Peptides (v.30, #4)

Identification and structural insights of three novel antimicrobial peptides isolated from green coconut water by Santi M. Mandal; Satyahari Dey; Mahitosh Mandal; Siddik Sarkar; Simone Maria-Neto; Octavio L. Franco (633-637).
Infections caused by pathogenic bacteria could cause an expressive negative impact on human health. A significant enhance in resistance to commercial antibiotics has been observed in all kinds of pathogenic bacteria. In order to find novel approaches to control such common infections, a wide number of defense peptides with bactericidal properties have been characterized. In this report, three peptides lower than 3 kDa were purified and identified from green coconut (Cocos nucifera L.) water by using reversed phase-high performance liquid chromatography (HPLC), showing molecular masses of 858 Da, 1249 Da and 950 Da. First one, named Cn-AMP1, was extremely efficient against both Gram-positive and Gram-negative bacteria, being MICs calculated for three peptides. All complete sequences were determined by MALDI-ToF analysis showing no identity in databanks. Moreover, peptide net charge and hydrophobicity of each peptide was in silico evaluated. Finally molecular modeling and dynamics were also applied generating peptides three-dimensional structures, indicating a better explanation to probable mechanisms of action. Cn-AMPs here reported show remarkable potential to contribute in the development of novel antibiotics from natural sources.
Keywords: Antibacterial activity; Green coconut water; Antimicrobial peptides; Mass spectrometry; Cocos nucifera;

Hepcidin gene is widely expressed in various fish, suggesting that this antimicrobial peptide is a very important component in the innate immune system. Large yellow croaker (Pseudosciaena crocea) is one of the important economic species of marine-cultured fish but knowledge of its innate immune mechanism is lacking. In this study, we characterize a P. crocea hepcidin gene named as PC-hepc. It consists of an open reading frame of 258 bases encoding 85 amino acids and has a conserved sequence in common with other known hepcidins. The genomic DNA of PC-hepc contains three exons and two introns, the same organization as other reported hepcidins, indicating that PC-hepc is one member of the hepcidin family in fish. The tissue-specific expression of PC-hepc gene in normal fish and the expression pattern in LPS-challenged fish at the time course of stimulation were investigated. The expression of PC-hepc mRNA was significantly increased in the spleen, heart and stomach but not significantly induced in the liver after LPS challenge. An interesting finding is the demonstration of high amounts of PC-hepc transcripts in the kidney in normal fish and their maintenance through 48 h exposure to LPS challenge. The synthetic PC-hepc demonstrated a rather wide spectrum of antimicrobial activity in vitro against bacteria and fungi tested, and particularly showed strong activity against the principal fish pathogens, Aeromonas hydrophila, Vibrio parahaemloyticus, Vibrio alginolyticus and Vibrio harvryi. The study indicates that PC-hepc may play a role with a tissue-specific mode in the innate immunity of P. crocea.
Keywords: Pseudosciaena crocea; PC-hepc; Gene expression; Synthetic peptide; Antimicrobial activity;

A peptide fragment derived from the T-cell antigen receptor protein α-chain adopts β-sheet structure and shows potent antimicrobial activity by Genghui Zhang; Xiaoyan Lin; Yi Long; Yanqiang Wang; Yueheng Zhang; Huaifeng Mi; Husheng Yan (647-653).
A 9-residue peptide, CP-1 (GLRILLLKV-NH2), is synthesized by solid-phase synthesis method. CP-1 is a C-terminal amidated derivative of a hydrophobic transmembrane segment (CP) of the T-cell antigen receptor (TCR) α-chain. CP-1 shows broad-spectrum antimicrobial activities against Gram-positive and Gram-negative bacteria with the minimal inhibitory concentration (MIC) values between 3 and 77 μM. Circular dichroism (CD) spectral data shows that CP-1 adopts a well-defined β-sheet structure in membrane-mimicking environments. CP-1 kills E. coli without lysing the cell membrane or forming transmembrane pores. However, CP-1 can penetrate the bacterial cell membranes and accumulate in the cytoplasm in both Gram-positive S. aureus and Gram-negative E. coli. Moreover CP-1 shows binding affinity for plasmid DNA. These results indicate that the killing mechanism of CP-1 likely involves the penetration into the cytoplasm and binding to intracellular components such as DNA.
Keywords: Antibiotic; Antimicrobial peptide; Peptide; β-Sheet; T-cell antigen receptor;

