Peptides (v.34, #2)

► [D4k]ascaphin-8 inhibited growth of Propionibacterium acnes isolates with high potency. ► [D4k]ascaphin-8 and [T5k] temporin-DRa modulated cytokine production by monocytes. ► The peptides show potential for topical treatment of acne vulgaris.The pathogenesis of acne vulgaris is multifactorial involving infection of the pilosebaceous unit with Propionibacterium acnes and a cytokine-mediated inflammatory response. Five frog skin-derived antimicrobial peptides ([D4k]ascaphin-8, [G4K]XT-7, [T5k]temporin-DRa, brevinin-2GU, and B2RP-ERa), chosen for their low hemolytic activity against human erythrocytes, were assessed for their effects on the growth of clinical isolates of P. acnes and on the release of pro-inflammatory and anti-inflammatory cytokines from peripheral blood mononuclear (PBM) cells. All peptides inhibited the growth of P. acnes with the highest potency exhibited by [D4k]ascaphin-8 (minimum inhibitory concentration, MIC = 3–12.5 μM). Release of TNF-α from concanavalin A (ConA)-stimulated PBM cells was significantly reduced by [D4k]ascaphin-8, [G4K]XT-7, brevinin-2GU, and B2RP-ERa (1 and 20 μg/ml) and by [T5k]temporin-DRa (20 μg/ml). Release of IFN-γ from unstimulated PBM cells was significantly reduced by [D4k]ascaphin-8 and brevinin-2GU (1 and 20 μg/ml). No peptide showed significant effects on Il-17 release. Release of the anti-inflammatory cytokines TGF-β, IL-4, and IL-10 from both unstimulated and ConA-treated PBM cells was significantly increased by [T5k]temporin-DRa and B2RP-ERa (1 and 20 μg/ml). The potent activities of [D4k]ascaphin-8 and [T5k]temporin-DRa in inhibiting the growth of P. acnes and the release of pro-inflammatory cytokines, and in stimulating the release of anti-inflammatory cytokines suggest a possible therapeutic role in the treatment of acne vulgaris.
Keywords: Acne vulgaris; Frog skin antimicrobial peptide; Propionibacterium acnes; Cytokine;

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb by Su A. Jang; Hyun Kim; Ju Young Lee; Ju Ri Shin; Da Jung Kim; Ju Hyun Cho; Sun Chang Kim (283-289).
► Novel antimicrobial peptides, Buf III analogs, are designed based on a potent cell-penetrating peptide buforin IIb. ► Buf III analogs, which contain the cell-penetrating motif QWPIG, have similar structure and mechanism of action with buforin IIb. ► The therapeutic index of Buf III analogs increase up to 7-fold compared to buforin IIb. ► Buf III analogs may be promising candidates for future development as novel antimicrobial agents.Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F6] and [V8]) in the proline hinge region with other hydrophobic residues ([W6] and [I8]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR]3 and KLLKQWPIG[KLLK]3, respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.
Keywords: Buforin IIb; Antimicrobial peptide; Cell-penetrating peptide; Mechanism of action; Specificity;

Gene cloning and functional characterization of four novel antimicrobial-like peptides from scorpions of the family Vaejovidae by Santos Ramírez-Carreto; Verónica Quintero-Hernández; Juana María Jiménez-Vargas; Gerardo Corzo; Lourival D. Possani; Baltazar Becerril; Ernesto Ortiz (290-295).
► We construct cDNA libraries from Vaejovis mexicanus smithi and Vaejovis subcristatus. ► We identify cDNAs coding for putative antimicrobial peptides from the libraries. ► We synthesize and characterize the activities of VmCT1, VmCT2, VsCT1 and VsCT2. ► VmCT1 and VmCT2 have broad-spectrum antibacterial activities with low hemolysis.From the cDNA libraries made from the venom glands of two scorpions belonging to the Vaejovidae family, four different putative non disulfide-bridged antimicrobial peptides were identified: VmCT1 and VmCT2 from Vaejovis mexicanus smithi plus VsCT1 and VsCT2 from Vaejovis subcristatus. These short peptides (with only 13 amino acid residues each) share important amino acid sequence similarities among themselves and with other reported antimicrobial peptides, but their biological activities vary dramatically. This communication reports the cloning, chemical synthesis and characterization of these peptides. Two peptides, VmCT1 and VmCT2 showed broad-spectrum antibacterial activity with minimum inhibitory concentrations MICs in the range of 5–25 μM and 10–20 μM respectively, whereas their hemolytic activity at these concentrations was low. Structure–function relationships that might determine the differences in activities are discussed.
Keywords: Antimicrobial peptide; cDNA library; Scorpion; Vaejovis;

