Peptides (v.33, #1)

Antimicrobial decapeptide KSL-W enhances neutrophil chemotaxis and function by Richard L. Williams; Herve Y. Sroussi; Kai Leung; Phillip T. Marucha (1-8).
► KSLW, an antimicrobial peptide, was demonstrated to be chemotactic for neutrophils in micromolar amounts. ► KSLW enhances F-actin polymerization in neutrophils. ► KSLW was shown to inhibit oxygen radical production in PMA- and LPS-stimulated neutrophils. ► Future studies may determine if KSLW regulates neutrophil phagocytosis, adhesion, and apoptosis.Mammalian cationic antimicrobial peptides have received increased attention over the last decade, due to their prokaryotic selectivity and decreased risk of microbial resistance. In addition, antimicrobial peptides display differential biological effects on mammalian immune cell function, such as migration, adhesion, and modulation of respiratory burst, which make them even more attractive as therapeutic agents. Synthetic combinatorial libraries provide a time-efficient and cost-effective source for these diverse molecules. The novel synthetic antimicrobial peptide, KSLW (KKVVFWVKFK-NH2), has been shown to display a broad spectrum of antimicrobial activity against Gram (+) and Gram (−) bacteria, fungi and viruses. In this study, we evaluated the alternative biological activity of the decapeptide on neutrophil migration and function. KSLW was demonstrated to be chemotactic for neutrophils in micromolar amounts, and neutrophil treatment with KSLW, after 1 min, resulted in significant increases in F-actin polymerization. KSLW was shown to inhibit oxygen radical production in PMA- and LPS-stimulated neutrophils. Future studies, to determine if KSLW regulates neutrophil phagocytosis, adhesion, and apoptosis, or examining the effect of KSLW on other mammalian cell types, such as cell populations of healing-impaired wounds, would provide significant insight for the potential therapeutic strategies offered by antimicrobial peptides.
Keywords: Antimicrobial peptide; Multifunctional; Neutrophil; Chemotaxis; Actin polymerization;

Peptidotriazoles with antimicrobial activity against bacterial and fungal plant pathogens by Imma Güell; Lluís Micaló; Laura Cano; Esther Badosa; Rafael Ferre; Emilio Montesinos; Eduard Bardají; Lidia Feliu; Marta Planas (9-17).
Display Omitted► Peptidotriazoles with an optimal biological profile were identified. ► They showed high antimicrobial activity and stability, low hemolysis, and no phytotoxicity. ► The synthesis involved the formation of the 1,2,3-triazole by a click reaction.We designed and prepared peptidotriazoles based on the antimicrobial peptide BP100 (LysLysLeuPheLysLysIleLeuLysTyrLeu-NH2) by introducing a triazole ring in the peptide backbone or onto the side chain of a selected residue. These compounds were screened for their in vitro growth inhibition of bacterial and fungal phytopathogens, and for their cytotoxic effects on eukaryotic cells and tobacco leaves. Their proteolytic susceptibility was also analyzed. The antibacterial activity and the hemolysis were influenced by the amino acid that was modified with the triazole as well as by the absence of presence of a substituent in this heterocyclic ring. We identified sequences active against the bacteria Xanthomonas axonopodis pv. vesicatoria, Erwinia amylovora, Pseudomonas syringae pv. syringae (MIC of 1.6–12.5 μM), and against the fungi Fusarium oxysporum (MIC < 6.2–12.5 μM) with low hemolytic activity (0–23% at 50 μM), high stability to protease digestion and no phytotoxicity. These peptidotriazoles constitute good candidates to design new antimicrobial agents.
Keywords: Antimicrobial peptides; Click reaction; Peptidomimetics; Heterocyclic peptides;

Toxicity study of antimicrobial peptides from wild bee venom and their analogs toward mammalian normal and cancer cells by Jiřina Slaninová; Veronika Mlsová; Hilda Kroupová; Lukáš Alán; Tereza Tůmová; Lenka Monincová; Lenka Borovičková; Vladimír Fučík; Václav Čeřovský (18-26).
► We tested toxicity of more than 60 peptides toward mammalian cells. ► Two normal cell lines (HUVEC and rat intestinal epithelial cells) were used. ► Three cancer cell lines (HeLa S3, CRC SW480 and CCRF-CEM T) were used. ► Most peptides are semi-selective for cancer cells, HeLa S3 cells are the most sensitive. ► The peptides are membranolytic.Recently, we have isolated and characterized remarkable antimicrobial peptides (AMPs) from the venom reservoirs of wild bees. These peptides (melectin, lasioglossins, halictines and macropin) and their analogs display high antimicrobial activity against Gram-positive and -negative bacteria, antifungal activity and low or moderate hemolytic activity. Here we describe cytotoxicity of the above-mentioned AMPs and some of their analogs toward two normal cell lines (human umbilical vein endothelial cells, HUVEC, and rat intestinal epithelial cells, IEC) and three cancer cell lines (HeLa S3, CRC SW 480 and CCRF-CEM T). HeLa S3 cells were the most sensitive ones (concentration causing 50% cell death in the case of the most toxic analogs was 2.5–10 μM) followed by CEM cells. For the other cell lines to be killed, the concentrations had to be four to twenty times higher. These results bring promising outlooks of finding medically applicable drugs on the basis of AMPs. Experiments using fluorescently labeled lasioglossin III (Fl-VNWKKILGKIIKVVK-NH2) as a tracer confirmed that the peptides entered the mammalian cells in higher quantities only after they reached the toxic concentration. After entering the cells, their concentration was the highest in the vicinity of the nucleus, in the nucleolus and in granules which were situated at very similar places as mitochondria. Experiments performed using cells with tetramethylrhodamine labeled mitochondria showed that mitochondria were fragmented and lost their membrane potential in parallel with the entrance of the peptides into the cell and the disturbance of the cell membrane.
Keywords: Antimicrobial peptides; Venom; Hymenoptera; Cancer cells; Toxicity; Confocal microscopy;