Purification, partial characterization and molecular cloning of the novel antiviral protein RC28 by Meng Gong; Frank Piraino; Naihong Yan; Jing Zhang; Minjie Xia; Jia Ma; Jingqiu Cheng; Xuyang Liu (654-659).
An antiviral protein, RC28, with anti-herpesvirus activity was purified from a PBS extract of Rozites caperata (Cortinarious caperata) by acetone precipitation followed by gel filtration and ion exchange chromatography. The molecular weight of this protein was 28.251 kDa as measured by MALDI-TOF mass spectrography. Our preparation of RC28 inhibited HSV-1 replication in vitro with an IC50 value of 0.078 mg/ml and a therapeutic index >32. The first 30 amino acid residues of RC28 were sequenced by Edman degradation to be MLTYRGKLNWYNYAVNEGFTLILPGXELKV. Based on that sequence, two degenerate primers were designed, the RC28 cDNA fragment was cloned by 3′-RACE, and the rest of the amino acid sequence was inferred from the cDNA sequence. The full-length peptide chain of RC28 has 235 amino acid residues, and was modified after its translation naturally. A search of the literature showed that this sequence has not been reported before and does not belong to any known protein family. A preliminary expression system was also constructed by inserting the cDNA into the PQE-30 vector, and transformed into Escherichia coli.
Keywords: Antiviral protein; Purification; Molecular cloning; Amino acid sequence; Expression;

Selective cancer cell cytotoxicity of enantiomeric 9-mer peptides derived from beetle defensins depends on negatively charged phosphatidylserine on the cell surface by Takashi Iwasaki; Jun Ishibashi; Hiromitsu Tanaka; Mitsuru Sato; Ai Asaoka; DeMar Taylor; Minoru Yamakawa (660-668).
Four enantiomeric 9-mer peptides named d-peptide A, B, C and D were designed and synthesized on the basis of 43-mer insect defensins from two beetles. The d-9-mer peptides maintained bacterial membrane disruptive activity similar to the original peptides and also showed various extents of growth inhibitory activity against different cancer cell lines. Of these peptides, d-peptide B exhibited the highest selective cancer cell cytotoxicity against the mouse myeloma cell line, P3-X63-Ag8.653. Flow cytometric and scanning electron microscopic analysis revealed d-peptide B disrupts mouse myeloma membrane construction, whereas no cytotoxic effect on normal leukocytes was observed. Moreover, a strong correlation between negatively charged phosphatidylserine (PS) density in cancer cell membrane surface and sensitivity to d-9-mer peptides were observed in various cancer cell lines. These results suggest that d-9-mer peptides have negative charge-dependent selective cancer cell cytotoxicity targeting PS in the cancer cell membrane. In addition, synergic growth inhibitory activity against mouse myeloma was observed in combinations of d-peptide B and dexamethasone. These results suggest d-9-mer peptides are promising candidates for novel anticancer drugs.
Keywords: Insect defensin; Enantiomeric peptides; Selective cytotoxicity; Myeloma; Phosphatidylserine; Negative charge;

Expression, purification and characterization of a group of lectin-like peptides from the spider Ornithoctonus huwena by Liping Jiang; Li Peng; Yongqun Zhang; Jinjun Chen; Dongyi Zhang; Songping Liang (669-674).
By sequencing random clones from the venom gland cDNA library of the spider Ornithoctonus huwena, a transcript, named SHL-Ib1, encoding a lectin-like peptide was cloned. The amino acid sequence of the putative mature peptide of SHL-Ib1 is identical, except for seven different residues, with that of SHL-I, a lectin found in the venom of O. huwena. The mature peptides of SHL-Ib1b and SHL-Ib1c are the mutants of SHL-Ib1 with two or three amino acid residues truncated at the C-terminal. The recombinant SHL-Ib1b and SHL-Ib1c were expressed successfully by the yeast expression system and purified by using a combination of ion-exchange and reverse phase high performance liquid chromatography (HPLC). The molecular masses of the two expressed peptides were identified by mass spectrometry, indicating that the C-terminals of the two peptides were not amidated. The two peptides can agglutinate human erythrocytes at minimal concentrations of 0.75 and 1.475 mg/ml, respectively. Structure modeling of SHL-Ib1 has given a clue to the low agglutination bioactivities of these recombinant toxins. These lectin-like peptides, due to the small molecular sizes, may have the advantage to investigate the binding mechanism of the lectin and have the potential to be the carrier for drug delivery.
Keywords: Ornithoctonus huwena; Spider toxin; Lectin; Spider venom; SHL-Ib1;

Molecular cloning and functional identification of a new K+ channel blocker, LmKTx10, from the scorpion Lychas mucronatus by Jun Liu; Yibao Ma; Shijin Yin; Ruiming Zhao; Shaozhong Fan; Youtian Hu; Yingliang Wu; Zhijian Cao; Wenxin Li (675-680).
Scorpions have a venom gland which is an important determinant in contributing to their successful survival for more than 400 million years. Their venoms contain a diversity of neurotoxins, which represent a tremendous hitherto partially unexplored resource not only for understanding ion channels but also for use in drug design and development. In this investigation, LmKTx10, a new toxin gene was identified from the venom of the scorpion Lychas mucronatus by constructing cDNA library method, and its product was expressed and characterized physiologically. The mature peptide has 38 residues including six conserved cysteines. The electrophysiological experiments further indicated that the recombinant LmKTx10 peptide has an interesting pharmacological profile: it blocks Kv1.3 channel with IC50  = 28 nM which is moderate Kv1.3 channel blocking activity compared to the other a-KTxs toxins, and exhibits good selectivity on Kv1.3 over Kv1.1 and Kv1.2, about 60 folds and 450 folds, respectively. These data not only enrich the family of K+ channel toxins from scorpion venoms but also present a potential drug template for selectively targeting the Kv1.3 channel.
Keywords: Lychas mucronatus; α-KTx; LmKTx10; Kv1.3;

