Peptides (v.27, #9)

Cytoskeleton alterations induced by Geodia corticostylifera depsipeptides in breast cancer cells by Marisa Rangel; Marisa P. Prado; Katsuhiro Konno; Hideo Naoki; José C. Freitas; Glaucia M. Machado-Santelli (2047-2057).
Crude extracts of the marine sponge Geodia corticostylifera from Brazilian Coast have previously shown antibacterial, antifungal, cytotoxic, haemolytic and neurotoxic activities. The present work describes the isolation of the cyclic peptides geodiamolides A, B, H and I (14) from G. corticostylifera and their anti-proliferative effects against sea urchin eggs and human breast cancer cell lineages. Its structure–activity relationship is discussed as well. In an initial series of experiments these peptides inhibited the first cleavage of sea urchin eggs (Lytechinus variegatus). Duplication of nuclei without complete egg cell division indicated the mechanism of action might be related to microfilament disruption. Further studies showed that the geodiamolides have anti-proliferative activity against human breast cancer cell lines (T47D and MCF7). Using fluorescence techniques and confocal microscopy, we found evidence that the geodiamolides A, B, H and I act by disorganizing actin filaments of T47D and MCF7 cancer cells, in a way similar to other depsipeptides (such as jaspamide 5 and dolastatins), keeping the normal microtubule organization. Normal cells lines (primary culture human fibroblasts and BRL3A rat liver epithelial cells) were not affected by the treatment as tumor cells were, thus indicating the biomedical potential of these compounds.
Keywords: Cancer cell; Cytoskeleton; Sea urchin egg; Marine sponge; Depsipeptide; Geodiamolide;

Antifouling activity of synthesized peptide analogs of the sponge metabolite barettin by Martin Sjögren; Ann-Louise Johnson; Erik Hedner; Mia Dahlström; Ulf Göransson; Hamid Shirani; Jan Bergman; Per R. Jonsson; Lars Bohlin (2058-2064).
Barettin (cyclo [(6-bromo-8-en-tryptophan) arginine]), a diketopiperazine isolated from the marine sponge Geodia barretti, is a potent inhibitor of barnacle larvae settlement with an EC50-value of 0.9 μM. In the present study, 14 analogs of barettin and its structural congener dipodazine were synthezised and tested for their ability to inhibit larval settlement. Two of the analogs have an intact barettin skeleton. The remaining analogs have a dipodazine skeleton (a diketopiperazine where arginine is replaced with glycine). Six of the tested synthetic analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine, which displayed even stronger activity than barettin (EC50-value 0.034 μM). The effect of benzo[g]dipodazine was also shown to be readily reversible, when cyprids were transferred to filtered seawater (FSW).
Keywords: Barettin; Dipodazine; Marine sponges; Geodia barretti; Synthesis; Cyprid settlement;

Arg-Ile-Tyr (RIY) derived from rapeseed protein decreases food intake and gastric emptying after oral administration in mice by Ewa D. Marczak; Kousaku Ohinata; Andrzej W. Lipkowski; Masaaki Yoshikawa (2065-2068).
We previously reported that a bioactive tripeptide Arg-Ile-Tyr (RIY), which has been isolated as an inhibitor for angiotensin I-converting enzyme from the subtilisin digest of rapeseed protein, decreased blood pressure. In this study, we also found that RIY dose-dependently decreased food intake at a dose of 150 mg/kg after oral administration in fasted ddY male mice. The anorexigenic action of RIY was blocked by a cholecystokinin-1 CCK1 receptor antagonist, lorglumide. RIY also decreased the gastric emptying rate at a dose of 150 mg/kg and the RIY-induced delay of gastric emptying was blocked by lorglumide. However, RIY had no affinity for CCK1 receptor. Taken together, RIY decreased food intake and gastric emptying by stimulating CCK release.
Keywords: Rapeseed; Food intake; Gastric emptying; CCK1 receptor;

The aim of this study was to elucidate the characteristics of glutathione S-transferase(GST)-activating compounds from medicinal plants. Among 265 kinds of medicinal plants, Phellodendron amurense showed the highest GST activity at 174.8%. The GST-activating compound of P. amurense was maximally extracted when treated with distilled water at 30 °C for 12 h. The compound was purified by ultrafiltration, Sephadex G-10 gel filtration chromatography, and reverse-phase HPLC. The purified GST-activating compound from P. amurense was a novel tetrapeptide with an amino acid sequence of Ala-Pro-Trp-Cys and its molecular weight was estimated to be 476 Da. It also displayed a clear detoxicative effect in 1-chloro-2,4-dinitrobenzene treated mice at a dosage of mg/kg body weight.
Keywords: Glutathione S-transferase-activating peptide; Oriental medicinal plant; Phellodendron amurense;

A mitogenic defensin from white cloud beans (Phaseolus vulgaris) by Jack Ho Wong; Xiao Qing Zhang; He Xiang Wang; Tzi Bun Ng (2075-2081).
A peptide, with a molecular mass of 7458 Da, was purified from the seeds of white cloud beans (Phaseolus vulgaris cv. ‘white cloud bean’). This peptide was isolated using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. The peptide had both antifungal and antibacterial activities. It reduced the activity of HIV-1 reverse transcriptase and it also inhibited translation in a cell-free rabbit reticulocyte lysate system. Its antifungal activity was retained after incubation with trypsin but was reduced when the ambient ionic strength was raised. The peptide elicited a mitogenic response from mouse splenocytes but did not stimulate nitric oxide production in mouse macrophages.
Keywords: White cloud bean; Defensin; HIV-1 reverse transcriptase; Inhibitory; Mitogenic; Isolation;

