Peptides (v.27, #1)

A one-enzyme strategy to release an antimicrobial peptide from the LFampin-domain of bovine lactoferrin by Jan G.M. Bolscher; Marieke I.A. van der Kraan; Kamran Nazmi; Hakan Kalay; Christian H. Grün; Wim van’t Hof; Enno C.I. Veerman; Arie V. Nieuw Amerongen (1-9).
Antimicrobial peptides have been found throughout living nature, yet antimicrobial sequences may still lie hidden within a wide variety of proteins. A rational strategy was developed to select interesting domains, based on the presumed common features of antimicrobial peptides, and to release these from accessible and safe proteins. In silico proteolysis simulations of bovine lactoferrin (bLF) with selected endoproteinases predicted the liberation of peptides that encompasses a cationic amphipathic α-helix. Three predicted peptides were synthesized and tested for their biological activity, demonstrating that one single enzyme was sufficient to obtain an antimicrobial peptide. The proof of principle demonstrated that a 32-mer fragment isolated from the endoproteinase AspN digestion of bLF possessed strong antimicrobial activity. Moreover, desalted crude digest had improved activity over native bLF. Hence, selective digestion of bLF increases its antimicrobial activity by release of antimicrobial stretches.
Keywords: Lactoferrin; Lactoferrampin; Antimicrobial peptide; Endoproteinase AspN; Proteolysis; Candida albicans; Escherichia coli;

Anabaenopeptins are commonly occurring bioactive peptides of cyanobacterial origin. Cyanobacteria (blue-green algae) are known to be capable of producing a large number of biologically active peptides, but the widespread occurrence of anabaenopeptins in particular, makes them ideal candidates for investigating the reasons that cyanobacteria produce such a complex spectrum of peptides and the wider implications of their natural function(s). Despite the identification of these peptides in cyanobacterial samples, little is known about the concentrations produced. For this reason, methods for the quantitative extraction of anabaenopeptins from lyophilized cyanobacterial cells were optimized. Higher yields of anabaenopeptins were obtained using aqueous methanol extraction than using water alone. However, repeat extractions using 50, 70 or 90% aqueous methanol did not result in significantly different total yields of the anabaenopeptin variants, ABPN-A and -B. Similarly, little difference was found in the quantification of purified ABPN-A and -B by high performance liquid chromatography with photodiode array detection (HPLC-PDA) when analyzed in methanol solutions of different concentrations. The effects of solvent concentration on the laboratory handling of ABPN-A and -B in glass and plastic containers were also investigated. Significantly lower concentrations of dissolved ABPN-A and -B were found when aqueous solutions came into contact with plastics, but not 50 or 100% methanol.
Keywords: Cyanobacteria; Anabaenopeptin; Extraction; Anabaena; Planktothrix;

Identification of antimicrobial peptide regions derived from genomic sequences of phage lysins by Shahar Rotem; Inna Radzishevsky; Roger T. Inouye; Matthew Samore; Amram Mor (18-26).
This study was designed to test the possibility that antimicrobial peptides could be derived from the genomic sequences of phage lysins. Using two lysins (D3 and ΦKZ) we selected and produced two putative peptides (X and Z, respectively) believed to possess antimicrobial properties based on their physicochemical characteristics. The data presented support this hypothesis in that the peptides and various analogs displayed antibacterial activity, bacteriostatic or bactericidal, either individually or upon combination. These putative peptides are believed to act by a mechanism of action resembling that of conventional antimicrobial peptides when judged by both structural and functional criteria. Thus, the peptides are shown to have the ability to form a helical structure, to bind to model bacterial membranes and permeabilize model liposomes. They also display rapid bactericidal kinetics and their antibacterial potency is increased upon amidation. The possible relevance of these results in contributing to potency of phage lysins is discussed. Such peptides may be used to design new potent antimicrobial compounds much needed in face of the ever threatening drug resistance problems.
Keywords: Structure–activity relationships; Antimicrobial peptides; Phage therapy; Pseudomonas aeruginosa; Multi-drug resistance;

A 15-kDa antifungal protein, designated ganodermin, was isolated from the medical mushroom Ganoderma lucidum. The isolation procedure utilized chromatography on DEAE-cellulose, Affi-gel blue gel, CM-Sepharose and Superdex 75. Ganodermin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. Ganodermin inhibited the mycelial growth of Botrytis cinerea, Fusarium oxysporum and Physalospora piricola with an IC50 value of 15.2 μM, 12.4 μM and 18.1 μM, respectively. It was devoid of hemagglutinating, deoxyribonuclease, ribonuclease and protease inhibitory activities.
Keywords: Antifungal protein; Ganoderma lucidum; Isolation;

A novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis by Da Yu; Zonggen Sheng; Xueqing Xu; Jianxu Li; Hailong Yang; Zhigang Liu; Huw H. Rees; Ren Lai (31-35).
A novel antimicrobial peptide named as ixosin was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as GLHKVMREVLGYERNSYKKFFLR by Edman degradation and its molecular weight was 2870.5 analyzed by fast atom bombardment (FAB) mass spectrometry. This is the first antimicrobial peptide from ticks that lacks cysteine in its primary structure. The cDNA encoding ixosin was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 79 amino acids including mature ixosin. Purified ixosin exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide.
Keywords: Ticks; Antimicrobial peptide; Ixodes sinensis; Salivary gland;

Amphibian skin secretions are rich in antimicrobial peptides that act as important components of an innate immune system. Here, we describe a novel “shotgun” skin peptide precursor cloning technique that facilitates rapid access to these genetically encoded molecules and effects their subsequent identification and structural characterization from the secretory peptidome. Adopting this approach on a skin secretion-derived library from a hitherto unstudied Chinese species of frog, we identified a family of novel antimicrobial peptide homologs, named pelophylaxins, that belong to previously identified families (ranatuerins, brevinins and temporins) found predominantly in the skin secretions from frogs of the genus Rana. These data further substantiate the scientifically robust nature of applying parallel transcriptome and peptidome analyses on frog defensive skin secretions that can be obtained in a non-invasive, non-destructive manner. In addition, the present data illustrate that rapid structural characterization of frog skin secretion peptides can be achieved from an unstudied species without prior knowledge of primary structures of endogenous peptides.
Keywords: Amphibian; Venom; Mass spectrometry; Peptide; Cloning;

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the “shotgun” cloning of novel brevinins by means of 3′-RACE, using a “universal” degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5′-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.
Keywords: Amphibian; Venom; Mass spectrometry; Peptide; Cloning;

Expression of functional scorpion neurotoxin Lqq-V in E.coli by Sami Banerjee; Ernest V. Curto; Matthew Beckman; George B. Brown; Juming Zhong; N. Rama Krishna (49-54).
We report the results on the expression in Escherichia coli of a functional neurotoxin LqqV from the scorpion Leiurus quinquestriatus quinquestriatus. The gene for LqqV was synthesized using recursive PCR and expressed as a poly-histidine-tagged fusion protein in thioredoxin mutant E. coli strain [AD494(DE3)pLysS], thus permitting disulfide-bond formation. When cultured at 37 °C, about 50% of the expressed protein is contained as a monomer in the soluble fraction of the E. coli extract. The fusion protein from the soluble fraction was purified and the His-tag was cleaved by thrombin, resulting in a yield of about 1.5 mg/liter. The globular structure of the purified protein was confirmed by NMR and CD spectroscopy. Patch-clamp measurements using native sodium channels in guinea pig ventricular myocytes reveal (1) a slowing of inactivation and (2) a decrease in peak current upon application of toxin, thus confirming the α-toxin activity of the purified recombinant protein.
Keywords: Scorpion neurotoxin; LqqV; E. coli; Recombinant expression; α-Toxin; Leiurus quinquestriatus quinquestriatus; Guinea pig ventricular myocytes;

Ion selectivity of scorpion toxin-induced pores in cardiac myocytes by Dale Elgar; Fons Verdonck; Anne Grobler; Carla Fourie; Johan du Plessis (55-61).
The lytic activity of parabutoporin (PP) and opistoporin 1 (OP1) on mammalian and bacterial membranes have been described. We investigated pore-formation and ion selectivity in cardiac myocytes by measuring the whole cell leak current by means of the patch clamp technique. Pore formation was observed as the induction of leak currents. Ion selectivity of the pores was indicated by the shift of the reversal potential (E rev) upon substitution of intra- and extra-cellular ions. Results were compared with the effect of gramicidin A (gramA). PP and OP1 induced a fluctuating leak current and indicate non-selectivity of PP-induced pores. PP- and OP1-induced pores are between 1.38 and 1.78 nm in diameter.
Keywords: Pore-forming; Peptides; Antimicrobial; Ion selectivity; Scorpion; Venom;