DNA-Bound peptides control the mRNA transcription through CDK7 by Xiaowen Lv; Jing Wang; Zhiyuan Dong; Feijie Lv; Yuchang Qin (681-688).
The degradation of intracytosolic proteins has been well described. However, the degradation pathway and physiological functions of the DNA-Bound peptides, which are free of degradation by peptidase of the post-ubiquitin–proteasome pathway, are still unclear. In this study, the DNA-Bound peptides were isolated from barley germ and two main fractions of about 25 different peptides were obtained. The DNA-Bound peptides were found to inhibit the proliferation of HeLa cells in a series of experiments. The DNA-Bound peptides also significantly inhibited in vitro and in vivo DNA transcription activity by regulating the expression and the corresponding functions of CDK7. Furthermore, signaling issues involving NFκB and ERK1/2 were observed. Such data suggests that DNA transcription could be inhibited by the DNA-Bound peptides via the CDK7 pathway. Thus we concluded that some of the post-proteasomal peptides were involved in the regulation of eukaryotic mRNA transcription.
Keywords: DNA-Bound peptides; Proteasome; Cyclin-dependent kinase-7; Transcription;

Controlled coupling of peptides at their C-termini by Bernd Peschke; Sonja Bak (689-698).
Fusion of two proteins has become an important tool in biotechnology. Whereas biotechnological methods easily can produce C-terminal to N-terminal fused compounds, methods to couple two proteins to each of their C-termini are not easily accessible. Herein, peptides are used as models for larger proteins. A method is described exploiting the possibility to attach different reactive handles to their C-termini using a reaction catalyzed by the enzyme carboxypeptidase Y (CPY). It is possible to attach pairs of reaction handles which can react with each other to each of the peptides to be coupled. In a second step, the two modified peptides can be linked together by a chemical reaction, such as an oxime-forming reaction or a copper(I) catalyzed [2+3]-cycloaddition reaction of an azide with an alkyne.
Keywords: Carboxypeptidase Y; C-terminus; Click chemistry; Dimerization; Enzyme; Oxime;

Lack of tolerance development with long-term administration of PEGylated cholecystokinin by Isabelle Verbaeys; Fabián León-Tamariz; Johan Buyse; Eddy Decuypere; Hans Pottel; Marnix Cokelaere (699-704).
Cholecystokinin (CCK) is a short acting satiating peptide hormone produced in the proximal small intestine. Daily CCK injection in rats initially inhibits food intake, but after several days, food intake is no longer affected, suggesting development of tolerance. Previously, we covalently coupled CCK to a 10 kDa polyethylene glycol (mPEG-OH) and showed that this conjugate, PEG-CCK9, produced a significantly longer anorectic effect than unmodified CCK9. The present study examined whether tolerance to the anorectic effect develops during long-term administration of PEG-CCK9. For 14 consecutive days, male Wistar rats (n  = 12) received a daily i.p injection of 8 μg kg−1 of PEG-CCK9 and a control group received a daily control injection of mPEG-OH. Body weight and food intake were monitored daily during the experiment. Effects on the pancreas were investigated. On each day, injection of PEG-CCK9 induced an anorectic effect lasting 3–6 h, but failed to significantly reduce daily total food intake compared to controls. The body weight gain of the PEG-CCK9-treated animals was not different from controls. The PEG-CCK9-treated group had a significantly higher pancreas weight, mainly due to hyperplasia. In conclusion, PEG-CCK9 continued to have a daily suppressive effect on food intake when administered for 14 consecutive days, showing there was no development of tolerance.
Keywords: Cholecystokinin; PEGylated Cholecystokinin; Satiety; Long-term administration; Tolerance; Pancreas proliferation;

The effect of energy restriction during pregnancy on obesity-related peptide hormones in rat offspring by Mirella Hietaniemi; Elina Malo; Maarit Jokela; Merja Santaniemi; Olavi Ukkola; Y. Antero Kesäniemi (705-709).
It has been proposed that fetal exposure to environmental stressors, such as undernutrition, during critical periods of development may lead to adaptations that permanently change the structure and function of the body. These adaptations may be important for immediate survival during fetal development, but can predispose to disease in later life. We designed a pilot study investigating the effect of fetal undernutrition on the obesity-related peptides adiponectin, ghrelin, leptin and resistin levels in rat. We also wanted to explore changes in lipid and insulin metabolism. Sprague–Dawley rats were randomly assigned to three dietary treatment groups on day 4 of gestation. The control group was fed ad libitum and the food-restricted rats received either 75% or 50% of ad libitum food intake until parturition. Serum levels of obesity-related peptides as well as lipid and insulin levels were measured from 1-month-old pups. Serum resistin concentrations were higher in both food-restricted groups and serum adiponectin concentration was lower in the 50% food-restricted group compared to the control group. Serum total cholesterol levels were significantly higher in both food-restricted groups. These results indicate that undernutrition during fetal development may lead to unfavorable changes in obesity-related peptide hormones as well as evoking adverse changes in serum cholesterol levels. The observed changes may predispose to insulin resistance and significantly increase the risk of developing cardiovascular disease in later life.
Keywords: Fetal; Undernutrition; Development; Rat; Peptide hormones; Resistin; Adiponectin;