Isolation and characterization of four antibacterial peptides from bovine hemoglobin by Naima Nedjar-Arroume; Véronique Dubois-Delval; Khalil Miloudi; Rachid Daoud; François Krier; Mostafa Kouach; Gilbert Briand; Didier Guillochon (2082-2089).
Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the α-chain of bovine hemoglobin: α107–141, α137–141, and α133–141, and one peptide to the β-chain: β126–145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.
Keywords: Antibacterial peptides; Bovine hemoglobin; Pepsin; Hydrolysate; Active peptides; Neokyotorphin;

Detection and identification of oligopeptides in Microcystis (cyanobacteria) colonies: Toward an understanding of metabolic diversity by Martin Welker; Blahoslav Maršálek; Lenka Šejnohová; Hans von Döhren (2090-2103).
Cyanobacteria and particularly Microcystis sp. (Chroococcales) are known to produce a multitude of peptide metabolites. Here we report on the mass spectral analysis of cyanobacterial peptides in individual colonies of Microcystis sp. collected in a drinking water reservoir. A total number of more than 90 cyanopeptides could be detected, 61 of which could be identified either as known peptides or new structural variants of known peptide classes. For 18 new peptides flat structures are proposed. New congeners differed from known ones mainly in chlorination (aeruginosins), methylation (microginins), or amino acid sequences (cyanopeptolins). The high number of peptides and especially the new peptides underline the capability of Microcystis strains as producers of a high diversity of potentially bioactive compounds.
Keywords: Cyanobacteria; Microcystis; Peptides; Metabolic diversity; MALDI-TOF mass spectrometry;

Pre-treatment of central venous catheters with the cathelicidin BMAP-28 enhances the efficacy of antistaphylococcal agents in the treatment of experimental catheter-related infection by Oscar Cirioni; Andrea Giacometti; Roberto Ghiselli; Cristina Bergnach; Fiorenza Orlando; Federico Mocchegiani; Carmela Silvestri; Alberto Licci; Barbara Skerlavaj; Margherita Zanetti; Vittorio Saba; Giorgio Scalise (2104-2110).
An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of the 27 residues cathelicidin peptide BMAP-28, quinupristin/dalfopristin (Q/D), linezolid, and vancomycin. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with BMAP-28. Thirty minutes later rats were challenged via the CVC with 1.0 × 106 CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC at a concentration equal to the MBC observed using adherent cells, or at a much higher concentration (1024 μg/mL) began 24 h later. The inhibition activities of all antibiotics against adherent bacteria were at least two-four-fold lower that against freely growing cells. When antibiotics were used in BMAP-28 pre-treated wells, they showed higher activities. The in vivo studies showed that when CVCs were pre-treated with BMAP-28 or with a high dose of antibiotics, biofilm bacterial load was reduced from 107 to 103  CFU/mL and bacteremia reduced from 103 to 101  CFU/mL. When CVCs were treated with both BMAP-28 and antibiotics, biofilm bacterial load was further decreased to 101  CFU/mL and bacteremia was not detected. These results suggest that CVC pre-treated with BMAP-28 represents an attractive choice for the treatment of device-related infections caused by staphylococci.
Keywords: CVC; Biofilm; Antibiotic-lock technique; BMAP-28; Antimicrobial peptides;

Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami by J. Michael Conlon; Nadia Al-Ghaferi; Bency Abraham; Hu Jiansheng; Pascal Cosette; Jérôme Leprince; Thierry Jouenne; Hubert Vaudry (2111-2117).
Seven peptides with antimicrobial activity were isolated in pure form from an extract of the skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevinin-2 (three peptides), and esculentin-1 (one peptide) families. Nigrocin-2GRb (GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide with highest potency against microorganisms (MIC = 3 μM against Escherichia coli, 12.5 μM against Staphylococcus aureus and 50 μM against Candida albicans) and the greatest hemolytic activity against human erythrocytes (LD50  = 40 μM). In contrast, nigrocin-2GRa (GLLSGILGAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relationships among Eurasian ranid frogs are less well understood than those of North American ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa brevipoda.
Keywords: Antimicrobial peptides; Frog skin; Brevinin; Esculentin; Nigrocin;

Odorous frogs of the sub-genus Odorrana are of oriental distribution, and are so called due to the foul smell of their defensive skin secretions released from specialized skin glands following stress or predator attack. Here we report the application of a “shotgun” skin secretion cDNA library cloning technique which can rapidly expedite identification of secretion bioactive peptides. From a library constructed from the skin secretion of the Large Chinese Odorous frog, Rana (Odorrana) livida, we have identified four novel peptides whose primary structures were deduced initially from cloned precursors. Subsequently, mature peptides were located in and structurally characterized from reverse phase HPLC fractions of skin secretion. Named lividins 1–4, these were found to be structural homologs of known antimicrobial peptide families from Rana frogs. Rapid identification of novel peptides can thus be rapidly achieved using this non-invasive, non-destructive technology and the extensive similarities revealed between antimicrobial peptide precursor organization and nucleic acid sequences would lend support to the hypothesis that they have a common ancestral origin.
Keywords: Amphibian; Molecular cloning; Antimicrobial; Peptide;