LC–MS/MS with 2D mass mapping of skin secretions’ peptides as a reliable tool for interspecies identification inside Rana esculenta complex by Tatyana Yu. Samgina; Vladimir A. Gorshkov; Konstantin A. Artemenko; Egor A. Vorontsov; Oleg V. Klykov; Sergey V. Ogourtsov; Roman A. Zubarev; Albert T. Lebedev (296-302).
R. esculenta skin peptidome differs from R. ridibunda and R. lessonae peptidomes. ► LC–MS/MS allows easy determination of frog skin peptides. ► 2D maps provide clear representation of peptidome differences.Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex. LC–MS/MS is an ideal analytical tool for the quantitative and qualitative analysis of these peptides. The results covering elemental composition of these peptides, their levels in the secretion, as well as their belonging to a certain family of peptides may be visualized by means of 2D mass maps. The proposed approach proved itself to be a perspective tool for the reliable identification of all 3 species constituting R. esculenta complex. Easy distinguishing between the species may be achieved using 2D maps as fingerprints. Besides this approach may be used to study hybridogenesis and mechanisms of hemiclonal transfer of genetic information, when rapid and reliable identification of species involved in the process is required.
Keywords: Rana esculenta complex; Skin secretion; Peptides; LC–MS/MS; 2D mass mapping;

Characterization of GnRH-related peptides from the Pacific oyster Crassostrea gigas by Laetitia Bigot; Céline Zatylny-Gaudin; Franck Rodet; Benoit Bernay; Pierre Boudry; Pascal Favrel (303-310).
► We characterized two GnRH forms in the oyster. ► Expression of GnRH peptides and encoding gene is specific to the CNS. ► Expression of GnRH gene depends of the reproduction and the nutritional status.Gonadotropin-releasing hormone (GnRH), a key neuropeptide regulating reproduction in vertebrates has now been characterized in a number of non-vertebrate species. Despite the demonstration of its ancestral origin, the structure and the function of this family of peptides remain poorly known in species as distant as lophotrochozoans. In this study, two GnRH-related peptides (Cg-GnRH-a and CgGnRH-G) were characterized by mass spectrometry from extracts of the visceral ganglia of the Pacific oyster Crassostrea gigas. These peptides showed a high degree of sequence identity with GnRHs of other mollusks and annelids and to a lesser extent with those of vertebrates or with AKH and corazonins of insects. Both the mature peptides and the transcript encoding the precursor protein were exclusively expressed in the visceral ganglia. Significant differences in transcriptional activity of Cg-GnRH encoding gene were recorded in the ganglia along the reproductive cycle and according to trophic conditions with a higher level in fed animals compared to starved animals. This suggests the involvement of Cg-GnRHs as synchronizers of nutritional status with energy requirements during reproduction in oyster. Evidence for a role of Cg-GnRHs as neuroregulators and as neuroendocrine factors in bivalve is discussed.
Keywords: Mollusk; Bivalve; Oyster; Neuroendocrinology; Reproduction; GnRH;

► Three new K+-channel specific toxins (referred to as BmKcugx, BmKcug1 and BmKcug2, respectively) were identified from the Chinese scorpion Mesobuthus martensii Karsch. ► BmKcugx represents a new subfamily of K+-channel toxins (α-KTx22). ► Genomic analysis showed that there are intronic number polymorphisms among the genes of BmKcugx, BmKcug1 and BmKcug2. ► An alternative splicing event was found with BmKcug2 transcript. ► Intron located in the 5′UTR of the BmKcug1 and BmKcug2 gene could markedly increase their expression level in the scorpion venom.K+-channel specific toxins from scorpions are powerful probes used in the structural and functional characterization of different subfamilies of K+-channels which are thought to be the most diverse ion channels. However, only a limited number of K+-channel toxins have been identified from scorpions so far; moreover, little is known about the mechanisms for the generation of a combinatorial peptide library in a venom gland of a scorpion. Here, we identified and characterized three new K+-channel toxin-like peptides from the scorpion Mesobuthus martensii Karsch, which were referred to as BmKcug1, BmKcug2 and BmKcugx, respectively. BmKcug1 and BmKcug2 are two new members of α-KTx1 subfamily, and have been classified as α-KTx1.14 and α-KTx1.15, respectively. BmKcugx represents a new subfamily of K+-channel specific toxins which was classified into α-KTx22. BmKcugx was thus classified as α-KTx22.1. Genomic analysis demonstrated that BmKcugx gene has two exons interrupted by an intron inserted in the signal peptide encoding region, whereas BmKcug1a (a close homologue of BmKcug1)/BmKcug2 gene was interrupted by two introns, located within the 5′UTR of the gene and in the signal peptide encoding region, respectively. Transcriptomic analysis for the venom glands of M. martensii Karsch indicated that the abundances of the transcripts of BmKcug1a and BmKcug2 are much higher than that of BmKcugx; it suggests that the intron in 5′UTR could markedly increase the expression level of the K+-channel toxins. Alignment of the genomic sequences of BmKcug1a and BmKcug2 revealed that an alternative splicing event occurred at the intron 1-exon 2 junction in the 5′UTR of BmKcug2 transcript.
Keywords: K+-channel specific toxins; Scorpion; Mesobuthus martensii Karsch; Intron; Alternative splicing; Genomic organization; α-KTx22;