Identification and characterization of antimicrobial peptides from skin of Amolops ricketti (Anura: Ranidae) by Hui Wang; Ran Ran; Haining Yu; Zhijun Yu; Yuhong Hu; Hongyuan Zheng; Duo Wang; Fan Yang; Renjie Liu; Jingze Liu (27-34).
► cDNAs encoding five novel antimicrobial peptide precursors were cloned from skin cDNA library of Amolops ricketti. They are the first antimicrobial peptides from A. ricketti. The actual presence and characterization of mature antimicrobial peptides was confirmed by two methods RP-HPLC and LC–MS/MS-based proteomics approach. We use the proteomic method in 4 different ways to prepare the sample for peptides identification. Incompletely digestion of protein can markedly increase peptide numbers of identification.As one of large amphibian group, there are a total of 45 species of Amolops in the world. However, the antimicrobial peptides (AMPs) existing in this genus has not been extensively studied. In this study, cDNAs encoding five novel AMP precursors were cloned by screening the skin-derived cDNA library of Amolops ricketti, a frog species that exists in southern and western parts of China. Protein sequence analysis led to the identification of five deduced peptides, three belonging to the brevinin-1 family and two belonging to the brevinin-2 family of amphibian AMPs. Thus, they were named as brevinin-1RTa (FLPLLAGVVANFLPQIICKIARKC), brevinin-1RTb (FLGSLLGLVGKVVPTLFCKISKKC), brevinin-1RTc (FLGSLLGLVGKIVPTLICKISKKC), brevinin-2RTa (GLMSTLKDFGKTAAKEIAQSLLSTASCKLAKTC), and brevinin-2RTb (GILDTLKEFGKTAAKGIAQSLLSTASCKLAKTC), respectively. The purification of brevinin-1RTa, brevinin-1RTb, and brevinin-2RTb was carried out by RP-HPLC, and confirmed by the LC–MS/MS-based proteomics approach. All of the peptides displayed different antimicrobial potency against a variety of microorganisms. In addition, brevinin-2RTa and brevinin-2RTb were found to have relatively low hemolytic activity (>400 μg/ml) against mammalian red blood cells in vitro, which could potentially be as candidates for developing novel anti-infection agents.
Keywords: Amphibian; Amolops ricketti; Frog skin; Antimicrobial peptides (AMPs);

Host-defense peptides from skin secretions of the tetraploid frogs Xenopus petersii and Xenopus pygmaeus, and the octoploid frog Xenopus lenduensis (Pipidae) by Jay D. King; Milena Mechkarska; Laurent Coquet; Jérôme Leprince; Thierry Jouenne; Hubert Vaudry; Koji Takada; J. Michael Conlon (35-43).
► Magainin, PGLa, and CPF peptides have been isolated from skin secretions of three Xenopus species. ► Xenopus petersii has a close but not conspecific phylogenetic relationships with Xenopus laevis. ► Xenopus pygmaeus does not belong in the laevis and muelleri species groups. ► Xenopus lenduensis expresses the full complement of four PGLa genes. ► CPF peptides show the greatest antimicrobial activity.Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of host-defense peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families from the tetraploid frogs, Xenopus petersii (Peters’ clawed frog) and Xenopus pygmaeus (Bouchia clawed frog), and the octoploid frog Xenopus lenduensis (Lendu Plateau clawed frog). Xenopsin-precursor fragment (XPF) peptides were not detected. The primary structures of the antimicrobial peptides from X. petersii demonstrate a close, but not conspecific relationship, with Xenopus laevis whereas the X. pygmaeus peptides show appreciable variation from previously characterized orthologs from other Xenopus species. Polyploidization events within the Xenopodinae (Silurana  +  Xenopus) are associated with extensive gene silencing (nonfunctionization) but unexpectedly the full complement of four PGLa paralogs were isolated from X. lenduendis secretions. Consistent with previous data, the CPF peptides showed the highest growth-inhibitory activity against bacteria with CPF-PG1 (GFGSLLGKALKIGTNLL.NH2) from X. pygmaeus combining high antimicrobial potency against Staphylococcus aureus (MIC = 6 μM) with relatively low hemolytic activity (LC50  = 145 μM).
Keywords: Antimicrobial peptide; Frog skin; Magainin; PGLa; Procaerulein; Allopolyploidy; Xenopus;

► BmKbpp possesses strong antimicrobial activity against the majority of tested bacteria and fungi with very weak hemolytic activity. ► BmKbpp markedly inhibits the superoxide production in granulocytes at low concentration. ► Both BmKbpp and the C-terminal region of BmKbpp possess bradykinin-potentiating activity. ► Multi-functionalization of a single peptide could be a new mechanism for the functional diversification of scorpion venom peptides.BmKbpp is a novel cationic and α-helical peptide from the Chinese scorpion Mesobuthus martensii Karsch, of which function or biological activity has not been characterized so far. Here we showed that BmKbpp possesses strong antimicrobial activity against both Gram-positive and Gram-negative bacteria with a MIC range from 2.3 μM to 68.2 μM for the majority of tested bacteria. BmKbpp also inhibits the growth of tested fungi with an IC50 range from 0.2 μM to 3.1 μM. Because BmKbpp potently inhibits the growth of some antibiotics-resistant pathogens, and shows very weak hemolytic activity, it has considerable potentials for therapeutic applications. Moreover, we found that BmKbpp markedly inhibits the superoxide production in granulocytes or HL-60 cells at the concentrations of submicromolar level; this suggests that BmKbpp can act as a signaling molecule involving innate immune regulation at low concentrations. The C-terminal region of BmKbpp (BmKbpp-C) shows 72% similarity to the peptide K-12, a bradykinin-potentiating peptide. We found that both BmKbpp and BmKbpp-C possess bradykinin-potentiating activity, and the activity of BmKbpp-C is stronger than that of BmKbpp. PCR amplification for the genomic gene of BmBpp showed that it is not a continuous sequence in the genome; it suggests that BmKbpp could come from a recombination event in transcript level. Taken together, our data suggest that multi-functionalization of a single peptide, which is probably mediated by trans-splicing, could be a new mechanism for the functional diversification of scorpion venom peptides.
Keywords: Scorpion; Mesobuthus martensii Karsch bradykinin-potentiating activity; Antimicrobial peptide; Trans-splicing; Non-disulfide-bridged Peptide; Peptide K-12;