The temporins are a family of hydrophobic, C-terminally α-amidated antimicrobial peptides that are synthesized in the skins of a wide range of species of frogs belonging to the genus Rana. In the present study, we investigated using RT-PCR the expression of preprotemporin mRNAs in extradermal tissues of Tago's brown frog Rana tagoi. cDNAs encoding temporin-1TGa (FLPILGKLLS10GIL.NH2), previously isolated from an extract of the skin of R. tagoi skin, were amplified and cloned from the stomach, liver, kidney, skeletal muscle. However, a net insertion of 10 nucleotides resulted in the presence of a premature stop codon in the open reading frame that was not present in the corresponding region of preprotemporin-1TGa from skin. The preprotemporin cDNA obtained from small intestine contained an additional 12 nucleotide insertion in the region that encodes the temporin sequence so that a novel peptide (FLPVILPVIG10KLLSGIL.NH2), termed temporin-1TGc, is specified. This cDNA also contained a premature stop codon in the open reading frame. Although it is unclear whether temporin-1TGc is produced in R. tagoi tissues, a synthetic replicate of the peptide of was biologically active, inhibiting the growth of Staphylococcus aureus (minimal inhibitory concentration = 37.5 μM) and producing hemolysis of human erythrocytes (LD50  = 50 μM).
Keywords: Antimicrobial peptide; Rana tagoi; Temporin;

The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin—a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.
Keywords: Amphibian; Peptide; Skin; Cloning; cDNA;

Bradykinin-related peptides from Phyllomedusa hypochondrialis by G.D. Brand; F.C. Krause; L.P. Silva; J.R.S.A. Leite; J.A.T. Melo; M.V. Prates; J.B. Pesquero; E.L. Santos; C.R. Nakaie; C.M. Costa-Neto; C. Bloch (2137-2146).
Bradykinin related peptides (BRPs) present in the water-soluble secretion and freshly dissected skin fragments of Phyllomedusa hypochondrialis were investigated by mass spectrometry techniques. Eighteen BRPs, along with their post-translational modifications, were characterized in the secretion by de novo MS/MS sequencing and direct MALDI imaging experiments of the frog skin. These molecules revealed strong sequence similarities to the main plasma kinin of some mammals and reptiles. Such a diversity of molecules, within the same peptide family, belonging to a single amphibian species may be related to functional specializations of these peptides and a variety of corresponding receptors that might be present in a number of different predators. Also, a novel analog, [Val]1,[Thr]6-bradykinyl-Gln,Ser had its biological activity positively detected in cell culture expressing the human bradykinin B2 receptor and in guinea pig ileum preparations.
Keywords: Imaging mass spectrometry; Frog skin; BRP; Phyllomedusa;

Antagonistic and agonistic activities of backbone cyclic (BBC) pheromone biosynthesis activating neuropeptide (PBAN) analogues were evaluated in an attempt to identify potent melanotropic antagonists, to gain an insight into their structure–activity relationship (SAR), and to discover molecules with selective and non-selective melanotropic and pheromonotropic properties. Eight potent melanotropic BBC antagonists and seven agonists were disclosed. SAR studies revealed that the structural requirements of the melanotropic and pheromonotropic agonists and antagonists are different. The cyclic structure of the BBC peptides was unimportant for antagonistic activity, and linearization retained their melanotropic and pheromonotropic antagonistic properties. Comparison of the antagonistic activities of the BBC and precyclic peptides with respect to both functions revealed eight selective antagonists (six that were selective melanotropic antagonists and two selective pheromonotropic antagonists) and four non-selective (melanotropic and pheromonotropic) antagonists. The selective melanotropic antagonists exhibited both, pure or mixed agonistic/antagonistic activities. The selective pheromonotropic compounds were pure antagonists. All non-selective compounds were pure antagonists. Comparison of the agonistic activities of the BBC peptides with respect to both functions revealed six selective melanotropic agonists and one non-selective agonistic compound. All compounds (whether selective or non-selective) exhibited pure agonistic activity. Discovery of the selective compounds hints at the possibility that the receptors that mediate the respective activities may have different properties.
Keywords: PBAN; MRCH; Cuticular melanization; Neuropeptide antagonists; Spodoptera littoralis; Insect neuropeptide;

The midgut plays a major role in digestion and absorption of nutrients in insects, and contains endocrine cells throughout the epithelial layer that express neuropeptides, including crustacean cardioactive peptide (CCAP). In the present study, we demonstrate regulation of digestive enzyme activities by CCAP in response to nutrient ingestion in the cockroach, Periplaneta americana. The midgut of the cockroach exhibits maximal α-amylase and protease activities 3 h after intake of either starch or casein, but not of non-nutrients. Similar time-dependent responses of CCAP expression in midgut endocrine cells were observed after feeding starch and casein, but not after non-nutrients. We also show that incubation of the dissected midgut with CCAP leads to an increase in α-amylase and protease activity in a time-dependent manner, with the maximal activity at 2 h. Taken together, our data indicate the existence of an inducible mechanism where endocrine cells in the midgut are stimulated to synthesize and secrete CCAP by nutrients, and CCAP then up-regulates the activity of digestive enzymes.
Keywords: Midgut; Endocrine cell; Digestive enzyme; Nutrition; Neuropeptide;