The changes in concentrations of two neuropeptides, neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) in different segments of the rat tail artery have been investigated (a) after 12 and 16 weeks of streptozotocin (STZ)-induced hyperglycemia that has been induced at the age of 10 weeks, and (b) in 52-week-old Goto-Kakizaki (GK) rats that were intolerant of glucose, and age-matched Wistar controls. In the control animals at 22, 26 and 52 weeks of age, the concentration of CGRP was significantly greater in distal, relative to proximal, segments of normal arteries, and this contrasted with the pattern of distribution of NPY, which was consistently greater in the proximal than the distal segments. STZ-induced diabetes caused significant reductions in the concentrations of NPY and CGRP in the middle and distal segments of the vessel after 12 and 16 weeks of hyperglycemia. In the glucose-intolerant Goto-Kakizaki (GK) rats, the noradrenalin and adrenalin levels increased significantly in the distal segment of the artery relative to controls; in contrast there was a significant fall in dopamine concentration. The only significant change in the level of NPY in 52-week-old GK rats was an increase in the proximal segment, suggesting that in Type II pre-diabetes, noradrenalin and its co-transmitter NPY are affected independently. The concentration of CGRP increased significantly in all segments of the artery of the 12-month-old GK rats relative to controls. The similarities and differences between these measurements in Type I and Type II diabetic models are discussed.
Keywords: Diabetes; Neuropathy; Autonomic nervous system; Sympathetic nervous system; Amines; Tail artery; Aging; Streptozotocin; Goto-Kakizaki rats;

Sympathetic nervous system-targeted neuropeptide Y overexpression in mice enhances neointimal formation in response to vascular injury by Suvi T. Ruohonen; Ken Abe; Mia Kero; Laura Toukola; Saku Ruohonen; Matias Röyttä; Markku Koulu; Ullamari Pesonen; Zofia Zukowska; Eriika Savontaus (715-720).
Sympathetic neurotransmitter neuropeptide Y (NPY) is associated with vascular remodelling, neointimal hyperplasia and atherosclerosis in experimental animal models and clinical studies. In order to study the role of sympathetic nerve-produced NPY in vascular diseases, transgenic mouse model overexpressing NPY in central and peripheral noradrenergic neurons under the dopamine-beta-hydroxylase (DBH) promoter was recently created (OE-NPYDBH mouse). This study aimed to examine the effect of NPY overexpression on arterial neointimal hyperplasia in an experimental model of vascular injury. Transgenic OE-NPYDBH mice and wildtype control mice of two different inbred strains (C57BL/6 and FVB/n) underwent a femoral artery surgery with a transluminar injury by a 0.38-mm guide wire insertion. Arteries were harvested 4 weeks from the surgery, and they were stained for basic morphology. Both strains of OE-NPYDBH mice, as compared with wildtype control mice, showed on average 50% greater formation of the neointima (P  < 0.01) and an increase in the medial area (P  = 0.05). The results suggest that moderately increased neuronal NPY causes the arteries to be more susceptible to femoral artery thickening after endothelial injury. The OE-NPYDBH mouse provides a novel tool to explore the role of NPY in the development of vascular disease related to metabolic disorders.
Keywords: Neuropeptide Y; Transgenic mouse; Vascular injury; Neointimal hyperplasia;

Neuropeptide Y augments cocaine self-administration and cocaine-induced hyperlocomotion in rats by Tia Maric; Anna Cantor; Holly Cuccioletta; Stephanie Tobin; Uri Shalev (721-726).
Food restriction and deprivation are known to modulate drug-related behaviors. However, the mechanisms through which metabolic manipulations intercede the rewarding effects of drug reward are unknown. Neuropeptide Y (NPY) is thought to be critically involved in the regulation of energy balance. Central administration of NPY induces feeding in sated animals, and importantly, is reported to increase the rewarding properties of food. NPY has also been shown to be involved in drug-related behavior. We have recently demonstrated that NPY injections augmented on-going heroin self-administration and induced a reinstatement of heroin seeking. The present study sought to support and expand our previous finding on NPY's role in addictive drugs-related behaviors by examining the effects of NPY on cocaine-induced locomotor hyperactivity and cocaine self-administration. In Experiment 1, rats received NPY injections (0.0, 2.5, 5.0 μg/rat, ICV), followed by cocaine administration (0.0, 1.0, 5.0, and 10.0 mg/kg, IP) and their locomotor activity was monitored over 90 min. In Experiment 2, rats were trained to self-administer cocaine (0.50 mg/kg/infusion) during one 3-h session per day for 12 days. Once trained, NPY (0.0, 4.0, 10.0 μg/rat, ICV) was administered 15 min prior to the self-administration session. Results revealed that NPY injections augmented cocaine-induced hyperactivity and moderately increased cocaine self-administration. Together with our previous findings, these results suggest that NPY is involved, albeit to a limited extent, in the augmenting effect of food deprivation on drug-related behaviors.
Keywords: Food deprivation; Cocaine; Neuropeptide Y; Self-administration; Locomotor activity;