► The first report of food-derived Val-Ala-Pro (VAP) tripeptide. ► Excellent ACE-inhibitory activity of VAP (IC50 value of 0.00534 mg/mL). ► VAP is a competitive ACE inhibitor. ► VAP is stable against ACE and digestive enzymes. ► Purification of VAP from hydrolysate of grass carp protein was achieved.Peptides inhibiting angiotensin-I converting enzyme (ACE, EC. are possible cures of hypertension. Food-derived ACE-inhibitory peptides are particularly attractive because of reduced side effects. Previously, we reported ACE-inhibitory activity of grass carp protein hydrolysates. In this work, we report steps for purifying the ACE-inhibitory peptide from the hydrolysate and its biochemical properties. Following steps of ultrafiltration, macroporous adsorption resin, and two steps of reversed phase high performance liquid chromatography (RE-HPLC), a single Val-Ala-Pro (VAP) tripeptide was identified. The tripeptide with excellent ACE-inhibitory activity (IC50 value of 0.00534 mg/mL) was a competitive ACE inhibitor and stable against both ACE and gastrointestinal enzymes of pepsin and chymotrypsin. This is the first report of food-derived VAP. The identified unique biochemical properties of VAP may enable the application of grass carp protein hydrolysates as a functional food for treatments of hypertension. The developed purification conditions also allow the production of VAP for pharmaceutical applications.
Keywords: Grass carp protein hydrolysate; ACE-inhibitory peptide; Purification; Inhibition pattern; Stability against gastrointestinal enzymes;

Renal actions of dendroaspis natriuretic peptide in rabbits by Soo Mi Kim; Sun Young Kim; Suhn Hee Kim; Kyung Woo Cho; Sung Zoo Kim (59-66).
► Our study provides physiological roles of DNP on renal function, i.e. DNP has more effective renal functions than ANP, possibly because of the degradation resistance of DNP against the endogenous peptidases in plasma or tissues. ► Glomeruli are the most favorable cellular site in the kidney for cGMP production by DNP via activation of particulate guanylyl cyclase (GC). ► Our study adds incremental new information to characterize the renal function of DNP which plays a pivotal role as a renal regulating peptide via specific natriuretic peptide receptors with a guanylyl cyclase domain.Dendroaspis natriuretic peptide (DNP) is one of four members of the natriuretic peptide family sharing functional and structural properties. The purpose of the present study was to elucidate the physiological role of DNP on renal functions and its cellular mechanism in the rabbit kidney. DNP (5 μg/kg/min) infused intravenously increased urine volume and urinary excretion of electrolytes. These renal actions induced by DNP were more pronounced than those caused by atrial natriuretic peptide (ANP). We compared profiles of 125I-ANP and 125I-DNP by reverse-phase HPLC during incubation in rabbit plasma at 37 °C for 1, 2, and 4 h. While 125I-ANP was quickly degraded within 1 h, 125I-DNP was still stable in plasma for 4 h. DNP induced the greatest cyclic guanosine monophosphate (cGMP) production in the glomeruli in a dose-dependent manner, when compared to other renal structures including cortical tubules, outer medullary tubules, and inner medullary tubules. Affinity cross-linking analysis revealed NPR-A is selective receptor for DNP in glomeruli. Forskolin, a stimulator of adenylyl cyclase, significantly decreased cGMP production in the renal glomeruli but not in the renal medulla. In summary, DNP is a more effective activator of renal functions than ANP, possibly because of the degradation resistance of DNP against the endogenous peptidases in plasma or tissues. These findings suggest that DNP plays a pivotal role as a renal regulating peptide via specific natriuretic peptide receptors with a guanylyl cyclase domain.
Keywords: DNP; Renal Function; cGMP; Glomerulus; Stability;

Carboxypeptidases A1 and A2 from the perfusate of rat mesenteric arterial bed differentially process angiotensin peptides by Hugo J.V. Pereira; Laura L. Souza; Claudio M. Costa-Neto; Maria Cristina O. Salgado; Eduardo B. Oliveira (67-76).
► Rat vascular CPA1 and CPA2 are novel angiotensin-processing enzymes. ► Rat CPA1 metabolizes Ang II to Ang-(1-7). ► Rat CPA2 catalyzes a stepwise conversion of Ang-(1-12) to Ang I. ► mRNA for CPA1 and CPA2 are expressed in various extrapancreatic tissues of the rat.Here we report the isolation of carboxypeptidases A1 and A2 (CPA1 and CPA2) from the rat mesenteric arterial bed perfusate, which were found to be identical with their pancreatic counterparts. Angiotensin (Ang) I, Ang II, Ang-(1-9) and Ang-(1-12) were differentially processed by these enzymes, worthy mentioning the peculiar CPA1-catalyzed conversion of Ang II to Ang-(1-7) and the CPA2-mediated formation of Ang I from Ang-(1-12). We detected gene transcripts for CPA1 and CPA2 in mesentery and other extrapancreatic tissues, indicating that these CPAs might play a role in the renin–angiotensin system in addition to their functions as digestive enzymes.
Keywords: Carboxypeptidase; Angiotensin I; Angiotensin II; Angiotensin-(1-7); Angiotensin-(1-12); ACE2;