Thymosin α1 suppresses proliferation and induces apoptosis in human leukemia cell lines by Ying-zhe Fan; Hui Chang; Ye Yu; Jing Liu; Rui Wang (2165-2173).
Thymosin α1(Tα1), a 28-amino acid peptide, is a well-known immune system enhancer for the treatment of various diseases. In the present investigation, the effects of Tα1 on the proliferation and apoptosis of human leukemia cell lines (HL-60, K562 and K562/ADM) were studied. The proliferation was significantly depressed after 96 h of treatment with Tα1, and obvious signs of apoptosis, i.e., cell morphology, nuclei condensation and Annexin V binding, were observed thereafter. Moreover, the up-regulation of Fas/Apol (CD95) and decrease in bcl-2 anti-apoptotic gene expression were observed in apoptotic cells. The expression and the function of P-glycoprotein (P-gp) can be slightly inhibited by Tα1. It is noteworthy that K562 and K562/ADM were more sensitive than HL-60 cells when subjected to Tα1. Furthermore, HepG-2, the human hepatoma cell line, displayed significant less sensitivity to Tα1 than all the human leukemia cell lines. d-Tubocurarine (TUB), a nicotinic acetylcholine receptors (nAChRs) antagonist, significantly antagonized the inhibition effects induced by Tα1, whereas atropine, a muscaric acetylcholine receptor antagonist, did not exhibit such effects. All the results indicate that Tα1 was able to significantly suppress proliferation and induce apoptosis in human leukemia cell lines.
Keywords: Thymosin α1; HL-60; K562; K562/ADM; d-Tubocurarine;

Discovery of a novel class of conotoxin from Conus litteratus, lt14a, with a unique cysteine pattern by Can Peng; Shaojun Tang; Canhui Pi; Junliang Liu; Fang Wang; Lei Wang; Wenliang Zhou; Anlong Xu (2174-2181).
Conus litteratus is a worm-hunting cone snail with a highly sophisticated neuropharmacological defense strategy using small peptides in its venom. By analyzing different clones in the cDNA library of venom ducts from C. litteratus, we identified the peptide lt14a which displays a characteristic signal peptide sequence in its precursor and a unique arrangement of Cys residues (–C–C–C–C–) in its mature peptide region. RT-PCR analysis suggested that lt14a is abundantly expressed throughout the whole venom duct. An intensive analysis in sequence suggested that lt14a is similar to α-conotoxin qc1.1 cloned from Conus quercinus. We conducted the chemical synthesis of lt14a. The synthetic lt14a has a remarkable biological activity to suppress pain and inhibits the neuronal-type nicotinic acetylcholine receptors.
Keywords: Cone snails; Conus; Conus litteratus; Venom duct; Conotoxin gene expression; Nicotinic acetylcholine receptor;

In the present study, we investigated the anti-nociceptive effect and the underlying mechanism of BmK AS, an active peptide purified from scorpion Buthus martensi Karsch. The results showed that BmK AS can significantly relieve formalin-induced two-phase spontaneous flinching response and carrageenan-induced mechanical hyperalgesia. Using the whole-cell patch-clamp recording, exposure of acutely isolated sensory neurons to 500 nM BmK AS produced a one-fold decrease in the number of action potentials (APs) evoked by a ramp of depolarizing current. To investigate the mechanism of action of BmK AS, isolated membrane current and Ca2+ influx on rat primary sensory neurons were examined. BmK AS produced insignificant effect on voltage-dependent I K and KCl or caffeine-induced Ca2+ influx, but caused remarkable suppressive effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) I Na. Further experiments showed that BmK AS reduced the peak TTX-R and TTX-S Na+ conductance in a dose-dependent manner, prompted the voltage-dependent activation, and caused a negative shift of the steady-state inactivation of TTX-R and TTX-S I Na. Thus, the present results indicate the anti-nociceptive response of BmK AS may be ascribed to its specific modulation of voltage-gated Na+ channels of sensory neurons.
Keywords: Scorpion toxin; Sodium channel; Anti-nociception;

Anxiolytic-like effects of nociceptin/orphanin FQ in the elevated plus maze and in the conditioned defensive burying test in rats by Giovanni Vitale; Rossana Arletti; Valentina Ruggieri; Carlo Cifani; Maurizio Massi (2193-2200).
Different reports suggest that nociceptin/orphanin FQ (N/OFQ) may have either anxiolytic- or anxiogenic-like effect in rodents. Since N/OFQ elicits hypolocomotion, which undergoes rapid tolerance, and hypolocomotion may be associated to emotional consequences, the present study was designed to investigate the effect of N/OFQ on anxiety after development of tolerance to its hypolocomotor effect. The effect of single or double intracerebroventricular (i.c.v.) injection of N/OFQ was evaluated on anxiety-related behaviors in rats, in the elevated plus maze (EPM) and conditioned defensive burying (CDB) tests. After single administration, N/OFQ displayed an anxiogenic-like pattern of response on the elevated plus maze but hypolocomotion was also observed. Conversely, in the CDB test, N/OFQ induced a clear-cut anxiolytic pattern. To produce tolerance to N/OFQ-induced hypolocomotion the peptide was administered by two i.c.v. injections separated by 120 min; in these conditions it decreased the expression of anxiety-related behaviors in both tests without affecting locomotor activity. The nociceptin/orphanin FQ peptide (NOP) receptor antagonist UFP-101 significantly reduced the effects of N/OFQ to control values in either tests. Corticosterone levels were significantly increased after a single N/OFQ administration (not in a dose-dependent manner) but this increase did not reach significance after double administration (1 nmol/rat). Our results support the idea that N/OFQ may act as an anxiolytic-like agent in the rat; the apparent anxiogenic-like effect observed following its single administration in the EPM may be consequent to its effect on locomotion.
Keywords: Nociceptin/orphanin FQ (N/OFQ); UFP-101; NOP receptor; Anxiety tests; Corticosterone; Rat;