GHRP-6 mimics ghrelin-induced stimulation of food intake and suppression of locomotor activity in goldfish by Satowa Yahashi; Ki Sung Kang; Hiroyuki Kaiya; Kouhei Matsuda (324-328).
► GHRP-6 is one of the GHSs, and it is able to activate GHS-R2a-1 in goldfish in vitro. ► GHRP-6 stimulates food intake and suppresses locomotor activity in goldfish. ► GHRP-6-induced action might be mediated via the GHS-R2a-1 and subsequently through vagal afferent in goldfish.Ghrelin was first identified and characterized from rat stomach as an endogenous ligand for the growth hormone secretagogue (GHS) receptor (GHS-R). Ghrelin also acts as an orexigenic factor and regulates energy balance in rodents. In goldfish, native ghrelin consists of 11 molecular variants, the major form being a 17-residue peptide with n-octanoic acid modification (n-octanoyl ghrelin17), and intraperitoneal (IP) administration of n-octanoyl ghrelin17 induces central actions such as stimulation of food intake and suppression of locomotor activity through capsaicin-sensitive afferents. Four types of GHS-Rs (1a-1, 1a-2, 2a-1 and 2a-2) have been identified in goldfish, and one GHS, GHRP-6, can activate only GHS-R2a-1 in vitro. However, there is no information about the effect of GHRP-6 on food intake and locomotor activity in goldfish in vivo. Therefore, in the present study, we examined whether IP-administered GHRP-6 would mimic the orexigenic action of n-octanoyl ghrelin17 and its suppression of locomotor activity. IP administration of GHRP-6 at 1 pmol/g body weight (BW) stimulated food intake, and was equipotent to the orexigenic action of n-octanoyl ghrelin17 at 10 pmol/g BW. IP-injected GHRP-6 at 1 pmol/g BW also induced a significant decrease of locomotor activity, as was the case for IP-injected n-octanoyl ghrelin17 at 10 pmol/g BW. The action of GHRP-6 was blocked by IP-preinjected capsaicin at 160 nmol/g BW. These results suggest that the central action of GHRP-6 might be mediated via the GHS-R2a-1-signaling pathway, and subsequently through capsaicin-sensitive afferents in goldfish.
Keywords: Capsaicin; Vagal afferent; Food intake; Goldfish; Ghrelin; GHRP-6; GHS; GHS-R; GHS-R2a-1; Locomotor activity; n-Octanoyl ghrelin17;

Leptins and leptin receptor expression in the goldfish (Carassius auratus). Regulation by food intake and fasting/overfeeding conditions by Ana Belén Tinoco; Laura Gabriela Nisembaum; Esther Isorna; María Jesús Delgado; Nuria de Pedro (329-335).
gLep-aI is ubiquitous expressed, while gLep-aII is mainly located in goldfish brain. ► The widespread distribution of leptin receptor suggests pleitropic leptin actions. ► The gLep-aI increases after food intake in liver, but not in hypothalamus. ► The leptin system expression appears to be independent of the feeding status. ► Leptin could signal short-term changes in food intake, as postprandial satiety.Leptin is a hormone involved in feeding and body weight regulation in vertebrates, but the relationship between energy status and leptin has not been clearly established in fish. The aim of this study was to investigate in a teleost, the goldfish (Carassius auratus), the tissue expression pattern of two leptins (gLep-aI and gLep-aII) and leptin receptor (gLepR); and the effect of feeding on expression of these genes. Leptin system expression in goldfish was firstly analyzed in fish under overfeeding (2 weeks) or fasting (1 week), and secondly, at different postfeeding times (0, 3, 6, 9 and 12 h). Goldfish has two Lep-a paralog genes, gLep-aI was widely expressed in central and peripheral tissues, whereas gLep-aII was preferentially expressed in brain. This different distribution pattern of leptins suggests that they can play different physiological roles in goldfish. The gLepR mRNA was ubiquitous expressed, with the highest expression in the telencephalon and hypothalamus. No significant differences in the leptin system expression were found among control, overfed and fasting groups, suggesting an apparent lack of correlation between nutritional status and leptin system in goldfish. Hepatic expression of gLep-aI significantly increased 9 h after feeding time, while hypothalamic leptin system expression did not change after feeding. In summary, leptin in goldfish could signal short-term changes in food intake, as postprandial satiety, but seems to be independent of fasting/overfeeding conditions in this teleost. The widespread distribution of leptins and leptin receptor in goldfish strongly supports that this hormone may have pleitropic actions in fish.
Keywords: Leptin; Leptin receptor; Goldfish; Feeding; Fasting; Overfeeding;

