d-Tyr2, Thr-sup-4]OVT decreased the pain threshold, whereas the local administration of OXT increased the pain threshold in a dose-dependent manner; (2) OXT receptor antagonist can attenuate the analgesic role induced intra-CdN administration of OXT; and (3) pain stimulation could increase OXT concentration in the CdN perfusion liquid. The data suggested that OXT in the CdN was involved in this pain process via OXT receptors.
Keywords: Oxytocin; Oxytocin receptor; Caudate nucleus; Pain modulation;

Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor by Hui Yang; Xiang-Jun Zeng; Hong-Xia Wang; Li-Ke Zhang; Xiao-Li Dong; Shubin Guo; Jie Du; Hui-Hua Li; Chao-Shu Tang (2108-2115).
Angiotensin II (Ang II) is an important regulator of cardiac function and injury in hypertension. The novel Ang IV peptide/AT4 receptor system has been implicated in several physiological functions and has some effects opposite to those of Ang II. However, little is known about the role of this system in Ang II-induced cardiac injury. Here we studied the effect of Ang IV on Ang II-induced cardiac dysfunction and injury using isolated rat hearts, neonatal cardiomyocytes and cardiac fibroblasts. We found that Ang IV significantly improved Ang II-induced cardiac dysfunction and injury in the isolated heart in response to ischemia/reperfusion (I/R). Moreover, Ang IV inhibited Ang II-induced cardiac cell apoptosis, cardiomyocyte hypertrophy, and proliferation and collagen synthesis of cardiac fibroblasts; these effects were mediated through the AT4 receptor as confirmed by siRNA knockdown. These findings suggest that Ang IV may have a protective effect on Ang II-induced cardiac injury and dysfunction and may be a novel therapeutic target for hypertensive heart disease.
Keywords: Angiotensin II; Angiotensin IV; Cardiac function; Hypertrophy; Fibroblast proliferation; AT4 receptor;

Vascular mechanisms involved in angiotensin II-induced venoconstriction in hypertensive rats by Rodrigo Azevedo Loiola; Liliam Fernandes; Rosângela Eichler; Rita de Cássia Tostes Passaglia; Zuleica Bruno Fortes; Maria Helena Catelli de Carvalho (2116-2121).
► Angiotensin II (Ang II)-induced constriction was evaluated in mesenteric venules and portal vein of hypertensive rats (SHR). ► In SHR portal vein, Ang II-constriction was reduced when compared to normotensive Wistar rats. ► AT2 receptor protein expression and mRNA levels were reduced in portal vein from SHR. ► In SHR, Ang II venoconstriction was mediated by AT1 receptors and was counterbalanced by kinin B2 receptor and COX metabolites. ► Different cellular and molecular mechanisms are involved in the venous control of normotensive and hypertensive rats.To investigate the venoconstrictor effect of angiotensin II (Ang II) in spontaneously hypertensive rats (SHR), we used preparations of mesenteric venular beds and the circular muscle of the portal veins. Vessels were tested with Ang II in the presence or absence of losartan, PD 123319, HOE 140, L-NAME, indomethacin, or celecoxib. In the mesenteric venular bed of SHR, the effect of Ang II (0.1 nmol) was nearly abolished by losartan and enhanced by HOE 140, indomethacin, and celecoxib, while PD123319 and L-NAME had no effect. In portal vein preparations, cumulative-concentration response curves (CCRC) to Ang II (0.1–100 nmol/L) exhibited a lower maximal response (Emax) in SHR compared to Wistar rats. AT1 receptor expression was similar in the two strains, while AT2 receptor levels were lower in SHR portal veins when compared to Wistar. In SHR portal veins, losartan shifted the CCRC to Ang II to the right, while indomethacin and HOE 140 increased the Emax to Ang II. PD 123319, celecoxib, and L-NAME had no effect. Taken together, our results suggest that Ang II-induced venoconstriction in SHR is mediated by activation of AT1 receptors and this effect may be counterbalanced by kinin B2 receptor and COX metabolites. Furthermore, our data indicate that there are different cellular and molecular mechanisms involved in the regulation of venous tonus of normotensive and hypertensive rats. These differences probably reflect distinct factors that influence arterial and venous bed in hypertension.
Keywords: Angiotensin II; Venous system; Hypertension; Angiotensin receptors;