In previous work, we observed that N/OFQ-induced hyperphagia is greater in DA rats, animals resistant to metabolic syndrome, than in WOKW animals, which are prone to this disease. We attributed this difference to the fact that these two strains have different Cart gene sequences and expression. As a preliminary approach to pursue this hypothesis, the present work focused on Cart gene expression by developing from DA and WOKW rats various congenic animals with exchanges of metabolic syndrome-related QTL's of different chromosomes (3, 5, 10 and 16), and analyzing their N/OFQ-induced (2.1, 4.2, and 8.4 nmol/rat) food intake in terms of their CART gene expression and N/OFQ hypothalamic immunostaining. Two groupings emerged, the first, with strains 3a, 3b, and 5a with elevated N/OFQ-induced feeding similar to that of the DA rats, and the second, with strains 16 and 10, with lower feeding, like the WOKW rats. There was a perfect correlation between Cart gene expression and N/OFQ-induced feeding data at 30 min for the strains DA, 3a, 3b, 5 in the first group, and 16 and WOKW for the second, but not for strain 10. As expected, the strains with low content of Cart gene expression had elevated N/OFQ-induced feeding, but contrary to expectations, strain 10, with the lowest Cart gene expression, exhibited low N/OFQ-induced feeding, on the order of that of the WOKW rats. A comparable trend was observed with N/OFQ hypothalamic immunostaining. This anomaly may be due to other satiety-related factors involved in N/OFQ-induced feeding.
Keywords: Nociceptin/orphanin FQ; Dark Agouti rats; Wistar Ottawa Karlsburg W rats; Cocaine-amphetamine regulated transcript peptide; Congenic rats;

Angiotensin AT2 receptor agonists act as anti-opioids via EP3 receptor in mice by Yuko Yamada; Kousaku Ohinata; Andrzej W. Lipkowski; Masaaki Yoshikawa (735-739).
Novokinin (Arg-Pro-Leu-Lys-Pro-Trp) is a vasorelaxing and hypotensive peptide acting through the angiotensin AT2 receptor. Centrally administrated novokinin (30 nmol/mouse) inhibited the antinociceptive effect of μ agonist morphine in mice, as evaluated by the tail-pinch test. The anti-opioid effect of novokinin was blocked by PD123319, an antagonist of the AT2 receptor. Angiotensin II (0.01 nmol/mouse, i.c.v.) and [p-aminophenylalanine6]-angiotensin II [p-NH2Phe6]-Ang II (0.1 nmol/mouse, i.c.v.), a highly selective AT2 receptor agonist, also inhibited the antinociceptive effect of morphine, and the effects were also blocked by PD123319. Angiotensin II did not suppress the antinociceptive effect induced by κ or δ agonists. Novokinin, angiotensin II and [p-NH2Phe6]-Ang did not have affinity for the μ receptor. The anti-opioid effects induced by these peptides were blocked by ONO-AE3-240, an antagonist of the EP3 receptor. These results suggest that the anti-opioid effects of AT2 agonists are mediated by the PGE2-EP3 receptor system downstream of the AT2 receptor.
Keywords: Anti-opioid effect; Angiotensin II; AT2 receptor; Prostaglandin E2; EP3 receptor; Tail-pinch test;

Endogenous opiate peptides in the spinal cord are involved in the analgesia of hypothalamic paraventricular nucleus in the rat by Jun Yang; Yu Yang; Jiegen Chu; Gen Wang; Hongtao Xu; Wen-Yan Liu; Cheng-Hai Wang; Bao-Cheng Lin (740-744).
Many studies have shown that hypothalamic paraventricular nucleus (PVN) plays a role in pain process, and endogenous opiate peptide system in the spinal cord is involved in nociception. This communication was designed to study the relationship between PVN and endogenous opiate system in the spinal cord in the rat. The results showed that in both the thoracic and the lumber spinal cord, microinjection of 100 ng l-glutamate sodium into PVN could increase leucine-enkephalin (L-Ek), β-endorphin (β-Ep), dynorphinA1–13 (DynA1–13) concentrations and PVN cauterization decreased L-Ek and β-Ep concentrations. Pretreatment of the spinal cord with 5 μg naloxone, an opiate receptor antagonist could partly reverse the analgesia induced by microinjection of 100 ng l-glutamate sodium into PVN. The data suggested that PVN analgesia might be involved in the endogenous opiate peptide system in the spinal cord independently.
Keywords: Hypothalamic paraventricular nucleus; Analgesia; Endogenous opiate peptide; Arginine vasopressin; Spinal cord;