Neuropeptide Y activates phosphorylation of ERK and STAT3 in stromal vascular cells from brown adipose tissue, but fails to affect thermogenic function of brown adipocytes by Kohei Shimada; Yuta Ohno; Yuko Okamatsu-Ogura; Masahiro Suzuki; Akihiro Kamikawa; Akira Terao; Kazuhiro Kimura (336-342).
► Role of NPY, a peptidergic neurotransmitter, in BAT function has not been fully understood. ► NPY had no effect on basal and norepinephrine-enhanced oxygen consumption rate in brown adipocyte. ► NPY failed to activate MAP kinase (ERK) in brown adipocyte. ► NPY stimulated ERK phosphorylation in stromal vascular cells from BAT. ► NPY also stimulated STAT3 phosphorylation in the stromal vascular cells.The thermogenic function of brown adipose tissue (BAT) is increased by norepinephrine (NE) released from sympathetic nerve endings, but the roles of NPY released along with NE are poorly elucidated. Here, we examined effect of NPY on basal and NE-enhanced thermogenesis in isolated brown adipocytes that express Y1 and Y5 receptor mRNA. Treatment of cells with NPY did not influence the basal and NE-enhanced rates of oxygen consumption and cAMP accumulation. Treatment with NPY also failed to induce ERK (Thr202/Tyr204) phosphorylation in the brown adipocytes. In contrast, treatment with NPY increased ERK phosphorylation in cultured stromal vascular cells from the BAT that express Y1 receptor mRNA. In the latter treatment with NPY also increased STAT3 (Ser727) phosphorylation. These results suggest that NPY mainly acts on stromal vascular cells in BAT and plays roles in the regulation of their gene transcription through ERK and STAT3 pathways, while NPY does not affect the thermogenic function of brown adipocytes.
Keywords: BAT; Norepinephrine; p44/p42 MAPK; NPY; STAT; Thermogenesis;

► The plasma CGRP levels in both PCOS and control subjects were measured and the correlations of insulin, gonadal hormone levels with CGRP in PCOS were analyzed. ► The plasma CGRP level in women with PCOS was higher than that of control subjects, and the hormonal and metabolic parameters was positive correlation with CGRP levels. ► Human granulosa cells expressed CGRP receptor and CGRP caused an elevation of T and E2 released from the human granulosa cells.The aim of the study was to evaluate the plasma level of calcitonin gene-related peptide (CGRP) in patients with polycystic ovary syndrome (PCOS) and its relationship to hormonal and metabolic parameters. We also observed the effect of CGRP on testosterone (T) and estradiol (E2) release in cultured human granulosa cells. PCOS subjects (n  = 215) and matched healthy control women (n  = 103) at age of 22–38 years were enrolled in this study. We analyzed plasma CGRP concentrations, relationship of plasma CGRP with insulin resistance (IR), body mass index (BMI), luteinizing hormone/follicle-stimulating hormone (LH/FSH) ratio and T. The T and E2 release levels of cultured human granulosa cells treated by CGRP were also measured. The results showed that plasma CGRP concentrations were significantly higher in women with PCOS than those of control subjects. In women with PCOS, there was a strong positive correlation between the plasma CGRP level with HOMA-IR, AUC-insulin, AUC-glucose, the ratio of LH/FSH and plasma T concentration. Human granulosa cells expressed CGRP receptor. Exogenous CGRP caused an elevation of T and E2 released from the human granulosa cells. These findings suggest that CGRP may participate in the pathophysiological process of PCOS.
Keywords: Calcitonin gene-related peptide (CGRP); Insulin resistance (IR); Polycystic ovary syndrome (PCOS); Testosterone (T);

Blockade of early and late retinal biochemical alterations associated with diabetes development by the selective bradykinin B1 receptor antagonist R-954 by Orlando Catanzaro; Emilio Labal; Ana Andornino; Jorgelina Aria Capponi; Irene Di Martino; Pierre Sirois (349-352).
► Chronic hyperglycemia in diabetes development associated with significant organ damage. ► Hyperglycemia increased in NO, kallikrein and vascular capillary permeability in diabetic rat retina 4 and 12 weeks after STZ. ► Hyperglycemia decreased Na/K ATPase activity in diabetic rat retina 4 and 12 weeks after STZ injection. ► Treatment for 5 days with BKB1-R antagonist R-954 highly reduced NO, kallikrein and capillary permeability and increased Na/K ATPase activity in the retina. ► BKB1-R receptor subtype over-expressed during STZ-induced diabetes development in rat retina.The chronic hyperglycemia measured alongside diabetes development is associated with significant long-term damage and failure of various organs. In the present study it was shown that hyperglycemia induced early and long term increases in nitric oxide (NO) levels, kallikrein activity and vascular capillary permeability measured as plasma extravasation, and decreases of Na/K ATPase activity in diabetic rat retina 4 and 12 weeks after streptozotocin (STZ) injection. Treatment of the animals for 5 consecutive days with a novel selective bradykinin B1 receptor (BKB1-R) antagonist R-954 (2 mg/kg s.c) at the end of the 4 and 12 week periods highly reduced NO, kallikrein and capillary permeability and increased Na/K ATPase activity in the retina. These results suggest that the BKB1-R receptor subtype is over-expressed during the streptozotocin-induced development of diabetes in rat retina as evidenced by the inhibitory effects of the BKB1-R antagonist R-954 on NO, kallikrein and vascular permeability increases as well as Na/K ATPase decreases. The beneficial role of the BKB1-R antagonist R-954 for the treatment of the diabetic retinopathy is also suggested.
Keywords: Diabetes; Retina; Nitric oxide; Kallikrein; Na/K ATPase; Vascular permeability; Streptozotocin; Bradykinin B1 receptor; R-954;