Characterization of the kallikrein–kinin system during the bovine ovulation process by Gustavo Freitas Ilha; Joabel Tonellotto dos Santos; Alisson Minozzo da Silveira; Karina Gutierrez; Camila de Campos Velho Gewehr; Sara Marchesan de Oliveira; Juliano Ferreira; Paulo Bayard Dias Gonçalves; João Francisco Coelho de Oliveira (2122-2126).
► Kallikrein–kinin system is produced in the ovary during ovulation in bovine. ► Bradykinin and kallikrein have a regulation in follicular fluid during ovulation. ► The B1R expression is induced in both follicular cells. ► The B2R is constitutively expressed in granulosa cells and induced in theca cells. ► The study uses an in vivo approach to study monovulatory species.The kallikrein–kinin system (KKS) has been described as an important mediator of physiologic processes. Kallikreins use kininogen (KNG) as substrate to generate bradykinin, the main active peptide of the KKS that acts through two types of receptors, the B1R and the B2R. The goal of this study was to characterize some components of the KKS in different compartments of the ovary during the bovine ovulation process. The KNG, B1R and B2R mRNA expression patterns were assessed in theca and granulosa cells, as well as the bradykinin concentration and kallikrein-like activity in follicular fluid of bovine periovulatory follicles. To obtain a periovulatory follicle (≥12 mm), twenty-seven cows were submitted to estrus synchronization protocol and ovariectomized by colpotomy at 0, 3, 6, 12 or 24 h after a GnRH-analog injection (gonadorelin; 100 μg, IM). Follicular fluid was aspirated for enzymatic assays while granulosa and theca cells were harvested for mRNA analysis. The mRNA expressions in follicular cells were evaluated by real-time RT-PCR and data representation related to the cyclophilin housekeeping gene. The bradykinin concentration and kallikrein-like activity were measured in follicular fluid by enzymatic immunoassay and selective substrate cleavage, respectively. The B2R expression in theca cells and B1R expression in theca and granulosa cells showed different profiles during the periovulatory period (P  < 0.05). The bradykinin concentration and kallikrein-like activity in the follicular fluid were different (P  < 0.05) due to the time during the ovulation process. KNG mRNA expression was similar for both follicular cell types (P  > 0.05). Taken together, these results provide an important characterization of the presence and possible KKS regulation during the bovine ovulation.
Keywords: Ovulation; Kallikrein–kinin system; Bradykinin; Ovary; Bovine;

Post-translational modifications of pro-opiomelanocrtin related hormones in medaka pituitary based on mass spectrometric analyses by Akikazu Yasuda; Yoshiro Tatsu; Yoshikazu Kawata; Toshifumi Akizawa; Yasushi Shigeri (2127-2130).
► The novel molecular forms of α-melanocyte-stimulating hormone (MSH) and β-MSH in medaka (Oryzias latipes var.) pituitary. ► The acetylation sites in triacetyl-α-MSH were determined as N,O-diacetylation of Ser residue at position 1 and O-acetylation of Ser residue at position 3. ► The Pro residue at position 15 was partially hydroxylated in the β-MSH molecule.Direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry analysis provides a selective detection of mass profile for the peptides contained into cell secretory granules. By this mass spectrometry with slice of pituitary, two novel molecular forms of pro-opimelanocrtin related hormone were found in the orange-red strain medaka (Oryzias latipes var.). The structures of [N,O-diacetyl Serine1, O-acetyl Serine3]-α-melanocyte-stimulating hormone (MSH) and [hydroxyproline15]-β-MSH, together with [phosphoserine15]-corticotropin-like intermediate lobe peptide, were determined for the first time using a collision-induced dissociation with electrospray ionization mass spectrometry. A combination of mass spectrometry analyses is thus a powerful tool to lead to the elucidation of the post-translational processing from the pre-prohormone.

This study involves the evaluation of protective efficacy of Pam3Cys administered on day −7 and +7 of Leishmania challenge (on day 0) alone and in combination with miltefosine (2.5 mg/kg for 5 days) against experimental visceral leishmaniasis.Display Omitted► Prophylactic efficacy of Pam3Cys was evaluated against VL experimental model. ► Protective effect of Pam3Cys on the therapeutic efficacy of miltefosine was seen. ► Miltefosine + Pam3Cys showed significant reduction in parasitic burden. ► Use of Pam3Cys with miltefosine provides a promising alternative for cure of VL.Prophylactic potential of synthetic bacterial lipopeptide and a TLR2 agonist, Pam3Cys was first evaluated against experimental visceral leishmaniasis in rodent model. After establishing the potential its effect on therapeutic efficacy of miltefosine was also studied. Pam3Cys showed 74.64% inhibition in parasitic establishment when administered by ip route at a dose of 100 μg/animal spaced at two weeks, i.e. on day −7 and +7 of challenge with Leishmania donovani amastigotes. However, when aforesaid dose of Pam3Cys was given with sub-curative dose of miltefosine (2.5 mg/kg for 5 days) its efficacy enhanced from 49.80% to 92.25%. These findings revealed that this lipopeptide has potential protective efficacy which significantly enhanced the therapeutic efficacy of miltefosine used at low dose against Leishmania infection and warrants detailed investigations on its possible immunopotentiatory actions.