Delta-opioid augments cardiac contraction through β-adrenergic and CGRP-receptor co-signaling by Vince T. Nguyen; Yewen Wu; Ashley N. Guillory; Bradley K. McConnell; Kenichi Fujise; Ming-He Huang (77-82).
► ICA cells exclusively express δ-opioid receptor in left ventricular myocardium. ► δ-opioid receptor stimulation of ICA cells augments myocardial contraction. ► This Effect is due to enhanced myocardial β-adrenergic/CGRP receptor co-signaling. ► This adrenopeptidergic co-signaling is coupled with cAMP-dependent PKA pathway.Cardiac epinephrine and calcitonin gene-related peptide (CGRP) are produced by intrinsic cardiac adrenergic cells (ICA cells) residing in human and animal hearts. ICA cells are neuroparicine cells expressing δ-opioid receptors (DOR). We hypothesized that δ-opioid stimulation of ICA cells enhances epinephrine and CGRP release, which results in the augmentation of heart contraction. Rats were injected with DOR-agonist DPDPE (100 μg/kg) with or without 10-min pretreatment with either β-adrenergic receptor (β-AR) blocker propranolol (2 mg/kg) or CGRP-receptor (CGRPR) blocker CGRP8–37 (300 μg/kg), or their combination. Hemodynamics were monitored with echocardiogram and systolic blood pressure (SBP) was monitored via a tail arterial catheter. Changes in left ventricular fraction-shortening (LVFS) and heart rate (HR) were observed at 5-min after DPDPE infusion. At 5-min DPDPE induced a 36 ± 18% (p  < 0.001) increase of the LVFS, which continues to increase to 51 ± 24% (p  < 0.0001) by 10 min, and 68 ± 19% (p  < 0.001) by 20 min. The increase in LVFS was accompanied by the decrease of HR by 9 ± 5% (p  < 0.01) by 5 min and 11 ± 6% (p  < 0.001) by 15 min post DPDPE infusion. This magnitude of HR reduction was observed for the remainder of the 20 min. Despite the HR-reduction, cardiac output was increased by 17 ± 8% (p  < 0.05) and 28 ± 5% (p  < 0.001) by 5- and 20-min post DPDPE administration, respectively. There was a modest (9 ± 9%, p  = 0.03) decrease in SBP that was not apparent until 20 min post DPDPE infusion. The positive inotropism of DPDPE was abrogated in animals pretreated with propranolol, CGRP8–37, or combined propranolol + CGRP8–37. Furthermore, in whole animal and cardiomyocyte cell culture preparations, DPDPE induced myocardial protein-kinase A (PKA) activation which was abrogated in the animals pretreated with propranolol + CGRP8–37. DOR agonists augment myocardial contraction through enhanced β-AR and CGRPR co-signaling.
Keywords: Contractility; δ-Opioid receptor; Heart; ICA cell;

Plasma C-type natriuretic peptide levels in healthy children by S. Del Ry; M. Cantinotti; M. Cabiati; C. Caselli; S. Storti; T. Prescimone; B. Murzi; A. Clerico; D. Giannessi (83-86).
► The childhood obesity epidemic compromises the health of the pediatric population by promoting premature development of atherosclerosis and metabolic syndrome. ► Given the involvement of CNP in these conditions it becomes extremely important to have age-specific reference intervals for this peptide. ► The present study reports the reference intervals for CNP plasma levels measured in healthy children (0–12 years). ► According to data obtained, at least 5 different reference intervals should be used: 0–3 days, 4–30 days, 1–12 months, 1–12 years and adults.C-type natriuretic peptide (CNP) is assuming increasing importance in cardiovascular disease, and in adults its plasma levels are related to clinical and functional disease severity. Data are scarce regarding the reference values for CNP in infancy. Aim of this study was to assess the reference intervals for CNP in human healthy newborns and infants. Plasma CNP was measured in 121 healthy children divided into: 41 newborns (age 0–3 days), 24 newborns (4–30 days), 22 infants (1–12 months) and 32 children (1–12 years). A group of 32 healthy adult subjects (age 64 ± 1 years) was also studied. CNP was measured by a specific radioimmunoassay. Between- and within-assay variability resulted ≤30 and 20%, respectively and analytical sensitivity 0.77 ± 0.05 pg/tube. Plasma CNP resulted significantly higher in children than in adult subjects (13.6 ± 1.2 pg/ml vs. 7.4 ± 1.0 pg/ml, p  = 0.030). When the results were analyzed as a function of the age the reference intervals for plasma CNP resulted: 11.6 ± 2.1 pg/ml for newborns (0–3 days), 16.4 ± 3.7 pg/ml for newborns (4–30 days), 15.4 ± 2.7 pg/ml for infants (1–12 months), 13.6 ± 2.3 pg/ml for children (1–12 years) [p  = 0.01 newborns (4–30 days) vs. adults; p  = 0.03 infants (1–12 months) vs. adults]. CNP showed the highest concentrations after 12 h of life with a peak between 4 and 5 days of life and with a progressive decline afterwards. According to these data at least five different reference intervals for CNP determinations should be used. These observations may be helpful for future clinical application of CNP in human children.
Keywords: C-type natriuretic peptide; Infants; Natriuretic peptides; Newborns; Reference values;

Effects of urotensin II on functional activity of late endothelial progenitor cells by Kaihong Yi; Min Yu; Libiao Wu; Xuerui Tan (87-91).
► Human late EPCs expressed UT receptor. ► UII increased the migratory, adhesive and in vitro vasculogenesis capacity and inhibited proliferative activity of late EPCs. ► Urantide abolished the effects of UII on late EPCs.Urotensin II (UII) is a potent vasoactive cyclic peptide which has multiple effects on the cardiovascular system. However, the effects of UII on late endothelial progenitor cells (EPCs) are still unclear. The aim of the present study is to investigate whether UII influences the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood by Ficoll density gradient centrifugation and treated with UII (10−10, 10−9, 10−8, 10−7 and 10−6  M), or vehicle control. Expression of urotensin II receptor (UT) in late EPCs was confirmed by indirect immunofluorescence staining. Late EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell chamber assay, and matrigel tube formation assay. Late EPCs adhesive assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Incubation with UII increased the migratory, adhesive and in vitro vasculogenesis capacity and inhibited the proliferative activity of late EPCs. Furthermore, these UII-mediated effects on late EPCs were attenuated by pretreatment with the UT antagonist urantide. These findings indicate that UII may exert multiple effects on functional activity of late EPCs through UT.
Keywords: Urotensin II; Late endothelial progenitor cells; Urotensin II receptor; Atherosclerosis;