Regional sampling and the effects of experimental heart failure in sheep: Differential responses in A, B and C-type natriuretic peptides by Christopher J. Charles; Timothy C.R. Prickett; Eric A. Espiner; Miriam T. Rademaker; A. Mark Richards; Timothy G. Yandle (62-68).
While regional plasma concentrations of the endocrine hormones atrial and brain natriuretic peptide (ANP and BNP) have been studied, there are few reports of regional changes in the largely paracrine C-type natriuretic peptide (CNP) and its amino terminal fragment NT-CNP. Accordingly, we have performed trans-organ arteriovenous sampling for measurement of plasma ANP, BNP, CNP and NT-CNP in anesthetized sheep before and after induction of experimental heart failure. ANP and BNP plasma concentrations are sourced from a single organ (the heart) and are subject to substantial extraction across most tissue beds. In contrast, our data demonstrate that multiple tissues including liver, heart, hind limb and kidney contribute to circulating CNP. Given that arteriovenous gradients for NT-CNP were similar, this is likely to represent de novo secretion. Circulating levels of CNP and NT-CNP were raised in heart failure but to a much lesser degree than ANP and BNP. There was no evidence of net extraction of CNP or NT-CNP across any tissue bed.
Keywords: Heart; Liver; Kidney; Arteriovenous gradient;

Adrenomedullin immunoreactivity in the human carotid body by Andrea Porzionato; Veronica Macchi; Anna Sandra Belloni; Anna Parenti; Raffaele De Caro (69-73).
We studied by immunocytochemistry the expression of AM in human carotid bodies, sampled at autopsy from 16 adult subjects (mean age ± S.D.: 44.3 ± 3.4 years) and from six fetuses (mean gestational age ± S.D.: 167 ± 11 days). No AM immunoreactivity was visible in the type II cells of both series. The percentage of immunoreactive type I cells was higher in the adult subjects (32.3 ± 7.7%) with respect to the fetuses (11.8 ± 2.7%, P  < 0.001). Dark cells showed a higher percentage of positive immunoreaction with respect to light cells, both in adult subjects (61.7 ± 13.4% versus 19.2 ± 5.2%) and in fetuses (25.3 ± 4.4% versus 6.2 ± 2.0%). AM may play a role in the regulation of chemoreceptor discharge through paracrine releasing action and/or vasodilator effect. The low expression of AM in fetuses may be ascribed to the absence of pulmonary respiration with lack of regulatory role of the carotid body during the prenatal period.
Keywords: Adrenomedullin; Immunohistochemistry; Carotid body;

Intermedin 1–53 in central nervous system elevates arterial blood pressure in rats by Yong-Sheng Ren; Jing-Hui Yang; Jing Zhang; Chun-Shui Pan; Jun Yang; Jing Zhao; Yong-Zheng Pang; Chao-Shu Tang; Yong-Fen Qi (74-79).
Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family identified from human and other vertebrate tissues. Preprointermedin can generate various mature peptides by proteolytic cleavage. Amino acid sequence analysis showed cleavage sites located between two basic amino acids at Arg93–Arg94 resulting in the production of prepro-IMD95–147, namely IMD1–53. The present study was designed to determine the effects of the IMD1–53 fragment in the central nervous system (CNS) on mean arterial blood pressure and heart rate in normal rats and its possible mechanism. Rats were given doses of adrenomedullin (ADM) or IMD1–53, intracerebroventricularly or intravenously, respectively, with continuous blood pressure and heart rate monitoring for 45 min. Analysis with CGRP receptor antagonist CGRP8–37, ADM receptor antagonist ADM22–52, and anti-prepro-IMD antibody showed that 0.1, 0.5, and 1.0 nmol/kg IMD1–53, caused a dose-dependent elevation in blood pressure, which was more prominent than the increase with equivalent IMD1–47 or ADM. As well, IMD1–53 caused a persistent increase in heart rate. The CNS action of IMD1–53 could be blocked by ADM22–52, CGRP8–37, or prepro-IMD antibody. In contrast to the CNS action, intravenous administration of IMD1–53 induced a depressor effect. These results suggest that IMD1–53 is an important regulatory factor in mean arterial blood pressure and heart rate through its central and peripheral bioaction.
Keywords: Intermedin; Central nervous system; Mean arterial pressure; Heart rate;

To elucidate whether aging influences the vascular contractile effect of urotensin II in rat thoracic aorta, and to evaluate the contribution of endothelial vasodilating substances in mediating the effect of urotensin II, the effect of urotensin II was examined in the vessels of young (2–3-month-old) and aged rat. Isolated rat aortic rings incubated in Krebs–Henseleit solution gassed with 95% O2/5% CO2 were stimulated with urotensin II, and the developed tension was measured. Urotensin II increased the developed tension, which was decreased by aging. In 2–3-months-old young aorta without endothelium, urotensin II (10−10 to 10−7) elicited a concentration-dependent aortic contraction to the maximal response almost equivalent to high KCl-induced contraction (79.4 ± 11.3% of KClmax). In the presence of endothelium, the urotensin II-induced vasoconstriction in young aorta was significantly attenuated to 33.3 ± 4.6% of KClmax. However, the contractile response was greater in the pretreatment with N G-nitro-l-arginine (l-NNA) (100 μM) (50.3 ± 8.4% of KClmax in endothelial denuded aorta), suggesting the vasorelaxing role of endothelial nitric oxide. In 25–27-months-old aged rat aorta, the urotensin II-mediated contraction was remarkably decreased, both in the presence (6.3 ± 2.0% of KClmax) and absence (11.7 ± 3.0% of KClmax) of endothelium. A cyclooxygenase inhibitor, diclofenac (10 μM), did not have any effect on the urotensin II-induced contraction. These results suggest that urotensin II can induce vascular smooth muscle contraction in rat aorta, and there was an aging-related decline in the urotensin II-induced contraction. Endothelial production of nitric oxide in response to urotensin II but not cyclooxygenase metabolites such as prostacyclin may play a role in reducing the vascular constriction especially in young aorta.
Keywords: Urotensin II; Nitric oxide; Endothelial cell; Vascular smooth muscle; Contraction; Aging;

Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis by B. Gasz; B. Rácz; E. Rőth; B. Borsiczky; A. Ferencz; A. Tamás; B. Cserepes; A. Lubics; F. Gallyas; G. Tóth; I. Lengvári; D. Reglődi (87-94).
Pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects, and one of the main factors leading to neuroprotection seems to be its anti-apoptotic effects. The peptide and its receptors are present also in the heart, but whether PACAP can be protective in cardiomyocytes, is not known. Therefore, the aim of the present study was to investigate the effects of PACAP on oxidative stress-induced apoptosis in cardiomyocytes. Our results show that PACAP increased cell viability by attenuating H2O2-induced apoptosis in a cardiac myocyte culture. PACAP also decreased caspase-3 activity and increased the expression of the anti-apoptotic markers Bcl-2 and phospho-Bad. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis.
Keywords: Cardiomyocyte; Apoptosis; PACAP; Cardioprotection;

Ion channel formation by Alzheimer's disease amyloid β-peptide (Aβ40) in unilamellar liposomes is determined by anionic phospholipids by Juan Marcos Alarcón; Julio A. Brito; Tamara Hermosilla; Illani Atwater; David Mears; Eduardo Rojas (95-104).
Incorporation of Alzheimer's disease amyloid β-proteins (AβPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide–membrane interactions involved in channel formation, we used cation reporter dyes to measure AβP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Aβ40, but not Aβ40-1 or Aβ28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Aβ40-induced changes in cation concentration, which we attribute to ion entry through Aβ40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AβP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Aβ40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AβP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AβP channels.
Keywords: Neurotoxicity; AβP40; Cation channels; Fluorescence; Phosphatidylserine; Phosphatidylcholine;

Phosphoproteomic analysis of the effect of cyclo-[His-Pro] dipeptide on PC12 cells by Alba Minelli; Ilaria Bellezza; Silvia Grottelli; Francesco Pinnen; Luigi Brunetti; Michele Vacca (105-113).
The effects of dipeptide cyclo-[His-Pro] (CHP), known to participate in the appetite behavior and food intake control, have been investigated using PC12 cells in culture as model system. We found that only in the presence of experimental conditions that cause cellular stress the cyclic dipeptide affect cellular proliferation and protects from apoptosis. It greatly enhances the phosphorylation of hsp27, α-B-crystallin, Cdc2, and p-38 MAPK, whereas it decreases the phosphorylation of MEK1, Cav 2, GSK3a, PKB/Akt, PKCδ, PKCγ, and Erk2. PKA and PKG are involved in ERK1/2 deactivation via a receptor that appears to be dually coupled to Gs and Gq protein subfamilies.
Keywords: Neuroprotection; Apoptosis; Proliferation; Kinexus KSS6.0 phospho-site screen; G-protein family; Protein kinases;

Administration of the anabolic androgenic steroid nandrolone decanoate affects substance P endopeptidase-like activity in the rat brain by Kristina Magnusson; Mathias Hallberg; Anna M.S. Kindlundh Högberg; Fred Nyberg (114-121).
The effect of the anabolic androgenic steroid, nandrolone decanoate, on substance P endopeptidase-like activity was examined in adult male Sprague–Dawley rats. Nandrolone decanoate (15 mg/kg day) or oil vehicle (sterile arachidis oleum) were administered by intramuscular injections during 14 days. Substance P endopeptidase, a predominantly cytosolic enzyme, generates the bioactive N-terminal fragment substance P1–7 from the enzyme substrate substance P. Nandrolone decanoate significantly reduced the substance P endopeptidase-like activity compared to control animals in hypothalamus (43% reduction), caudate putamen (44%), substantia nigra (32%) and the ventral tegmental area (27%). It was previously reported that both hypothalamus and caudate putamen contained significantly higher levels of substance P after nandrolone administration. The higher concentration of substance P in these regions could to an extent be attributed to the reduction in substance P endopeptidase-like activity. This result elucidates the important role of peptidase activity in the regulation of the substance P transmitter system. The present study provides additional support for the hypothesis that alterations in the substance P system in certain brain areas may contribute to some of the personality changes reported in connection with AAS abuse.
Keywords: Anabolic androgenic steroids; Nandrolone decanoate; Substance P endopeptidase; Rat brain; Enzyme;