Effects of chronic ethanol administration on brain interstitial fluid levels of Methionine-enkephalin as measured by microdialysis in vivo by Akihiko Urayama; Kevin King; Frederich S. Gaskin; Susan A. Farr; William A. Banks (2201-2206).
The level of Met-enkephalin in the brain is inversely correlated with ethanol consumption and is controlled partially through efflux activity of peptide transport system-1 (PTS-1) at the blood–brain barrier (BBB). Prolonged alcohol drinking can perturb aspects of this system, including a loss of control of Met-enkephalin levels at the transcriptional and translational levels, and impaired release of Met-enkephalin from tissue sources. Met-enkephalin levels in whole brain homogenates often first paradoxically increase after a few days of ethanol drinking and then decrease with the development of physical dependence. Which of those various changes drives the others is unclear. To clarify these interactions, we here determined the levels of Met-enkephalin in striatal interstitial fluid (ISF) by microdialysis, striatal tissue homogenates, and serum after chronic ethanol treatment and alcohol withdrawal. Mice received ethanol (5%) in liquid diet for 7 days (ethanol-treated) and others withdrawn for a day following 7-day treatment (withdrawal). There was a significant (P  < 0.05) difference in the levels of Met-enkephalin in striatal microdialysate between the control (79.1 ± 5.9 pg/ml) and ethanol-treated group (94.9 ± 4.3 pg/ml), which was lost by withdrawing ethanol (83.9 ± 3.8 pg/ml). In contrast, ethanol treatment did not affect Met-enkephalin levels in the striatal tissue. In the ethanol-treated group, there was a significant (P  < 0.05) reduction of the levels of Met-enkephalin in serum to 70.5% of control levels. This decrease was restored to the level of control by withdrawing ethanol. These reversible changes in ISF and serum are readily explained by the known changes in the efflux activity of PTS-1 at the BBB.
Keywords: Methionine-enkephalin; Microdialysis; Blood–brain barrier; Peptide transport system-1; Striatum; Alcoholism;

In vivo inhibition of neuropeptide FF agonism by BIBP3226, an NPY Y1 receptor antagonist by Quan Fang; Jia Guo; Feng He; Ya-li Peng; Min Chang; Rui Wang (2207-2213).
BIBP3226 {(R)-N 2-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide} was recently shown to display relatively high affinities for neuropeptide FF (NPFF) receptors and exhibit antagonist activities towards NPFF receptors in vitro. The present study was undertaken to investigate the antagonistic effects of BIBP3226 on several in vivo pharmacologic profiles induced by exogenous NPFF and NPVF. (1) BIBP3226 (5 nmol) injected into the third ventricle completely antagonized the hypothermic effects of NPFF (30 nmol) and NPVF (30 nmol) after cerebral administration in mice; (2) BIBP3226 (5 nmol, i.c.v.) prevented the anti-morphine actions of NPFF (10 nmol, i.c.v.) in the mouse tail-flick assay; (3) in urethane-anaesthetized rats, both NPFF (200 nmol/kg, i.v.) and NPVF (200 nmol/kg, i.v.) increased the mean arterial blood pressure, which were significantly reduced by pretreatment with BIBP3226 (500 nmol/kg, i.v.). Collectively, these data suggest that BIBP3226, a mixed antagonist of NPY Y1 and NPFF receptors, shows in vivo antagonistic effects on NPFF receptors. In addition, it seems to be clear that the in vivo pharmacological profiles of NPFF are mediated directly by NPFF receptors.
Keywords: BIBP3226; Neuropeptide FF (NPFF); NPFF receptors; Antagonist; In vivo;

Interaction of the tachykinin NK3 receptor agonist senktide with behavioral effects of cocaine in marmosets (Callithrix penicillata) by Maria A. de Souza Silva; Eldon L. Mello; Christian P. Müller; Gerhard Jocham; Rafael S. Maior; Joseph P. Huston; Carlos Tomaz; Marilia Barros (2214-2223).
Brain neuropeptide transmitters of the tachykinin family are involved in the organization of many behaviors. However, little is known about their contribution to the behavioral effects of drugs of abuse. Recently, antagonism of the tachykinin NK3-receptor (NK3-R), one of the three tachykinin receptors in the brain, was shown to attenuate the acute and chronic behavioral effects of cocaine in rats and the acute effects in non-human primates. In order to expand these findings we investigated the effects of the NK3-R agonist, succinyl-[Asp6, Me-Phe8]SP6-11 (senktide), on the acute behavioral effects of cocaine in marmoset monkeys (Callithrix penicillata) using a figure-eight maze procedure. Animals were pretreated with senktide (0, 0.1, 0.2, 0.4 mg/kg, s.c.), and received either a treatment with cocaine (10 mg/kg) or saline (i.p.). Cocaine increased locomotor activity and the duration of aerial scanning behavior, but reduced exploratory activity, bodycare activity, the frequency of aerial scanning, and terrestrial glance behavior. Senktide blocked the effects of cocaine on locomotor activity, but enhanced the cocaine effects on exploratory activity, aerial scanning frequency, and terrestrial glance behavior. Senktide alone did not significantly influence monkey behavior in this study. These data expand previous findings suggesting a complex role of the NK3-R in the acute behavioral effects of cocaine in non-human primates.
Keywords: Cocaine; Tachykinin NK3-receptor; Senktide; Marmoset; Behavior; Sensitivity;