► Ghrelin stimulates angiogenesis in rat cardiac microvascular endothelial cells. ► MEK/ERK and PI3K/Akt signal pathways are involved in ghrelin-induced angiogenesis. ► GHSR1a mediates ghrelin activation of ERK, Akt, and in vitro angiogenesis. ► Ghrelin may play an important role in myocardial angiogenesis.Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), is thought to exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function such as cell proliferation, migration, survival and angiogenesis. However, the effect of ghrelin on angiogenesis and the corresponding mechanisms have not yet been extensively studied in cardiac microvascular endothelial cells (CMECs) isolated from left ventricular myocardium of adult Sprague-Dawley (SD) rats. In our study, we found that ghrelin and GHSR are constitutively expressed in CMECs. Ghrelin significantly increases CMECs proliferation, migration, and in vitro angiogenesis. The ghrelin-induced angiogenic process was accompanied by phosphorylation of ERK and Akt. MEK inhibitor PD98059 abolished ghrelin-induced phosphorylation of ERK, but had no effect on Akt phosphorylation. PI3K inhibitor LY294002 abolished ghrelin-induced phosphorylation of Akt, but had no effect on ERK phosphorylation. Ghrelin-induced angiogenesis was partially blocked by treatment with PD98059 or LY294002. In addition, this angiogenic effect was almost completely inhibited by PD98059 + LY294002. Pretreatment with GHSR1a blocker [D-Lys3]-GHRP-6 abolished ghrelin-induced phosphorylation of ERK, Akt and in vitro angiogenesis. In conclusion, this is the first demonstration that ghrelin stimulates CMECs angiogenesis through GHSR1a-mediated MEK/ERK and PI3K/Akt signal pathways, indicating that two pathways are required for full angiogenic activity of ghrelin. This study suggests that ghrelin may play an important role in myocardial angiogenesis.
Keywords: Ghrelin; GHSR; Cardiac microvascular endothelial cells; Angiogenesis;

Regulation of gene expression and biochemical changes in small intestine of newborn diabetic rats by exogenous ghrelin by Ayse Karatug; Ozlem Sacan; Zeynep Mine Coskun; Sehnaz Bolkent; Refiye Yanardag; Neslihan Turk; Sema Bolkent (101-108).
► The experimental model of type 2 diabetic rats given ghrelin. ► Changes in cholecystokinin, somatostatin, apelin mRNA and peptide levels. ► The effects on antiapoptotic and antioxidant system. ► Ghrelin may be an important hormone for the treatment of diabetes.The aim of this study was to investigate (i) the cholecystokinin, somatostatin and apelin mRNA levels, (ii) the changes in levels and localization of these peptides, (iii) relation between these peptides, (iv) antiapoptotic effects and (v) antioxidant effects of ghrelin. The rats were divided into four groups second day after birth. These groups were respectively treated with physiological saline, ghrelin (100 μg/kg/day), streptozotocin (100 mg/kg), ghrelin and streptozotocin. After four weeks, small intestine and blood samples were taken from rats. Cholecystokinin mRNA and peptide, somatostatin mRNA, release to duodenal lumen of apelin peptide and apelin mRNA signals decreased in ghrelin-treated diabetic rats compared to the diabetic group. There was no statistically significant difference among the four groups for somatostatin and apelin peptides. Caspase-3 signals were not observed only in diabetic group treated with ghrelin. Caspase-8 signals were increased while PCNA signals were decreased in diabetic group given ghrelin compared to diabetic group. Small intestine CAT, SOD, GPx and GST activities and GSH levels were decreased and LPO, PC levels were increased in diabetic rats. Administration of ghrelin to diabetic rats caused an increase in intestinal CAT, SOD, GPx and GST activities and GSH levels, while PC levels decreased. As a result, we observed positive changes in diabetic rats treated with ghrelin in both microscopic and biochemical studies. We can suggest that ghrelin may be an important hormone for the treatment of diabetes.
Keywords: Ghrelin; Gene expression; Antioxidant system; Small intestine; Type 2 diabetes;

Display Omitted► In type 2 diabetes, AG normalizes ET-1-induced aortic contraction by suppressing ETA-R/ERK activities through normalization of the imbalance between Jab1-modified ETA-R and O-GlcNAcylated ETA-R.Circulating levels of endothelin (ET)-1 are increased in the diabetic state, as is endogenous ETA-receptor-mediated vasoconstriction. However, the responsible mechanisms remain unknown. We hypothesized that ET-1-induced vasoconstriction is augmented in type 2 diabetes with hyperglycemia through an increment in advanced glycation end-products (AGEs). So, we investigated whether treatment with aminoguanidine (AG), an inhibitor of AGEs, would normalize the ET-1-induced contraction induced by ET-1 in strips of thoracic aortas isolated from OLETF rats at the chronic stage of diabetes. In such aortas (vs. those from age-matched genetic control LETO rats): (1) the ET-1-induced contraction was enhanced, (2) the levels of HIF1α/ECE1/plasma ET-1 and plasma CML-AGEs were increased, (3) the ET-1-stimulated ERK phosphorylation mediated by ETA-R was increased, (4) the expression level of Jab1-modified ETA-R protein was reduced, and (5) the expression level of O-GlcNAcylated ETA-R protein was increased. Aortas isolated from such OLETF rats that had been treated with AG (50 mg/kg/day for 10 weeks) exhibited reduced ET-1-induced contraction, suppressed ET-1-stimulated ERK phosphorylation accompanied by down-regulation of ETA-R, and increased modification of ETA-R by Jab1. Such AG-treated rats exhibited normalized plasma ET-1 and CML-AGE levels, and their aortas exhibited decreased HIF1α/ECE1 expression. However, such AG treatment did not alter the elevated levels of plasma glucose or insulin, or systolic blood pressure seen in OLETF rats. These data from the OLETF model suggest that within the timescale studied here, AG normalizes ET-1-induced aortic contraction by suppressing ETA-R/ERK activities and/or by normalizing the imbalance between Jab1 and O-GlcNAc in type 2 diabetes.
Keywords: Diabetes; Endothelin-1; ETA-R; Jab1; O-GlcNAc; Otsuka Long–Evans Tokushima Fatty rat;