Cortistatin modulates the expression and release of corticotrophin releasing hormone in rat brain. Comparison with somatostatin and octreotide by Giuseppe Tringali; Maria Cristina Greco; Lucia Lisi; Giacomo Pozzoli; Pierluigi Navarra (353-359).
► We investigated the effects of cortistatin on CRH production from isolated rat brain regions. ► We also compared the action of cortistatin with those of somatostatin and octreotide. ► Cortistatin inhibited the release and gene expression of CRH from rat hypothalamus. ► Cortistatin inhibited basal and stimulated CRH release from rat hippocampus. ► Somatostatin and octreotide showed a different action profile from cortistatin.Cortistatin (CST) is an endogenous neuropeptide characterized by remarkable structural and functional resemblance to somatostatin (SST), both peptides sharing the ability to bind and activate all five SST receptor subtypes. Evidence is also available showing that CST exerts biological activities independently from SST, perhaps via the activation of specific receptors that remain to be fully characterized at present. Here we have investigated the effects of CST on the gene expression and release of corticotrophin releasing hormone (CRH) from rat hypothalamic and hippocampal explants; moreover, we compared the effects of CST with those of SST and octreotide (OCT) in these models. We found that: (i) CST inhibits the expression and release of CRH from rat hypothalamic and hippocampal explants under basal conditions as well as after CRH stimulation by well known secretagogues; (ii) SST does not modify basal CRH secretion from the hypothalamus or the hippocampus, while it is able to reduce KCl-stimulated CRH release from both brain areas; (iii) OCT inhibits both basal and KCl-induced CRH secretion from rat hypothalamic explants, while it has no effect on CRH release from the hippocampus, either under basal conditions or after stimulation by high K+ concentrations; (iv) at variance with CST; SST and OCT have not effect whatsoever on veratridine-induced CRH release from the hypothalamus. In conclusion the present findings provide in vitro evidence in support of the hypothesis that CST plays a role in the regulation of endocrine adaptive responses to stress.
Keywords: Cortistatin; Corticotrophin releasing hormone; Somatostatin; Octreotide; Rat hypothalamus; Rat hippocampus;

Somatostatin modulates generation of inspiratory rhythms and determines asphyxia survival by Josué O. Ramírez-Jarquín; Sergio Lara-Hernández; Juan J. López-Guerrero; Miguel A. Aguileta; Ana J. Rivera-Angulo; Alicia Sampieri; Luis Vaca; Benito Ordaz; Fernando Peña-Ortega (360-372).
► Somatostatin modulates the preBötzinger complex. ► Somatostatin modulates the generation of eupnea, gasps and sighs. ► An endogenous somatostatinergic tone regulates respiratory rhythms generation. ► Somatostatin modulates the preBötzinger complex through somatostatin receptor 2. ► Blockade of somatostatin receptor 2 inhibits gasping and autoresuscitation.Breathing and the activity of its generator (the pre-Bötzinger complex; pre-BötC) are highly regulated functions. Among neuromodulators of breathing, somatostatin (SST) is unique: it is synthesized by a subset of glutamatergic pre-BötC neurons, but acts as an inhibitory neuromodulator. Moreover, SST regulates breathing both in normoxic and in hypoxic conditions. Although it has been implicated in the neuromodulation of breathing, neither the locus of SST modulation, nor the receptor subtypes involved have been identified. In this study, we aimed to fill in these blanks by characterizing the SST-induced regulation of inspiratory rhythm generation in vitro and in vivo. We found that both endogenous and exogenous SST depress all preBötC-generated rhythms. While SST abolishes sighs, it also decreases the frequency and increases the regularity of eupnea and gasping. Pharmacological experiments showed that SST modulates inspiratory rhythm generation by activating SST receptor type-2, whose mRNA is abundantly expressed in the pre-Bötzinger complex. In vivo, blockade of SST receptor type-2 reduces gasping amplitude and consequently, it precludes auto-resuscitation after asphyxia. Based on our findings, we suggest that SST functions as an inhibitory neuromodulator released by excitatory respiratory neurons when they become overactivated in order to stabilize breathing rhythmicity in normoxic and hypoxic conditions.
Keywords: Neuromodulation; Somatostatin; Respiratory rhythm; Gasping; Auto-resuscitation;