Identification of mature nocistatin and nociceptin in human brain and cerebrospinal fluid by mass spectrometry combined with affinity chromatography and HPLC by Tessy Joseph; Tat Leang Lee; Chou Ning; Yuji Nishiuchi; Terutoshi Kimura; Hiroyuki Jikuya; Keli Ou; Yau Chin Chin; Shinro Tachibana (122-130).
Nocistatin (NST) and nociceptin/orphanin FQ (NCP) are two important bio-peptides derived from the precursor protein prepronociceptin (ppNCP), involved in several central nervous system (CNS) functions including pain transmission. Since the actual form of human NST in CNS is not fully characterized, we studied the structure of NST from human brain tissue and cerebrospinal fluid (CSF) samples. NST and NCP were isolated from human brain and CSF samples by affinity chromatography combined with HPLC. Mass spectrometry was used for the identification and characterization of the peptides. The total NST immunoreactivity was detected as 11.5 ± 2.3 pmol/g tissue for the brain and 0.44 pmol/ml for the pooled CSF sample after the HPLC purification by radioimmunoassay. The presence of two different forms of mature nocistatin (NST-17 and NST-30) and a possible N-terminal methionine cleaved NST-29 were confirmed by both radioimmunoassay and mass spectrometry. Affinity chromatography, HPLC and mass spectrometry methods used in this study were highly sensitive and suitable for identification of actual chemical structures and quantification of very small amounts of peptides in biological samples. The present findings may help further for search for new treatment of neuropathic pain, which is often poorly managed by current therapies.
Keywords: Affinity chromatography; Cerebrospinal fluid; Human brain; MALDI-TOF mass; Nocistatin; Nociceptin;

Enzymatic degradation studies of endomorphin-2 and its analogs containing N-methylated amino acids by Anna Janecka; Rafal Kruszynski; Jakub Fichna; Piotr Kosson; Tomasz Janecki (131-135).
In this paper, we describe the synthesis of novel endomorphin-2 analogs, containing N-methylated amino acids, consecutively in each position. The receptor-binding profile of the new analogs and their stability against enzymatic cleavage by commercially available peptidases, carboxypeptidase Y and aminopeptidase M, and a rat brain homogenate are reported. The best analog of this series, [Sar2]endomorphin-2, was almost equipotent with the parent peptide in the μ-receptor-binding assay and was also highly resistant to enzymatic degradation. This analog may be a suitable candidate for the in vivo antinociceptive studies.
Keywords: Constrained amino acids; μ-Receptor agonist; Binding studies; Enzymatic hydrolysis; Brain homogenate;

C-terminal amide to alcohol conversion changes the cardiovascular effects of endomorphins in anesthetized rats by Ye Yu; Chang-lin Wang; Yun Cui; Ying-zhe Fan; Jing Liu; Xuan Shao; Hong-mei Liu; Rui Wang (136-143).
Endomorphin1-ol (Tyr-Pro-Trp-Phe-ol, EM1-ol) and endomorphin2-ol (Tyr-Pro-Phe-Phe-ol, EM2-ol), with C-terminal alcohol (-ol) containing, have been shown to exhibit higher affinity and lower intrinsic efficacy in vitro than endomorphins. In the present study, in order to investigate the alterations of systemic hemodynamic effects induced by C-terminal amide to alcohol conversion, responses to intravenous (i.v.) or intracerebroventricular (i.c.v.) injection of EM1-ol, EM2-ol and their parents were compared in the system arterial pressure (SAP) and heart rate (HR) of anesthetized rats. Both EM1-ol and EM2-ol induced dose-related decrease in SAP and HR when injected in doses of 3–100 nmol/kg, i.v. In terms of relative vasodepressor activity, it is interesting to note that EM2-ol was more potent than endomorphin2 [the dose of 25% decrease in SAP (DD25) = 6.01 ± 3.19 and 13.99 ± 1.56 nmol/kg, i.v., respectively] at a time when responses to EM1-ol were less potent than endomorphin1. Moreover, decreases in SAP in response to EM1-ol and EM2-ol were reduced by naloxone, atropine sulfate, l-NAME and bilateral vagotomy. It indicated that the vasodepressor responses were possibly mediated by a naloxone-sensitive, nitric oxide release, vagus-activated mechanism. It is noteworthy that i.c.v. injections of -ol derivatives produced dose-related decreases in SAP and HR, which were significantly less potent than endomorphins and were attenuated by naloxone and atropine sulfate. In summary, the results of the present study indicated that the C-terminal amide to alcohol conversion produced different effects on the vasodepressor activity of endomorphin1 and endomorphin2 and endowed EM2-ol distinctive hypotension characters in peripheral (i.v.) and central (i.c.v.) tissues. Moreover, these results provided indirect evidence that amidated C-terminus might play an important role in the regulation of the cardiovascular system.
Keywords: Endomorphin1-ol; Endomorphin2-ol; C-terminal amide to alcohol conversion; DD25; System arterial pressure; Heart rate;