Measurement of salivary resistin, visfatin and adiponectin levels by Irene Mamali; Nikolaos D. Roupas; Anastasia K. Armeni; Anastasia Theodoropoulou; Kostas B. Markou; Neoklis A. Georgopoulos (120-124).
► Hormonal determination in saliva offers several advantages. ► We introduced a method for measuring resistin, visfatin and adiponectin in saliva. ► Salivary resistin and adiponectin significantly correlated with serum levels. ► No significant correlation between serum and salivary visfatin levels was observed. ► The determination of adipokines in saliva could be useful in clinical research.Hormonal determination in saliva offers several advantages. Peptides enter the salivary glands either by active transport mechanisms or are expressed and secreted by the salivary glands themselves. The collection of saliva is a noninvasive, easily repeatable and less stressful technique than blood withdrawal. The purpose of the present study was to introduce a method for measuring salivary resistin, visfatin and adiponectin levels and to evaluate their associations with serum levels. Resistin, visfatin and adiponectin levels were measured in serum and saliva of 50 healthy adult volunteers (17 male and 33 female) using commercial enzyme immunoassay kits for serum with minor modifications. The present study documented the determination of resistin and adiponectin levels in saliva and the significant correlation of salivary levels with serum levels (r  = 0.441, p  < 0.01 and r  = 0.347, p  < 0.05, respectively). Moreover, the identification of visfatin in saliva was achieved, but no significant correlation with serum visfatin levels was observed. To our knowledge, this is the first study to report the determination of resistin and visfatin in saliva and the significant correlation of salivary resistin with serum levels, while it confirmed the significant association between salivary and serum adiponectin. The introduction of salivary determinations of adipokines could contribute to the elucidation of the physiology and the role of the specific adipokines in various clinical conditions (obesity, insulin resistance, inflammation, reproduction, energy imbalance and stress response).
Keywords: Resistin; Visfatin; Adiponectin; Saliva; Adipose tissue; Adipokines;

Gastrin releasing peptide-29 requires vagal and splanchnic neurons to evoke satiation and satiety by Susan A. Wright; Martha C. Washington; Carlos Garcia; Ayman I. Sayegh (125-131).
► Gastrin-releasing peptide-29 reduces MS (10% sucrose) and prolongs IMI. ► We tested MS and IMI by GRP-29 in VGX and CMGX rats. ► We also tested, by myotomy, if the duodenum is the site of action. ► CMGX and VGX/CMGX attenuated MS and all surgeries attenuated the IMI. ► MS and IMI by GRP-29 require duodenal vagal and splanchnic nerves.We have shown that gastrin-releasing peptide-29 (GRP-29), the large molecular form of GRP in rats, reduces meal size (MS, intake of 10% sucrose solution) and prolongs the intermeal interval (IMI). In these studies, we first investigated possible pathways for these responses in rats undergoing total subdiaphragmatic vagotomy (VGX, removal of vagal afferent and efferent innervation of the gut), celiaco-mesenteric ganglionectomy (CMGX, removal of splanchnic afferent and efferent innervation of the gut) and combined VGX and CMGX. Second, we examined if the duodenum communicates the feeding signals (MS and IMI) of GRP-29 (0, 0.3, 1.0, 2.1, 4.1, 10.3 and 17.2 nmol/kg) with the feeding control areas of the hindbrain by performing duodenal myotomy (MYO), a procedure that severs some layers of the duodenal wall including the vagal, splanchnic and enteric neurons. We found that GRP-29 (2.1, 4.1, 10.3, 17.2 nmol/kg) reduced the size of the first meal (10% sucrose) and (1, 4.1, 10.3 nmol/kg) prolongs the first IMI but did not affect the subsequent meals or IMIs. In addition, CMGX and combined VGX/CMGX attenuated reduction of MS by GRP-29 and all surgeries attenuated the prolongation of the IMI. Therefore, reduction of MS and prolongation of IMI by GRP-29 require vagal and splanchnic nerves, and the duodenum is the major conduit that communicates prolongation of IMI by GRP-29 with the brain.
Keywords: Bombesin; Sympathetic nerve; Meal size; Intermeal interval; Enteric nervous system;

Central apelin-13 inhibits food intake via the CRF receptor in mice by Shuang-Yu Lv; Yan-Jie Yang; Yao-Jun Qin; Jia-Run Mo; Ning-Bo Wang; Yi-Jing Wang; Qiang Chen (132-138).
► Apelin-13 inhibited food and water intake in satiated mice during dark period. ► Apelin-13 inhibited food and water intake in fasted mice during dark period. ► Apelin-13 had no role in food and water intake in satiated mice during light period. ► The apelin-13(F13A) and α-helical CRF9–41 blocked the effect of apelin-13 on feeding. ► The deamino(CH2)5Tyr(Me)AVP could not antagonize the action of apelin-13 on feeding.Apelin, the novel identified peptide, is the endogenous ligand for the APJ. Previous studies have reported the effect of apelin on food intake, however the action of acute central injected apelin on food intake in mice remains unknown. The present study was designed to investigate the mechanism as well as the effect of central apelin-13 on food intake in mice. During the dark period, the cumulative food intake was significantly decreased at 4 h after the intracerebroventricular (i.c.v.) injection of 1 and 3 μg/mouse apelin-13 and the period food intake was significantly reduced during 2–4 h after treatment. In the fasted mice, the cumulative food intake was significantly decreased at 2 and 4 h after injection of 3 μg/mouse apelin-13. The cumulative water intake was significantly reduced by apelin-13 (3 μg/mouse) at 4 h after injection in freely feeding and fasted mice. However, during light period, apelin-13 had no influence on food and water intake in freely feeding mice. The APJ receptor antagonist apelin-13(F13A) (6 μg/mouse) and the corticotrophin-releasing factor (CRF) receptor antagonist α-helical CRF9–41 (3 μg/mouse) could reverse the inhibitory effect on cumulative food intake/0–4 h induced by apelin-13 (3 μg/mouse) in freely feeding mice during the dark period, whereas the anorexic effect could not be antagonized by the arginie vasopressin (AVP) receptor antagonist deamino(CH2)5Tyr(Me)AVP (0.5 μg/mouse). Taken together, these results suggest that central apelin-13 inhibits food intake in mice and it seems that APJ receptor and CRF receptor, but not AVP receptor, might be involved in this process.
Keywords: Apelin-13; Food intake; APJ receptor; CRF receptor; AVP receptor;