Effect of arginine vasopressin in the nucleus raphe magnus on antinociception in the rat by Jun Yang; Jian-Min Chen; Wen-Yan Liu; Cao-You Song; Cheng-Hai Wang; Bao-Cheng Lin (2224-2229).
Previous work has shown that arginine vasopressin (AVP) regulates antinociception through brain nuclei rather than the spinal cord and peripheral organs. The present study investigated the nociceptive effect of AVP in the nucleus raphe magnus (NRM) of the rat. Microinjection of AVP into the NRM increased pain threshold in a dose-dependent manner, while local administration of AVP-receptor antagonist-d(CH2)5Tyr(Et)DAVP decreased the pain threshold. Pain stimulation elevated AVP concentration in the NRM perfuse liquid. NRM pretreatment with AVP-receptor antagonist completely reversed AVP's effect on pain threshold in the NRM. The data suggest that AVP in the NRM is involved in antinociception.
Keywords: Arginine vasopressin; Vasopressin receptor antagonist; Nucleus raphe magnus; Antinociception; Rat;

Effects of i.c.v. losartan on the angiotensin II-mediated vasopressin release and hypothalamic fos expression in near-term ovine fetuses by Lijun Shi; Caiping Mao; Jiawei Wu; Paul Morrissey; Juanxiu Lee; Zhice Xu (2230-2238).
Our previous studies have shown that central administration of angiotensin (ANG II) causes arginine vasopressin (AVP) release in the fetus at 70–90% gestation. This is evidence that the hypothalamic-neurohypophysial system is relatively mature before birth. However, few data exist regarding central ANG receptor mechanisms-mediated AVP response during fetal life. To determine roles of brain ANG receptor subtypes in this response, AT1 and AT2 receptor antagonists, losartan and PD123319, were investigated in the brain in chronically prepared ovine fetuses at the last third of gestation. Application of losartan intracerebroventricularly (i.c.v.) at 0.5 mg/kg suppressed central ANG II-stimulated plasma AVP release. Losartan at 5 mg/kg (i.c.v.) demonstrated a significant enhancement of AVP increase to i.c.v. ANG II. Associated with the increase of plasma vasopressin levels, c-fos expression in the hypothalamic neurons was significantly different between the low and high doses of losartan. The low dose losartan markedly reduced the dual immunoreactivity for FOS and AVP in the supraoptic nuclei and paraventricular nuclei after i.c.v. ANG II, whereas the high dose losartan together with ANG II, significantly increased the co-localization of positive FOS in the AVP-containing neurons than that induced by i.c.v. ANG II alone. Central ANG II induced fetal plasma vasopressin increase was not altered by PD123319. The data suggest that losartan in the fetal brain has remarkably different effects based on the doses administrated on central ANG II-related neuroendocrine effects at the late gestation, and that the AT1 mechanism is critical in the regulation of fetal body fluid homeostasis related to plasma AVP levels.
Keywords: AT1 receptor; Losartan; Fetal vasopressin; Hypothalamic nuclei;

Bilateral electrolytic lesions of the paraventricular nucleus of the hypothalamus (PVN) produce hyperphagia with excess weight gain. The orexigenic neuropeptide Y (NPY) system and the anorexigenic melanocortin system act in the PVN to regulate food intake, and participate in mediating the anorexic effects of leptin. We hypothesized that changes in the responsiveness of these systems may contribute to the hyperphagia observed in PVN-lesioned rats. Adult female Sprague–Dawley rats received either sham or electrolytic lesions in the PVN immediately followed by implantation of a guide cannula into the third cerebroventricle. Twenty-five days following surgery groups of sham and hyperphagic PVN-lesioned rats were injected intracerebroventicularly (icv) with either 118 pmole or 470 pmole of NPY and food intake was measured for 3 h. Food intake in response to NPY was nearly three-fold higher in PVN-lesioned rats as compared to sham rats. However, the response to 5 μg leptin icv was not different in lesioned versus sham rats. The effect of the melanocortin agonist MTII on food intake was tested in additional rats beginning either 7–14 days or 30–40 days following surgery. Doses of 0.1 nmole or 1.0 nmole of MTII were injected immediately before lights-off and food intake was measured at 2 h, 24 h and 48 h post-injection. Suppression of food intake in PVN-lesioned rats was not different from that in sham-lesioned rats. These data suggest that hyper-responsiveness to NPY may account in part for the hyperphagia observed in PVN-lesioned rats. Furthermore, based on the similarities of responses of PVN-lesioned and sham control rats to the anorexigenic agents MTII and leptin and the hypersensitivity of lesioned rats to NPY, we conclude that the PVN is not essential for NPY stimulation of food intake or for melanocortin suppression of food intake and that NPY and melanocortin receptors outside of the PVN are sufficient to produce these effects.
Keywords: Obesity; Food intake; Melanocortin system;