Novel anti-inflammatory peptides as cosmeceutical peptides by Youn-A Kang; Jung-Im Na; Hye-Ryung Choi; Jee-Woong Choi; Hee-Young Kang; Kyoung-Chan Park (2134-2136).
► Prostaglandin (PG) E2 is the primary mediator of UVB induced photoinflammation. ► We screened an internal library for dipeptides that inhibited UVB induced PGE2 synthesis but showed no cytotoxicity toward human keratinocytes. ► We identified three highly active inhibitory sequences, LE (Leu + Glu), MW (Met + Trp) and MY (Met + Tyr). ► We found LE and MW significantly decreased UVB induced erythema. ► LE and MW are candidate cosmeceutical peptides that can protect skin from UVB induced photoinflammation.Ultraviolet (UV) radiation induced inflammation plays an important role in the aging of human skin. Prostaglandin (PG) E2 is the primary mediator of UVB induced photoinflammation. We screened an internal library for dipeptides that inhibited UVB induced PGE2 synthesis but showed no cytotoxicity toward human keratinocytes. We identified three highly active inhibitory sequences, LE (Leu + Glu), MW (Met + Trp) and MY (Met + Tyr). To evaluate their efficacy in human skin, 24 sites of abdomen skin were irradiated with a 308 nm excimer laser (300 mJ/cm2), after which 2% LE, MW, MY or a control were applied to the irradiated sites for 24 h. The erythema index (EI) was measured before and 24 h after treatment. The results showed that LE and MW significantly decreased UVB induced erythema (p  = 0.041 and p  = 0.036, respectively), but ME did not. Overall, LE and MW are candidate cosmeceutical peptides that can protect skin from UVB induced photoinflammation.
Keywords: Cosmeceuticals; Inflammation; Peptides;

The evolution of pro-opiomelanocortin: Looking for the invertebrate fingerprints by Davide Malagoli; Alice Accorsi; Enzo Ottaviani (2137-2140).
► Pro-opiomelanocortin (POMC)-derived peptides are involved in neuro endocrine functions in all vertebrates. ► POMC-derived and POMC-like peptides are present in invertebrate taxa. ► Discrepancies exist between POMC distribution and phylogeny of metazoans. ► The evolution of POMC-derived molecules remains open to debate.The presence and role of the pro-opiomelanocortin (POMC) gene and encoded peptides in invertebrates are here summarized and discussed. Some of the POMC-derived peptides show a significant similarity regarding their functions, suggesting their appearance before the split of protostomian–deuterostomian lineages and their maintenance during evolution. The basic mechanisms that govern the exchange of information between cells are usually well conserved, and this could have also been for POMC-derived peptides, that are mainly involved in fundamental functions such as immune and neuroendocrine responses. However, the presence and functions that POMC-derived peptides exhibit in taxonomically distant models, are not always reflected by the expected gene homology, leaving the problem of POMC evolution in invertebrates in need of additional study.
Keywords: Pro-opiomelanocortin (POMC); POMC-products; Immune response; Stress response; Evolution;

A new look at the renin–angiotensin system—Focusing on the vascular system by Aurelie Nguyen Dinh Cat; Rhian M. Touyz (2141-2150).
► All the elements of the RAS are present in the vasculature, indicating that the vascular system may act independently from the systemic RAS to generate Ang II. ► Vascular renin probably derives primarily from circulating kidney-derived renin. ► New components of the RAS have been identified in the vascular wall, including (P)RR, Ang-(1-7), Ang III, Ang IV, ACE2 and ProAng-12. ► Ang II generated from immune and inflammatory cells contributes to Ang II levels in the vascular wall. ► Activation of the RAS in perivascular adipose tissue contributes to the vascular RAS and influences vascular function. ► Functionally the vascular RAS may amplify vascular effects of the circulating RAS.The renin–angiotensin system (RAS), critically involved in the control of blood pressure and volume homeostasis, is a dual system comprising a circulating component and a local tissue component. The rate limiting enzyme is renin, which in the circulating RAS derives from the kidney to generate Ang II, which in turn regulates cardiovascular function by binding to AT1 and AT2 receptors on cardiac, renal and vascular cells. The tissue RAS can operate independently of the circulating RAS and may be activated even when the circulating RAS is suppressed or normal. A functional tissue RAS has been identified in brain, kidney, heart, adipose tissue, hematopoietic tissue, gastrointestinal tract, liver, endocrine system and blood vessels. Whereas angiotensinsinogen, angiotensin converting enzyme (ACE), Ang I and Ang II are synthesized within these tissues, there is still controversy as to whether renin is produced locally or whether it is taken up from the circulation, possibly by the (pro)renin receptor. This is particularly true in the vascular wall, where expression of renin is very low. The exact function of the vascular RAS remains elusive, but may contribute to fine-tuning of vascular tone and arterial structure and may amplify vascular effects of the circulating RAS, particularly in pathological conditions, such as in hypertension, atherosclerosis and diabetes. New concepts relating to the vascular RAS have recently been elucidated including: (1) the presence of functionally active Ang-(1-7)-Mas axis in the vascular system, (2) the importance of the RAS in perivascular adipose tissue and cross talk with vessels, and (3) the contribution to vascular RAS of Ang II derived from immune and inflammatory cells within the vascular wall. The present review highlights recent progress in the RAS field, focusing on the tissue system and particularly on the vascular RAS.
Keywords: Tissue RAS; Renin; Circulating RAS; Vascular tone; Ang-(1-7); Vascular cells;