Electrophysiological effects of ghrelin on pedunculopontine tegmental neurons in rats: An in vitro study by Juhyon Kim; Kazuki Nakajima; Yutaka Oomura; Matthew J. Wayner; Kazuo Sasaki (745-757).
Ghrelin is a potent stimulant for growth hormone (GH) secretion and feeding. Recent studies further show a critical role of ghrelin in the regulation of sleep-wakefulness. Pedunculopontine tegmental nucleus (PPT), which regulates waking and rapid eye movement (REM) sleep, expresses GH secretagogue receptors (GHS-Rs). Thus, the present study was carried out to examine electrophysiological effects of ghrelin on PPT neurons using rat brainstem slices, and to determine the ionic mechanism involved. Whole cell recording revealed that ghrelin depolarizes PPT neurons dose-dependently in normal artificial cerebrospinal fluid (ACSF). The depolarization persisted in tetrodotoxin-containing ACSF, although action potentials did not occur. Application of [d-Lys3]-GHRP-6, a selective antagonist for GHS-Rs, almost blocked the ghrelin-induced depolarization. Furthermore, the ghrelin-induced depolarization was reduced in high K+ ACSF or low Na+ ACSF, and abolished in high K+–low Na+ ACSF or in a combination of low Na+ ACSF and recordings with Cs+-containing pipettes. An inhibitor of Na+/Ca2+ exchanger had no effect on the depolarization. Most of the PPT neurons recorded were characterized by an A-current or both the A-current and a low threshold Ca2+ spike, and they were predominantly cholinergic as revealed by nicotinamide adenine dinucleotide phosphate-diaphorase staining. These results suggest that ghrelin depolarizes PPT neurons postsynaptically and dose-dependently via GHS-Rs, and that the ionic mechanisms underlying the ghrelin-induced depolarization include a decrease of K+ conductance and an increase of non-selective cationic conductance. The results also support the notion that ghrelin plays a role in the regulation of sleep-wakefulness.
Keywords: Ghrelin; Pedunculopontine tegmental nucleus; Cholinergic neurons; Sleep; Wakefulness; Ionic mechanism; [d-Lys3]-GHRP-6;

Purification and properties of ghrelin from the intestine of the goldfish, Carassius auratus by Tohru Miura; Keisuke Maruyama; Hiroyuki Kaiya; Mikiya Miyazato; Kenji Kangawa; Minoru Uchiyama; Seiji Shioda; Kouhei Matsuda (758-765).
In goldfish, intraperitoneal (IP) or intracerebroventricular (ICV) administration of synthetic ghrelin consisting of 12- or 19-amino-acid residues, deduced from its precursor cDNA, with an octanoic acid modification at the third N-terminal serine residue (Ser3), stimulates growth hormone release and food intake. However, native ghrelin generated from its precursor has not yet been identified in this species. Therefore, we purified ghrelin from the goldfish intestine using acid extraction, cation-exchange and reverse-phase high-performance liquid chromatography combined with immune-affinity purification. In order to confirm ghrelin activity in the fractions at each purification step, we examined the effect of each fraction on intracellular Ca2+ mobilization in rat growth hormone secretagogue-receptor (GHS-R)-expressing cells. We characterized the goldfish ghrelin as 11 molecular forms consisting of 14-, 17-, 18- and 19-amino-acid residues with acylation at Ser3, and the 17-residue form was predominant. We then synthesized 17-residue forms with octanoic acid modification (octanoyl ghrelin17) and without acylation (des-acyl ghrelin17) at Ser3, and examined their biological activity. Octanoyl ghrelin17, but not des-acyl ghrelin17, increased the intracellular Ca2+ concentration in rat GHS-R-expressing cells with a potency similar to those of synthetic ghrelin consisting of 12 residues (octanoyl ghrelin12) and octanoyl rat ghrelin. IP and ICV administration of octanoyl ghrelin17 and octanoyl ghrelin12, but not des-acyl ghrelin17, increased food intake in goldfish. The present findings indicate that native goldfish ghrelin consists of 11 molecular variants, the major form being a 17-residue peptide. This dominant form with acylation is implicated in the regulation of food intake in goldfish.
Keywords: Goldfish; Ghrelin; Purification; Orexigenic action; Acylation;

Full-length complementary deoxyribonucleic acid sequences encoding pituitary adenylate cyclase activating polypeptide (PACAP)/PACAP-related peptide (PRP) and preprosomatostatin 1 (PPSS 1) were cloned from Atlantic cod (Gadus morhua) hypothalamus using reverse transcription and rapid amplification of complementary deoxyribonucleic acid ends. Semi-quantitative reverse transcriptase polymerase chain reaction shows that PRP/PACAP mRNA and PPSS 1 mRNA are widely distributed throughout cod brain. During development, PRP/PACAP and PPSS 1 were detected at the 30-somite stage and pre-hatching stage, respectively, and expression levels gradually increased up to the hatched larvae. PPSS 1, but not PRP/PACAP, appeared to be affected by food availability during early development. In juvenile cod, PPSS 1 expression levels increased and remained significantly higher than that of control fed fish throughout 30 days of starvation and during a subsequent 10 days refeeding period. In contrast, PRP/PACAP expression levels were not affected by 30 days of food deprivation, but a significant increase in expression levels was observed during the 10 days refeeding period in the experimental food-deprived group as compared to the control fed group. Our results suggest that PRP/PACAP and PPSS 1 may be involved in the complex regulation of growth, feeding and metabolism during food deprivation and refeeding in Atlantic cod.
Keywords: Atlantic cod; Pituitary adenylate cyclase activating polypeptide; Somatostatin; Expression; Ontogeny; Food deprivation; Refeeding;