Ghrelin promotes the differentiation of human embryonic stem cells in infarcted cardiac microenvironment by Meijuan Gao; Jin Yang; Guoqiang Liu; Rui Wei; Lin Zhang; Haining Wang; Guang Wang; Hongwei Gao; Guian Chen; Tianpei Hong (373-379).
► Ghrelin benefits to the survival of hESCs in infarcted cardiac microenvironment. ► Ghrelin facilitates hESCs differentiated into cardiac progenitor cells. ► Ghrelin administration and hESC transplantation do not improve heart function.Ghrelin is broadly expressed in myocardial tissues, where it exerts different functions. It also has been found to have a wide variety of biological functions on cell differentiation and tissue development. The aim of this study was to investigate the effect of ghrelin on human embryonic stem cell (hESC) differentiation in infarcted cardiac microenvironment. The hESCs grown on feeder layers expressed several pluripotential markers including alkaline phosphatase (AKP). Four weeks after transplantation into rat infarcted hearts, the hESCs and their progeny cells survived and formed intracardiac grafts were 54.7% and 19.6% respectively in ghrelin- and phosphate-buffered saline (PBS)-treated groups. Double immunostaining with anti-human Sox9 and anti-HNA or anti-human fetal liver kinase-1 (Flk1) and anti β-tubulin showed that the human grafts were in development. However, double positive stains were only found in the ghrelin-treated group. In addition, the hESC injection protocol was insufficient to restore heart function of the acute myocardial infarction model. Our study, therefore, provides a new insight of ghrelin on promoting hESC survival and differentiation in rat infarcted cardiac microenvironment. This may give a clue for therapy for myocardial infarction by hESCs or progeny cells.
Keywords: Ghrelin; Embryonic stem cells; Differentiation; Myocardial infarction;

Angiotensin-(1–7) abrogates mitogen-stimulated proliferation of cardiac fibroblasts by LaTronya T. McCollum; Patricia E. Gallagher; E. Ann Tallant (380-388).
► Angiotensin-(1–7) [Ang-(1–7)] inhibited DNA, protein, and collagen synthesis in cardiac fibroblasts. ► Ang-(1–7) increased dual specificity phosphatase DUSP1 and reduced ERK1/ERK2 MAP kinases. ► Ang-(1–7) decreased COX-2 and prostaglandin E synthase (PGES) to reduce prostaglandins.Previous studies showed that angiotensin-(1–7) [Ang-(1–7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1–7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1–7) that participate in fibroblast proliferation. Ang-(1–7) reduced 3H-thymidine, -leucine and -proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1–7) was blocked by the AT(1–7) receptor antagonist [d-Ala7]-Ang-(1–7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1–7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and -ERK2. In contrast, Ang-(1–7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1–7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1–7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1–7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies.
Keywords: Angiotensin-(1–7); Cardiac fibrosis; Mitogen-activated protein kinases; Dual specificity phosphatase; Cyclooxygenase; Prostaglandin E synthase;

Release patterns of copeptin and troponin in Tako-Tsubo cardiomyopathy by Christof Burgdorf; Andreas Schubert; Heribert Schunkert; Volkhard Kurowski; Peter W. Radke (389-394).
► Elevated serum copeptin levels can be found in approximately 50% of patients with acute Tako-Tsubo cardiomyopathy. ► Copeptin levels prior to cardiac catheterization do not differ significantly between patients with Tako-Tsubo cardiomyopathy and matched control patients with acute myocardial infarction. ► Patients with Tako-Tsubo cardiomyopathy and a typical (apical) ballooning pattern show significantly lower levels of copeptin as compared to patients with an atypical (midventricular) wall motion abnormality.Copeptin, in addition to troponin, has recently been suggested for non-invasive differentiation between Tako-Tsubo cardiomyopathy (TTC) and acute myocardial infarction (AMI). In order to test this hypothesis, we investigated release patterns of pituitary copeptin and cardiac troponin in 49 patients with TTC and 49 age-, gender-, and ECG-matched control patients with AMI. Elevated copeptin levels (i.e. > 12 pmol/l) at cardiac catheterization were found in 23/49 (47%) TTC patients and 25/49 (51%) AMI patients. Of these, median copeptin levels were almost identical in both cohorts (34.1 vs. 35.4 pmol/l). Subgroup analysis according to ECG changes revealed that AMI patients with ST-segment elevation had 3.6-fold higher copeptin levels than AMI patients without ST-segment elevation (p  < 0.05). Furthermore, in patients with TTC and atypical (midventricular) ballooning on left ventricular angiography, copeptin levels were 5.7-fold higher than in TTC patients with a typical (apical) type of ballooning (p  < 0.01). Elevated troponin T levels (i.e. > 0.01 μg/l) at catheterization were detectable in 47/49 (96%) TTC patients and 45/49 (92%) AMI patients; however, peak levels did not differ significantly between both cohorts (median 0.35 vs. 0.27 μg/l). Subgroup analysis according to ECG changes revealed 2-fold higher peak troponin T levels in TTC patients presenting with ST-segment elevation than non-ST-segment elevation (p  < 0.05). In conclusion, copeptin does not seem to significantly increase non-invasive differentiation between TTC and AMI. At present, coronary angiography, specifically in patients with ST-segment elevation at presentation remains absolutely mandatory.
Keywords: Tako-Tsubo cardiomyopathy; Acute myocardial infarction; Copeptin; Troponin;

Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2 by Xinjin Guo; Qiang Meng; Qi Liu; Changyuan Wang; Huijun Sun; Taiichi Kaku; Kexin Liu (395-403).
► The methodology of transfected cells involved is simple, highly reproducible and results in low cytotoxicity. ► Transfected cells were used as an in vitro model to study the drug absorption/reabsorption mechanisms of PEPT1/PEPT2. ► HeLa-hPEPT1 and HeLa-hPEPT2 cells were used as an in vitro model to predict targets of the DDI between JBP485 and other substrates (such as cephalexin or lisinopril) of PEPT1 and PEPT2 in vivo.The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis–Menten constant (K m) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K m values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K i value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H+ dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug–drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.
Keywords: JBP485; Gly-Sar; Transfected cells; PEPT1; PEPT2; Uptake;

[ t Bu-d-Gly5]NPS, a pure and potent antagonist of the neuropeptide S receptor: In vitro and in vivo studies by C. Ruzza; A. Rizzi; V. Camarda; A. Pulga; G. Marzola; M. Filaferro; C. Novi; V. Ruggieri; E. Marzola; G. Vitale; S. Salvadori; R. Guerrini; G. Calo’ (404-411).
► The pharmacological profile of [ t Bu-d-Gly5]NPS was assessed in vitro and in vivo. ► In vitro [ t Bu-d-Gly5]NPS behaved as a pure and potent (pA2 7.17) NPSR antagonist. ► In vivo in mice [ t Bu-d-Gly5]NPS fully prevented the stimulant effects of NPS. ► In vivo in rats [ t Bu-d-Gly5]NPS antagonized the anxiolytic like actions of NPS. ► [ t Bu-d-Gly5]NPS is a novel and useful tool for investigating the NPS/NPSR system.Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, the NPSR ligand [ t Bu-d-Gly5]NPS was generated and in vitro characterized as a pure antagonist at the mouse NPSR. In the present study the pharmacological profile of [ t Bu-d-Gly5]NPS has been investigated. [ t Bu-d-Gly5]NPS activity was evaluated in vitro in the calcium mobilization assay at the rat NPSR and in vivo in the locomotor activity and righting reflex tests in mice and in the elevated plus maze and defensive burying assays in rats. In vitro, [ t Bu-d-Gly5]NPS was inactive per se while it inhibited the calcium mobilization induced by 30 nM NPS (pKB 7.42). In Schild analysis experiments [ t Bu-d-Gly5]NPS (0.1–10 μM) produced a concentration-dependent rightward shift of the concentration–response curve to NPS, showing a pA2 value of 7.17. In mouse locomotor activity experiments, supraspinal injection of [ t Bu-d-Gly5]NPS (1–10 nmol) dose dependently counteracted NPS (0.1 nmol) stimulant effects. In the mouse righting reflex assay [ t Bu-d-Gly5]NPS (0.1–10 nmol) fully prevented the arousal-promoting action of the natural peptide (0.1 nmol). Finally, [ t Bu-d-Gly5]NPS (3–30 nmol) was able to completely block NPS (1 nmol) anxiolytic-like actions in rat elevated plus maze and defensive burying assays. Collectively, the present results demonstrated that [ t Bu-d-Gly5]NPS behaves both in vitro and in vivo as a pure and potent NPSR antagonist. This compound represents a novel and useful tool for investigating the pharmacology and neurobiology of the NPS/NPSR system.
Keywords: Neuropeptide S; [ t Bu-d-Gly5]NPS; Calcium mobilization; Locomotor activity; Righting reflex; Anxiety;

Supraspinal injection of Substance P attenuates allodynia and hyperalgesia in a rat model of inflammatory pain by Carmela Parenti; Giuseppina Aricò; Giuseppe Ronsisvalle; Giovanna M. Scoto (412-418).
Display Omitted► Central injection of Substance P prevented inflammatory pain. ► Analgesia induced by supraspinal Substance P is opioid-mediated. ► Substance P-NK1 receptor system is involved in inflammatory pain modulation.The neuropeptide Substance P (SP), that has a high affinity for the neurokinin 1 (NK1) receptor, is involved in modulation of pain transmission. Although SP is thought to have excitatory actions and promote nociception in the spinal cord, the peptide induces analgesia at the supraspinal level. The aim of this study was to evaluate the role of supraspinal SP and the NK1 receptor in inflammatory pain induced by injection of carrageenan in the hind paw of the rat. There are two nociceptive behavioral responses associated with this pain state: mechanical allodynia and heat hyperalgesia. Because the NK1 receptor colocalizes with the MOP receptor in supraspinal sites involved in pain modulation, we also decided to study the possible involvement of the opioid system on SP-induced analgesia. We found that treatment with SP, at doses of 3.5, 5 and 7 μg/5 μl/rat i.c.v., clearly showed inhibition of allodynia and hyperalgesia. Pretreatment with the selective NK1 antagonist L-733,060 (10 mg/kg i.p.) blocked the SP-induced analgesia, suggesting the involvement of the NK1 receptor. This SP-induced analgesia was significantly reduced by administration of the opioid antagonist naloxone (3 mg/kg s.c.). This reduction occurred when SP was administered either before or after the carrageenan injection. These results suggest a significant antinociceptive role for SP and the NK1 receptor in inflammatory pain at the supraspinal level, possibly through the release of endogenous opioids.
Keywords: NK1 receptor; Supraspinal level; Naloxone; Hyperalgesia; Allodynia; Inflammatory pain;