It has been recently reported that acute intracerebroventricular injection of 1 nmol/rat of the non-selective melanocortin 3 and 4 receptor (MC3/4) agonist MTII reduces ethanol intake in female AA alcohol-preferring rats and alters opioid peptide levels in the ventral tegmental area of rats. To better understand the role of the MC system in the control of ethanol intake, we tested the acute and chronic effects of lateral ventricular (LV) injections of 0.01–1 nmol MTII, of 0.1–1 nmol of the MC3/4R receptor antagonist agouti related peptide (AgRP), and 0.1–0.5 nmol of the MC3/4R receptor antagonist SHU9119 on food, water, and 10% ethanol intake in Marchigian-Sardinian alcohol-preferring (msP) rats, which spontaneously ingest pharmacologically relevant quantities of ethanol both under short and long term access conditions. The data showed that with 2 h/day ethanol access, LV MTII injections reduced intake of food and ethanol intakes. When food, water, and ethanol were available ad libitum and 0.01 nmol MTII was given by daily LV injection, however, ethanol intake was reduced for only the first 2 days, whereas food intake was reduced for all 5 days of treatment. Finally, acute LV injection of neither AgRP nor SHU9119 affected ethanol intake under ad libitum conditions, although both antagonists significantly increased food and water intake. In conclusion, these data fail to support a role for endogenous MC3/4R in the control of spontaneous ethanol intake in the msP rat. MC3/4R agonism, however, reduced ethanol intake in association with reduced food intake, suggesting that MTII might reduce nutrient-related controls of ethanol intake rather than, or in addition to, reward-related controls of ethanol intake.
Keywords: Alcohol intake; Melanocortin agonist and antagonists; Marchigian-Sardinian alcohol-preferring rats;

Effects of neuropeptide Y antagonists on food intake in rats: Differences with cold-adaptation by Erika Pétervári; Márta Balaskó; Boglárka Uzsoki; Miklós Székely (150-156).
Hyperphagia followed both central neuropeptide Y (NPY) administration and the presumed increase of endogenous NPY activity after food deprivation. NPY induced greater hyperphagia in cold-adapted than non-adapted rats; fasting of comparable severity caused similar hyperphagia in the two groups. NPY-receptor-antagonist d-Tyr27,36,d-Thr32-NPY(27,36) or functional NPY-antagonist d-myo-inositol-1,2,6-trisphosphate attenuated the hyperphagic effect of both NPY and fasting in non-adapted rats. However, while completely preventing the NPY-hyperphagia, they did not influence the fasting-induced hyperphagia in cold-adapted rats. With cold-adaptation the sensitivity to NPY and to its antagonists increases, but the hypothalamic NPY loses from its fundamental role in the regulation of food intake, and the hyperphagia seen in cold-adaptation may need some other explanation.
Keywords: Neuropeptide Y; NPY-receptor-antagonist; α-Trinositol; Food intake; Cold-adaptation;

Blockade of central GLP-1 receptors prevents CART-induced hypophagia and brain c-Fos expression by Susan Aja; Cassandra Ewing; Jennifer Lin; Jayson Hyun; Timothy H. Moran (157-164).
Central administration of both CART and GLP-1 reduces feeding and increases c-Fos in brain areas associated with food intake. To determine whether aspects of CART's effects were mediated through GLP-1's action, we examined whether the GLP-1 receptor antagonist des-His1-Glu9-exendin-4 (EX) blocked CART-induced feeding inhibition, and c-Fos activation. An i.c.v. dose of 100 μg EX blocked the feeding inhibitory action of 1 μg of CART i.c.v. and prevented CART-induced c-Fos expression at multiple hindbrain and hypothalamic sites. These data suggest that i.c.v. CART administration activates a central release of GLP-1 to inhibit feeding and produce widespread neural activation.
Keywords: c-Fos; Cocaine–amphetamine-regulated transcript; Exendin; Food intake; Hindbrain; Glucagon-like peptide-1;