Oxytocin alleviates oxidative renal injury in pyelonephritic rats via a neutrophil-dependent mechanism by Neşe Karaaslan Bıyıklı; Halil Tuğtepe; Göksel Şener; Ayliz Velioğlu-Öğünç; Şule Çetinel; Şükrü Midillioğlu; Nursal Gedik; Berrak Ç. Yeğen (2249-2257).
Background: Urinary tract infection (UTI) may cause inflammation of the renal parenchyma and may lead to impairment in renal function and scar formation. Oxidant injury and reactive oxygen species (ROS) have been found responsible in the pathogenesis of UTI. The neurohypophyseal hormone oxytocin (OT) facilitates wound healing and is involved in the modulation of immune and inflammatory processes. We investigated the possible therapeutic effects of OT against Eschericia coli induced pyelonephritis in rats both in the acute and chronic setting. Methods: Twenty-four Wistar rats were injected 0.1 ml solution containing E. coli ATCC 25922 1010 colony forming units/ml into left renal medullae. Six rats were designed as sham group and were given 0.1 ml 0.9% NaCl. Pyelonephritic rats were treated with either saline or OT immediately after surgery and at daily intervals. Half of the pyelonephritic rats were decapitated at the 24th hour of E. coli infection, and the rest were followed for 7 days. Renal function tests (urea, creatinine), systemic inflammation markers [lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-α)] and renal tissue malondialdehyde (MDA) as an end product of lipid peroxidation, glutathione (GSH) as an antioxidant parameter and myeloperoxidase (MPO) as an indirect index of neutrophil infiltration were studied. Results: Blood urea, creatinine, and TNF-α levels were increased, renal tissue MDA and MPO levels were elevated and GSH levels were decreased in both of the pyelonephritic (acute and chronic) rats. All of these parameters and elevation of LDH at the late phase were all reversed to normal levels by OT treatment. Conclusion: OT alleviates oxidant renal injury in pyelonephritic rats by its anti-oxidant actions and by preventing free radical damaging cascades that involves excessive infiltration of neutrophils.
Keywords: Eschericia coli; Oxytocin; Glutathione; Lipid peroxidation; Myeloperoxidase; TNF-α;

Time-dependent expression of renal vaso-regulatory molecules in LPS-induced endotoxemia in rat by Naoto Yamaguchi; Subrina Jesmin; Sohel Zaedi; Nobutake Shimojo; Seiji Maeda; Satoshi Gando; Akio Koyama; Takashi Miyauchi (2258-2270).
To elucidate roles of microvascular factors in the pathogenesis of renal complications during endotoxemia, that is characterized by renal vasoconstriction and systemic hypotension/generalized non-renal vasodilation, we profile the expression pattern and time-course of three key vaso-regulators, namely endothelin (ET)-1, nitric oxide (NO), and angiotensin II (Ang II). We hypothesize that disruption of the overall balance between vasodilatation and vasoconstriction in the kidney, during the early phase of sepsis, contribute to its (kidney) predisposition to acute renal failure. Adult male Wistar rats were rendered endotoxemic at different time points (1, 3, 6 and 10 h) by a single i.p. injection of lipopolysaccharide (LPS) (15 mg/kg) dissolved in saline. Control group was injected vehicle only (saline). Both systolic and diastolic blood pressures significantly decreased at different time points after LPS administration. Surprisingly, renal histopathological evaluation showed no remarkable changes in LPS-induced endotoxemia. However, overall, levels of the vaso-regulators and, where applicable, their respective receptors were upregulated: (1) plasma ET-1 increased 25-fold and peaked, as renal ET-1 mRNA, at 3 h; renal ET-1 protein and its receptors, ET type A (ETA) receptor (vasoconstrictive) and ET type B (ETB) receptor (vasodilatatory) increased in a time-dependent fashion, (2) Ang II increased by 53% compared to control, peaking at 6 h. However, while levels of Ang II type 1 (AT1) receptor increased over time after LPS injection, those of Ang II type 2 (AT2) receptor were downregulated, (3) data of NO system (NO-NOS), the key vasodilator, were the most intriguing. Whereas levels of renal NO increased time-dependently following LPS administration, with a 2240-fold increase in renal iNOS expression, levels of eNOS, were almost unchanged. In conclusion, the present study overall reveals intriguing and complex dynamics between levels of vasoconstrictors and vasodilators during the early phase of LPS-induced endotoxemia. These shifts in molecular expressions are likely triggered by compensatory mechanisms aimed at counteracting the undesirable and dominant effects of one group of vaso-regulatory moiety over the other.
Keywords: Sepsis; Kidney; Endothelin; Nitric oxide; Angiotensin II;