Central and peripheral apelin receptor distribution in the mouse: Species differences with rat by George R. Pope; Emma M. Roberts; Stephen J. Lolait; Anne-Marie O’Carroll (139-148).
► APJ is well characterized in the rat but is not well described in mouse tissues. ► We show the first detailed anatomical distribution of APJ mRNA and protein in the mouse. ► There is a species difference in central APJ distribution and in the pituitary gland. APJ distribution in peripheral tissues appears comparable between rat and mouse. ► The apelin system may have a more wide-ranging central role in the rat than the mouse.The G protein-coupled apelin receptor (APJ) binds the endogenous peptide apelin and has been shown to have roles in many physiological systems. Thus far, distribution studies have predominantly been conducted in the rat and there is limited knowledge of the cellular distribution of APJ in mouse or human tissues. As recent functional studies have been conducted in APJ knock-out mice (APJ KO), in this study we undertook to characterize APJ mRNA and I125[Pyr1]apelin-13 binding site distribution in mouse tissues to enable correlation of distribution with function. We have utilized in situ hybridization histochemistry (ISHH) using APJ riboprobes, which revealed strong hybridization specifically in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus and in the anterior pituitary, with marginally lower levels in the posterior pituitary. In the periphery, strong hybridization was observed in the lung, heart, adrenal cortex, renal medulla, ovary and uterus. Autoradiographic binding to APJ with I125[Pyr1]apelin-13 exhibited significant binding in the anterior pituitary, while lower levels were observed in the posterior pituitary and PVN and SON. In the periphery, strong receptor binding was observed in tissues exhibiting intense riboprobe hybridization, indicating a good correlation between receptor transcription and translation. While the distribution of APJ mRNA and functional protein in the mouse shows similarities to that of the rat, we report a species difference in central APJ distribution and in the pituitary gland.
Keywords: APJ; Apelin; In situ hybridization histochemistry; Autoradiography;

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells by Kazunori Kageyama; Gen Hasegawa; Kanako Akimoto; Satoshi Yamagata; Naoki Tamasawa; Toshihiro Suda (149-155).
► There is a close interaction between the HPG axis and the HPA axis. ► CRF and Ucn2 can modulate GnRH mRNA and protein levels via each specific CRF receptor subtype in N39 cells. ► Differential regulation of GnRH by CRF family peptides contributes to the stress response and homeostasis in the GnRH cells.Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo–pituitary–adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF1 receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF2 receptor) with little or no binding affinity for the CRF1 receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF1 receptor and CRF2 receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF1 receptor, while Ucn2 increased the levels via the CRF2 receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.
Keywords: Corticotropin-releasing factor; Gpr147; Hypothalamus; Stress; Urocortin;

► NPFF inhibits amphetamine-induced CPP. ► NPFF receptors antagonist, RF9 reverses anxiety-like effect of amphetamine withdrawal. ► Modulation of NPFF receptors modifies the expression of amphetamine-induced CPP and amphetamine withdrawal anxiety.Many data indicate that endogenous opioid system is involved in amphetamine-induced behavior. Neuropeptide FF (NPFF) possesses opioid-modulating properties. The aim of the present study was to determine whether pharmacological modulation of NPFF receptors modify the expression of amphetamine-induced conditioned place preference (CPP) and amphetamine withdrawal anxiety-like behavior, both processes relevant to drug addiction/abuse. Intracerebroventricular (i.c.v.) injection of NPFF (5, 10, and 20 nmol) inhibited the expression of amphetamine CPP at the doses of 10 and 20 nmol. RF9, the NPFF receptors antagonist, reversed inhibitory effect of NPFF (20 nmol, i.c.v.) at the doses of 10 and 20 nmol and did not show any effect in amphetamine- and saline conditioned rats. Anxiety-like effect of amphetamine withdrawal was measured 24 h after the last (14 days) amphetamine (2.5 mg/kg, i.p.) treatment in the elevated plus-maze test. Amphetamine withdrawal decreased the percent of time spent by rats in the open arms and the percent of open arms entries. RF9 (5, 10, and 20 nmol, i.c.v.) significantly reversed these anxiety-like effects of amphetamine withdrawal and elevated the percent of time spent by rats in open arms at doses of 5 and 10 nmol, and the percent of open arms entries in all doses used. NPFF (20 nmol) pretreatment inhibited the effect of RF9 (10 nmol). Our results indicated that stimulation or inhibition of NPFF receptors decrease the expression of amphetamine CPP and amphetamine withdrawal anxiety, respectively. These findings may have implications for a better understanding of the processes involved in amphetamine dependence.
Keywords: Amphetamine; Conditioned place preference; Withdrawal anxiety; Elevated plus-maze test; NPFF; RF9;