Distribution of beacon immunoreactivity in the rat brain by Fei Wang; De-Run Tian; Nan Tian; Hui Chen; Yu-Shun Shi; Jaw-Kang Chang; Jun Yang; Lan Yuan; Ji-Sheng Han (165-171).
Beacon is a novel peptide isolated from the hypothalamus of Israeli sand rat. In the present study, we determined the distribution of beacon in the rat brain using immunohistochemical approach with a polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47–73). The hypothalamus represented the major site of beacon-immunoreactive (IR) cell bodies that were concentrated in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Additional immunostained cells were found in the septum, bed nucleus of the stria terminalis, subfornical organ and subcommissural organ. Beacon-IR fibers were seen with high density in the internal layer of the median eminence and low to moderate density in the external layer. Significant beacon-IR fibers were also seen in the nucleus of the solitary tract and lateral reticular formation. The beacon neurons found in the PVN were further characterized by double label immunohistochemistry. Several beacon-IR neurons that resided in the medial PVN were shown to coexpress corticotrophin-releasing hormone (CRH) and most labeled beacon fibers in the external layer of median eminence coexist with CRH. The topographical distribution of beacon-IR in the brain suggests multiple biological activities for beacon in addition to its proposed roles in modulating feeding behaviors and pituitary hormone release.
Keywords: Beacon; Corticotrophin-releasing hormone; Rat brain; Feeding behavior;

Regional brain cholecystokinin changes as a function of rough-and-tumble play behavior in adolescent rats by Jeffrey Burgdorf; Jaak Panksepp; Margery C. Beinfeld; Roger A. Kroes; Joseph R. Moskal (172-177).
Brain cholecystokinin (CCK) levels have been shown to be elevated in animals defeated during adult social aggression. The present experiment evaluated whether similar effects are evident in prolonged bouts of juvenile social-play fighting, which tend to switch from largely positive to some negative affect after approximately 15 min into a half-hour play session, as indexed by a gradual shift from positively valenced 50 kHz ultrasonic vocalizations (USVs) to negatively valenced 20 kHz USVs. Given the role of CCK in both positive and negative emotional events, we examined levels of CCK-8 in tissue homogenates from 14 brain areas in animals 6 h after a 30 min play bout compared to no-play control animals tested similarly in isolation for 30 min. As with patterns observed following adult defeat, significantly higher CCK levels were evident after play in the posterior neo-cortex compared to no-play control animals (+26%). Levels of CCK were also elevated in the midbrain (+35%). However, unlike in adult aggression, CCK levels were reduced in the hypothalamus (−40%) and basal forebrain (−24%) as compared to no-play animals. Posterior cortex CCK levels were positively correlated to the duration that each animal was pinned (r  = +.50) which suggests that elevated CCK in the posterior cortex may be related to the negative aspects of play. Hypothalamic CCK levels were negatively related to dorsal contacts and pins (r's = −.57), and suggest that the lower CCK levels may reflect the more positive valenced aspects of play. The data indicate that CCK utilization in the brain is dynamically responsive to rough-and-tumble play.
Keywords: Affect; Aggression; Brain; Cholecystokinin; Emotion; Play; Rat;

Expression of urocortin 3/stresscopin in human adrenal glands and adrenal tumors by Kazuhiro Takahashi; Kazuhito Totsune; Masayuki Saruta; Tsuyoshi Fukuda; Takashi Suzuki; Takuo Hirose; Yutaka Imai; Hironobu Sasano; Osamu Murakami (178-182).
Urocortin 3 (Ucn 3)/stresscopin (SCP) is a novel peptide of the corticotropin-releasing factor (CRF) family and is a specific ligand for the CRF type 2 receptor. In the present study, we studied expression of Ucn3/SCP in the normal adrenal and adrenal tumors by radioimmunoassay and reverse transcriptase-polymerase chain reaction (RT-PCR). High concentrations of immunoreactive (IR)-Ucn3 were present in the normal portions of adrenal glands (4.2 ± 0.51 pmol/g wet weight, mean ± S.E.M., n  = 14), and the levels were higher than those in the brain. IR-Ucn3 was also detected in the tumor tissues of aldosterone-secreting adenomas (6.2 ± 0.6 pmol/g wet weight, n  = 10), cortisol-secreting adenomas (5.0 ± 1.2 pmol/g wet weight, n  = 4), and pheochromocytomas (1.9 ± 0.4 pmol/g wet weight, n  = 7). Reverse phase high performance liquid chromatography showed that IR-Ucn3 in normal portions of adrenal glands and aldosterone-secreting adenomas was eluted mainly in the positions of Ucn3 and SCP with several minor peaks eluting earlier. The RT-PCR showed expression of Ucn3 mRNA in normal portions of adrenal gland (positive ratio; 4/4), aldosterone-secreting adenomas (3/4), cortisol-secreting adenomas (1/3) and pheochromocytomas (6/7). These findings indicate that Ucn3 is produced in normal adrenal and adrenal tumors (both adrenocortical tumors and pheochromocytomas), and suggest that Ucn3 acts as an autocrine or paracrine regulator in normal adrenal and adrenal tumors.
Keywords: Urocortin; Urocortin 3; Stresscopin; Corticotropin-releasing factor; Adrenal; Pheochromocytoma;

Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha by Persefoni Klimentzou; Maria Paravatou-Petsotas; Christos Zikos; Alexander Beck; Margarita Skopeliti; Jan Czarnecki; Ourania Tsitsilonis; Wolfgang Voelter; Evangelia Livaniou; Gregory P. Evangelatos (183-193).
Since conserved mammalian polypeptides are believed to exhibit enhanced immunogenicity in avian species, hens were immunized against the poorly immunogenic, highly conserved mammalian polypeptide prothymosin alpha (ProTα), i.e. against either non-conjugated ProTα (isolated from bovine thymus) or ProTα conjugated to keyhole limpet hemocyanin (ProTα/KLH). The antibodies Y were isolated from the egg yolk and evaluated through suitable dot-blot and ELISA systems in parallel with antibodies G isolated from the antiserum of rabbits immunized against the same immunogens. As revealed, antibodies Y and G of low titer and/or affinity were obtained against non-conjugated ProTα, while antibodies Y against ProTα/KLH had a better apparent titer, could better discriminate between ProTα and the closely related bioactive peptide thymosin alpha 1, and were obtained at much larger quantities than the corresponding antibodies G.
Keywords: Hen antibodies Y; Rabbit antibodies G; Highly conserved mammalian polypeptides; Prothymosin alpha; Prothymosin alpha conjugated to keyhole limpet hemocyanin;

NMR studies for identifying phosphopeptide ligands of the HIV-1 protein Vpu binding to the F-box protein β-TrCP by Nathalie Evrard-Todeschi; Josyane Gharbi-Benarous; Gildas Bertho; Gaël Coadou; Simon Megy; Richard Benarous; Jean-Pierre Girault (194-210).
The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and, its interaction with the ubiquitin ligase SCF-β-TrCP triggers the HIV-1 receptor CD4 degradation by the proteasome. The interaction between β-TrCP protein and ligands containing the phosphorylated DpSGXXpS motif plays a key role for the development of severe disease states, such as HIV or cancer. This study examines the binding and conformation of phosphopeptides (P1, LIERAEDpSG and P2, EDpSGNEpSE) from HIV protein Vpu to β-TrCP with the objective of defining the minimum length of peptide needed for effective binding. The screening step can be analyzed by NMR spectroscopy, in particular, saturation transfer NMR methods clearly identify the residues in the peptide that make direct contact with β-TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra provided clear evidence that the two peptides efficiently bound β-TrCP receptor protein. To better characterize the ligand–protein interaction, the bound conformation of the phosphorylated peptides was determined using transferred NOESY methods, which gave rise to a well-defined structure. P1 and P2 can fold in a bend arrangement for the DpSG motif, showing the protons identified by STD-NMR as exposed in close proximity at the molecule surface. Ser phosphorylation allows electrostatic interaction and hydrogen bond with the amino acids of the β-TrCP binding pocket. The upstream LIER hydrophobic region was also essential in binding to a hydrophobic pocket of the β-TrCP WD domain. These findings are in good agreement with a recently published X-ray structure of a shorter β-Catenin fragment with the β-TrCP complex.
Keywords: Human immunodeficiency virus type 1; Vpu; Phosphorylated peptide P-Vpu; STD-NMR; TRNOESY; Restrained molecular dynamics; Bound structure; Binding fragment;

A unique feature of cerebral endothelial cells (CECs) is the formation of the blood–brain barrier (BBB), which contributes to the stability of the brain microenvironment. CECs are capable of producing several substances mediating endothelium-dependent vasorelaxation or vasoconstriction, regulating BBB permeability, and participating in the regulation of cell–cell interactions during inflammatory and immunological processes. The chemical nature of these mediators produced by CECs ranges from gaseous anorganic molecules (e.g. nitric oxide) through lipid mediators (e.g. prostaglandins) to peptides. Peptide mediators are a large and diverse family of bioactive molecules which can elicit multiple effects on cerebral endothelial functions. In this review, we summarize current knowledge of peptide mediators produced by CECs, such as adrenomedullin, angiotensin, endothelin and several others and their role in the regulation of BBB functions.
Keywords: Blood–brain barrier; Adrenomedullin; Angiotensin; Endothelin; Cytokine; Substance P;