Structure–activity relationships of novel peptide agonists of the human bradykinin B2 receptor by Simon Bélanger; Veronica Bovenzi; Jérome Côté; Witold Neugebauer; Muriel Amblard; Jean Martinez; Bernard Lammek; Martin Savard; Fernand Gobeil (777-787).
The nonapeptide bradykinin (BK) is involved in the genesis of inflammation, edema and in pain mediation. As such, much effort has gone into the development of peptide/non-peptide antagonists to counteract these processes. However, there is an increasing awareness of the potential value of chemically stable BK agonists in the treatment of diabetes and cardiovascular diseases. In this study, a structure–activity relationship study of BK was performed to develop potent and stable peptide mimetics active at the human B2 receptors (hB2R). Twenty-three analogues were produced with substitutions at positions 1, 3, 5, 7, 8 and/or 9 of BK. In vitro binding (on transiently transfected HEK-293T cells) and biological activities (vasomotricity tests on human umbilical veins, MAPK assays on HEK-293T cells) of novel BK peptide derivatives at hB2R were determined alongside with previously reported synthetic agonists (e.g. RMP-7, JMV1609, FR190997). Some peptides were also tested in vivo in rats and rabbits using blood pressure assays. Two compounds, [Hyp3, Thi5, Cha8]-BK and [Hyp3, Thi5, NChg7, Thi8]-BK, exhibited equivalent (or even greater) in vitro affinities and potencies to BK at the naturally expressed and recombinant hB2R. Their potency and duration of action in vivo were highly superior to BK, thus inferring that they can withstand intravascular proteolysis. These novel compounds show promise as candidates for investigating the pharmacology of BK receptors and developing potential therapeutical applications.
Keywords: Bradykinin; GPCR; Peptide agonists; Bioassays; Human;

Novel kinin B1 receptor agonists with improved pharmacological profiles by Jérôme Côté; Martin Savard; Veronica Bovenzi; Simon Bélanger; Josée Morin; Witold Neugebauer; Annie Larouche; Céléna Dubuc; Fernand Gobeil (788-795).
There is some evidence to suggest that inducible kinin B1 receptors (B1R) may play beneficial and protecting roles in cardiovascular-related pathologies such as hypertension, diabetes, and ischemic organ diseases. Peptide B1R agonists bearing optimized pharmacological features (high potency, selectivity and stability toward proteolysis) hold promise as valuable therapeutic agents in the treatment of these diseases. In the present study, we used solid-phase methodology to synthesize a series of novel peptide analogues based on the sequence of Sar[dPhe8]desArg9-bradykinin, a relatively stable peptide agonist with moderate affinity for the human B1R. We evaluated the pharmacological properties of these peptides using (1) in vitro competitive binding experiments on recombinant human B1R and B2R (for index of selectivity determination) in transiently transfected human embryonic kidney 293 cells (HEK-293T cells), (2) ex vivo vasomotor assays on isolated human umbilical veins expressing endogenous human B1R, and (3) in vivo blood pressure tests using anesthetized lipopolysaccharide-immunostimulated rabbits. Key chemical modifications at the N-terminus, the positions 3 and 5 on Sar[dPhe8]desArg9-bradykinin led to potent analogues. For example, peptides 18 (SarLys[Hyp3,Cha5, dPhe8]desArg9-bradykinin) and 20 (SarLys[Hyp3,Igl5, dPhe8]desArg9-bradykinin) outperformed the parental molecule in terms of affinity, functional potency and duration of action in vitro and in vivo. These selective agonists should be valuable in future animal and human studies to investigate the potential benefits of B1R activation.
Keywords: B1 receptor; Kinins; Peptides; Biological assays; Human;