Prognostic value of copeptin: One-year outcome in patients with traumatic brain injury by Guo-Feng Yu; Qiang Huang; Wei-Min Dai; Yuan-Qing Jie; Xiao-Feng Fan; An Wu; Yao Lv; Yun-Ping Li; Xin-Jiang Yan (164-169).
► Plasma copeptin levels increased in patients with traumatic brain injury. ► Copeptin could possibly serve as a novel biomarker in traumatic brain injury. ► Copeptin may be a good prognostic factor for 1-year functional outcome and mortality in patients with traumatic brain injury.High plasma copeptin level has been associated with one-month mortality after traumatic brain injury. However, not much is known regarding its relation with long-term outcome. Thus, we investigated the ability of copeptin to predict 1-year outcome in patients with traumatic brain injury. One hundred and six healthy controls and 106 patients with acute severe traumatic brain injury were included. Plasma samples were obtained on admission. Its concentration was measured by enzyme-linked immunosorbent assay. Forty-eight patients (45.3%) suffered from unfavorable outcome (Glasgow Outcome Scale score of 1–3) and 31 patients (29.2%) died in 1 year after traumatic brain injury. Upon admission, plasma copeptin level in patients was substantially higher than that in healthy controls. A forward stepwise logistic regression selected plasma copeptin level as an independent predictor for 1-year unfavorable outcome and mortality of patients. A receiver operating characteristic curve analysis showed plasma copeptin level predicted 1-year unfavorable outcome and mortality obviously. The predictive value of the copeptin concentration was thus similar to that of Glasgow Coma Scale score for the prediction of unfavorable outcome and mortality after 1 year. In a combined logistic-regression model, copeptin improved the area under curve of Glasgow Coma Scale score for the prediction of unfavorable outcome and mortality after 1 year, but the differences were not significant. Thus, copeptin level is a useful, complementary tool to predict functional outcome and mortality 1 year after traumatic brain injury.
Keywords: Copeptin; Traumatic brain injury; 1-Year outcome;

Inhibition of intraerythrocytic proteasome retards the generation of hemorphins by Chang Zheng Song; Qing Wei Wang; Hong Liu; Chang Cheng Song (170-173).
► Hemorphins are immunoprecipitated from culture supernatant of human erythrocytes. ► Bortezomib inhibits the chymotrypsin-like activity of proteasome within erythrocyte. ► Intraerythrocytic proteasomal inhibition leads to a decline in hemorphin production.Hemorphins are a set of hemoglobin-derived opioid peptides. The production mechanism of these structural overlap peptides remains unclear. Based on the sequences of hemorphins, it could be inferred that hemorphins are probably generated by cleavage of hemoglobin β chain at sites favored by the chymotrypsin-like protease. 20S proteasome possesses the chymotrypsin-like activity and still persists in mature erythrocytes. This study attempts to clarify whether the intraerythrocytic proteasome involves in the formation of hemorphins. Hemorphins containing hemorphin-7 and V-hemorphin-7 are isolated by immunoprecipitation from culture supernatant of human erythrocytes. Bortezomib inhibits the chymotrypsin-like activity of intraerythrocytic proteasome and prevents the yield of hemorphins in a dose-dependent manner. The present study suggests that intraerythrocytic proteasome contributes to the generation of hemorphins.
Keywords: Erythrocyte; Hemorphin; Hemoglobin; Proteasome; Bortezomib;

Angiotensin-(1–7) Mas-receptor deficiency decreases peroxisome proliferator-activated receptor gamma expression in adipocytes by Érica Guilhen Mario; Sérgio Henrique S. Santos; Adaliene Versiane M. Ferreira; Michael Bader; Robson Augusto S. Santos; Leida Maria Botion (174-177).
► The lack of Ang-(1–7) action through Mas receptor increases the levels of serum NEFA. ► Mas-deficient mice present decreased response to the antilipolytic effect of insulin. ► Ang-(1–7) Mas receptor deficiency decreases lipogenic genes expression. ► Ang-(1–7) Mas receptor deficiency is accompanied by a decrease in PPARγ expression.The renin–angiotensin system is an important link between metabolic syndrome and cardiovascular diseases. Besides angiotensin II, other angiotensin peptides such as angiotensin-(1–7), have important biological activities. It has been demonstrated that angiotensin-(1–7), acting through the G protein-coupled receptor encoded by the Mas protooncogene have important actions on the cardiovascular system. However, the role of angiotensin-(1–7)-Mas axis in lipidic profile is not well established. In the present study, the adipocyte metabolism was investigated in wild type and FVB/N Mas-deficient male mice. The gene expression of peroxisome proliferator-activated receptor gamma, acetyl-CoA carboxylase and the amount of fatty acid synthase protein were reduced in the Mas-knockout mice. Serum nonesterified fatty acids of Mas-knockout showed a 50% increase in relation to wild type group. Basal and isoproterenol-stimulated lipolysis was similar between the groups, however, a significant decrease of the glycerol release (lipolytic index) in response to insulin was observed in wild type animals, while no effect of the insulin action was observed in a Mas-knockout group. The data suggest that the lack of angiotensin-(1–7) action through Mas receptor alters the response of adipocytes to insulin action. These effects might be related to decreased expression of PPARγ.

► Antioxidant and antimicrobial peptides derived from fish waste and muscle are reviewed. ► Technological approaches for the production of bioactive peptides are highlighted. ► Large-scale methods suitable for assaying of these peptides are outlined. ► The possible roles of these peptides in reducing many human diseases and their applications innutraceuticals and pharmaceuticals industries are investigated.Fishes are rich sources of structurally diverse bioactive compounds. In recent years, much attention has been paid to the existence of peptides with biological activities and proteins derived from foods that might have beneficial effects for humans. Antioxidant and antimicrobial peptides isolated from fish sources may be used as functional ingredients in food formulations to promote consumer health and improve the shelf life of food products. This paper presents an overview of the antioxidant and antimicrobial peptides derived from various fishes. In addition, we discuss the extraction of fish proteins, enzymatic production, and the techniques used to isolate and characterize these compounds. Furthermore, we review the methods used to assay the bioactivities and their applications in food and nutraceuticals.
Keywords: Fish-derived peptides; Antioxidant peptides; Antimicrobial peptides; Protein hydrolysates;