Orexins increase penicillin-induced epileptic activity by Selim Kortunay; Haydar Ali Erken; Gülten Erken; Osman Genç; Melike Şahiner; Sebahat Turgut; Günfer Turgut (419-422).
► Orexins are excitatory neuropeptides. ► We investigate the effects of orexins on penicillin-induced epilepsy in rats. ► Orexins cause dramatically increase penicillin-induced epileptic activity.Orexins have been implicated with physiological function including sleep-wake cycle, energy homeostasis, drinking behavior, analgesia, attention, learning and memory but their effects on excitability are controversial. We investigated the effects of intracortical injections of orexin A (100 pmol) and B (100 pmol) on the electrophysiological manifestation of epileptic seizures induced by cortical penicillin application in adult male rats. In comparison to saline, orexin A and B enhanced significantly the spike number, spike amplitude and spectral power values induced by cortical penicillin. Our findings indicates that orexins enhances the hyperexcitable and hypersyncronic cortical epileptic activity induced by focal application of penicillin-G.
Keywords: EEG; Epilepsy; Orexin; Penicillin; Power spectrum; Seizure;

Detection of small bioactive peptides from Atlantic herring (Clupea harengus L.) by Daniela M. Pampanin; Eivind Larssen; Fiona Provan; Morten Sivertsvik; Peter Ruoff; Magne O. Sydnes (423-426).
► Bioactive peptide identification in Atlantic herring residual material. ► Bioactive peptides related to cardiosystem, antioxidant, opioid, immunomodulation. ► Identification was performed using MS, bioinformatics and an in-house database. ► The approach used is good for mapping the bioactive peptide potential.Recent research has shown that fish residual materials contain a range of components with interesting biological activity. Therefore, there is a great potential in the marine bioprocess industry to utilize these by-products as starting material for generating more valuable products. The aim of the present study was to search for bioactive peptides (in particular small natural bioactive peptides with molecular weight lower than 10 kDa) in Atlantic herring (Clupea harengus L.) by-products such as skin and more general residual materials. By such means a range of peptides with claimed interesting biological activities was found. Herein the activity of the detected bioactive peptides and strategies for isolating peptide fragments containing the bioactive motif is discussed. Identification of bioactive peptides in crude peptide/protein sources (skin and residual materials) was performed directly using a combination of mass spectrometry (Orbitrap), bioinformatics and database search. This method was a good angle of approach in order to map the potential in new species and species that have been very little studied.

► Renin expression in the adrenal gland regulates aldosterone production. ► The adrenal RAS acts as amplification system for circulating angiotensin. ► The adrenal gland expresses a transcript that encodes for a cytosolic renin isoform.In the adrenal gland all components of the renin–angiotensin system (RAS) are expressed in both the adrenal cortex and the adrenal medulla. In this review evidence shall be presented that a local secretory RAS exists in the adrenal cortex that stimulates aldosterone production and serves as an amplification system for circulating angiotensin (ANG) II. The regulation of the secretory adrenal RAS clearly differs from the regulation of the circulatory RAS in terms of renin expression as well as of renin secretion. For example under potassium load the activity of the renal and circulatory RAS is suppressed whereas the activity of the adrenal RAS is stimulated. Thus the activity of the adrenal RAS but not of the circulating RAS correlates well with the regulation of aldosterone production by potassium. The present review also summarizes the knowledge about the expression and functions of an additional renin transcript that has recently been discovered. This transcript encodes for a non-secretory cytosolic renin isoform. The cytosolic renin may be a basis for the existence of an intracellular renin system in the adrenal gland that has long been proposed. The present state of knowledge shall be discussed indicating that such an intracellular system modulates cell survival and cell death such as apoptosis and necrosis or cell functions such as aldosterone production.
Keywords: Renin; Adrenal gland; Angiotensin; Secretion; Cytosolic renin;

Display Omitted► Ligands of bradykinin B1 and B2 receptors have been extended at their N-terminus. ► A certain loss of affinity was observed, more so for agonists than antagonists. ► The full set of fluorescent agonist and antagonist ligands has been characterized. ► Angiotensin converting enzyme was imaged using a fluorescent bradykinin analog. ► Metal- or drug-containing conjugates show the prospect of functional cargoes.Peptide agonists and antagonists of both bradykinin (BK) B1 and B2 receptors (B1R, B2R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ɛ-aminocaproyl (ɛ-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B1R: B-10376: CF-ɛ-ACA-Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-BK; B2R: B-10380: CF-ɛ-ACA-d-Arg-[Hyp3, Igl5, d-Igl7, Oic8]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B2R agonists CF-ɛ-ACA-BK, AF350-ɛ-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B1R agonist B-10378 (CF-ɛ-ACA-Lys-des-Arg9-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ɛ-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.
Keywords: Bradykinin receptors; Fluorescent ligands; Structure–activity relationship; Cell imaging; Receptor endocytosis; Angiotensin converting enzyme;