Effects of adrenomedullin on systolic and diastolic myocardial function by Ana Patrícia Fontes-Sousa; Ana Luísa Pires; Catarina Santos Carneiro; Carmen Brás-Silva; Adelino F. Leite-Moreira (796-802).
Adrenomedullin (AM) effects were studied in rabbit papillary muscles by adding increasing concentrations (10−10 to 10−6  M) either alone or after pre-treatment with l-NNA, indomethacin, AM22–52 (AM receptor antagonist), CGRP(8–37) (CGRP receptors antagonist), KT5720 (PKA inhibitor), as well as after endocardial endothelium (EE) removal. Passive length–tension relations were constructed before and after a single concentration of AM (10−6  M). AM concentration-dependently induced negative inotropic and lusitropic effects, and increased resting muscle length (RL). At 10−6  M, AT, dT/dt max and dT/dt min decreased 20.9 ± 4.9%, 18.3 ± 7.3% and 16.7 ± 7.8%, respectively, and RL increased to 1.010 ± 0.004  L/L max. Correcting RL to its initial value resulted in a 26.6 ± 6.4% decrease of resting tension, indicating decreased muscle stiffness, also patent in the down and rightward shift of the passive length–tension relation. The negative inotropic effect of AM was dependent on its receptor, CGRP receptor, PKA, the EE and NO, while the effects of AM on myocardial stiffness were abolished by EE damage and NO inhibition. This latter effect represents a novel mechanism of acute neurohumoral modulation of diastolic function, suggesting that AM is an important regulator of cardiac filling.
Keywords: Adrenomedullin; Inotropism; Lusitropism; Myocardial stiffness;

The purpose of the present study was to elucidate the possible role of calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin-2/intermedin (IMD) on food intake regulation in goldfish (Carassius auratus). We examined the effects of intracerebroventricular (ICV) administration of these related hormones on food intake. Food-deprived goldfish were subjected to ICV injections of CGRP, AM and IMD and their food intake were quantified. CGRP at 10 ng/g body weight (bw) significantly decreased food intake as compared to saline-treated fish. IMD at 10 and 50 ng/g bw both significantly decreased food intake as compared to saline group. No significant differences were observed after AM administration. Our results suggest, for the first time in fish, a role for both CGRP and IMD in the central regulation of feeding in fish.

BC-264 a CCK2 agonist reverses chronically developed habituated predatory fear freezing behavior in PVG hooded rats. However, the acute effects of BC264 have not been previously examined. The effects of BC-264 (0.1–30 μg/kg) on the mean percentage of PVG hooded rat freezing during an initial first time 20 min cat exposure were calculated. At higher doses (15 or 30 μg/kg) but not lower doses (0.1–1 μg/kg) BC264 statistically significantly decreased freezing compared to control (p  < 0.001).
Keywords: PVG hooded rat; CCK; Predatory fear;

Corticotropin-releasing factor (CRF), produced in the hypothalamic paraventricular nucleus (PVN) in response to stress, stimulates the synthesis and secretion of adrenocorticotropin (ACTH) via CRF receptor type 1 (CRF1 receptor) in the anterior pituitary (AP) of mammals. CRF is critical for the circadian rhythmicity of the hypothalamic–pituitary–adrenal axis and the augmented release of ACTH from the pituitary in response to the stress. A higher molecular weight form of immunoreactive β-endorphin, putative proopiomelanocortin (POMC), is increased in CRF-knockout mice (CRF KO), suggesting the important role of CRF in the processing of POMC. In fact, CRF is able to modulate the processing of POMC through changes in prohormone convertase (PC)-1 expression levels. Multiple forms of ACTH-related peptides containing unprocessed ones are present in some cases of ACTH-producing tumors, presumably without action of PC-1 under the control of CRF. Following CRF-activated stimulation of the receptor signaling, CRF1 receptor is down-regulated and desensitized. In fact, CRF facilitates the degradation of CRF1 receptor mRNA via the protein kinase A pathway. Prolonged agonist activation of CRF1 receptor leads to a loss of responsiveness, or desensitization of the receptor. G protein-coupled receptor kinase 2 is involved in desensitization of CRF1 receptor by CRF in the corticotroph.
Keywords: Corticotropin-releasing factor; ACTH; Proopiomelanocortin; Cyclic AMP; Receptor;

δ-hemolysin, an update on a membrane-interacting peptide by Julien Verdon; Nicolas Girardin; Christian Lacombe; Jean-Marc Berjeaud; Yann Héchard (817-823).
δ-hemolysin is a hemolytic peptide produced by Staphylococcus, and it has been studied for nearly 50 years. Therefore, it has become a model in the study of peptides interacting with membranes. In this review, we report some recent findings and compare them with previous works. δ-hemolysin is a 26 amino acid peptide, somewhat hydrophobic and presenting a zero net charge. Study of its structure has shown that δ-hemolysin is α-helical and amphipathic, such as many antimicrobial peptides (e.g. magainin and melittin). However, δ-hemolysin had not displayed any reported antimicrobial activity until a recent publication showed its high potency against Legionella. Its mode of action is based on direct interaction with target membranes. In accordance with its concentration, δ-hemolysin may slightly perturb a membrane or lead to cell lysis. Peptide charge plays an important role in its interaction with membranes, as is shown in the study of peptide variants. Some positively charged variants become highly hemolytic and even active against Escherichia coli and Staphylococcus aureus. Finally, it has recently been demonstrated that peptide preferentially binds to lipid-disordered domains. It has been postulated that as a result, enrichment in lipid-ordered domains might increase peptide concentration in lipid-disordered domains and thereby improve its activity.
Keywords: Antimicrobial; Pore; Hemolysis; Staphylococcus; Bacteria; Erythrocyte; Detergent; Curvature strain;