Human VIP-α: A long-acting, biocompatible and biodegradable peptide nanomedicine for essential hypertension by Hayat Önyüksel; Florence Séjourné; Hideyuki Suzuki; Israel Rubinstein (2271-2275).
We have previously shown that self-association of human vasoactive intestinal peptide with sterically stabilized liposomes (VIP-α) alters peptide conformation from random coil in aqueous solution to α-helix. This, in turn, protects the peptide from hydrolysis and amplifies and prolongs its bioactivity. The purpose of this study was to determine whether a single, intravenous injection of low-dose human VIP-α normalizes systemic arterial pressure in anesthetized spontaneously hypertensive hamsters for a prolonged period of time in a selective fashion. We found that intravenous injection of human VIP-α, VIP alone (each, 1.0 nmol) and empty liposomes had no significant effects on mean arterial pressure (MAP) in normotensive hamsters. By contrast, human VIP-α (0.01–1.0 nmol) evoked a significant concentration-dependent decrease in MAP to the normative range in spontaneously hypertensive hamsters that lasted throughout the observation period (6 h; p  < 0.05). VIP alone and empty liposomes had no significant effects on MAP in these animals. We conclude that a single, low-dose intravenous injection of human VIP-α normalizes systemic arterial pressure in spontaneously hypertensive hamsters for a prolonged period of time in a selective fashion. We suggest that human VIP-α should be further developed as a long-acting, biocompatible and biodegradable peptide nanomedicine for essential hypertension.
Keywords: Microcirculation; Vasomotor tone; Amphipathic peptide; Conformation; DSPE-PEG; Sterically stabilized liposomes; Hamster;

T4-induced cardiac hypertrophy disrupts cyclic GMP mediated responses to brain natriuretic peptide in rabbit myocardium by Elizabeth Katz; Qihang Zhang; Harvey R. Weiss; Peter M. Scholz (2276-2283).
Brain natriuretic peptide (BNP) affects the regulation of myocardial metabolism through the production of cGMP and these effects may be altered by cardiac hypertrophy. We tested the hypothesis that BNP would cause decreased metabolism and function in the heart and cardiac myocytes by increasing cGMP and that these effects would be disrupted after thyroxine-induced cardiac hypertrophy (T4). Open-chest control and T4 rabbits were instrumented to determine local effects of epicardial BNP (10−3  M). Function of isolated cardiac myocytes was examined with BNP (10−8–10−7  M) with or without KT5823 (10−6  M, cGMP protein kinase inhibitor). Cyclic GMP levels were measured in myocytes. In open-chest controls, O2 consumption was reduced in the BNP area of the subepicardium (6.6 ± 1.3 ml O2/min/100 g versus 8.9 ± 1.4 ml O2/min/100 g) and subendocardium (9.4 ± 1.3 versus 11.3 ± 0.99). In T4 animals, functional and metabolic rates were higher than controls, but there was no difference between BNP-treated and untreated areas. In isolated control myocytes, BNP (10−7  M) reduced percent shortening (PSH) from 6.5 ± 0.6 to 4.3 ± 0.4%. With KT5823 there was no effect of BNP on PSH. In T4 myocytes, BNP had no effect on PSH. In control myocytes, BNP caused cGMP levels to rise from 279 ± 8 to 584 ± 14 fmol/105 cells. In T4 myocytes, baseline cGMP levels were lower (117 ± 2 l) and were not significantly increased by BNP. Thus, BNP caused decreased metabolism and function while increasing cGMP in control. These effects were lost after T4 due to lack of cGMP production. These data indicated that the effects of BNP on heart function operated through a cGMP-dependent mechanism, and that this mechanism was disrupted in T4-induced cardiac hypertrophy.
Keywords: Natriuretic peptides; Cardiac hypertrophy; Myocardial oxygen consumption; Cardiac myocyte function; Cyclic GMP;

Hemopressin, a hemoglobin fragment, dilates the rat systemic vascular bed through release of nitric oxide by Howard Lippton; Bin Lin; Bulent Gumusel; Norman Witriol; Anatolio Wasserman; Martha Knight (2284-2288).
The present study was undertaken to investigate the effects of intravenous (i.v.) administration of rat hemopressin (rHP), 30–1000 μg/kg, on systemic arterial pressure (SAP), cardiac output (CO) and systemic vascular resistance (SVR) in the anesthetized rat. Bolus i.v. injections of rHP produced mild decreases in SAP that were dose-dependent. Since CO was not altered, the decreases in SAP reflect reductions in SVR. The systemic vasodilator response to rHP was not subject to tachyphylaxis. The systemic vasodilator response to rHP was abolished by l-nitro-arginine methylester (l-NAME) but was not altered by meclofenamate. In addition, rHP lacked direct contractile and relaxant activity on isolated rat aortic rings (AA) and pulmonary arterial rings (PA). The present data suggest rHP dilates the rat systemic vascular bed through the endogenous release of nitric oxide (NO) independent of the formation of cyclooxygenase products including prostacyclin. It is possible rHP acts as an endogenous vasodilator substance to regulate local blood flow during clinical states of altered red cell turnover, microvascular disease and hemolysis.
Keywords: Hemopressin; Systemic arterial pressure; Systemic vascular resistance; Nitric oxide; Rat; Prostacyclin; Hemoglobin products; Angiotensin converting enzyme;

The action of urocortins on body temperature in rats by G. Telegdy; A. Adamik; G. Tóth (2289-2294).
The actions of individual urocortins on colon temperature were studied in rats. Urocortin 1, urocortin 2 or urocortin 3 was injected into the lateral brain ventricle in conscious rats and the colon temperature was measured at different times following injection, for up to 6 h. In order to study the possible role of prostaglandins, the animals were treated with either a urocortin together with the pyrazolone derivative noraminophenazone to inhibit the action of cyclooxygenase in initiating hyperthermia, or with noraminophenazone 30 min following urocortin administration to act on existing hyperthermia. Noraminophenazone was administered intramuscularly in a dose of 50 mg/kg. Urocortin 1 caused a dose-related increase in colon temperature, maximal action being observed at a dose of 2 μg with the maximal increase in body temperature at 4 h. Noraminophenazone prevented the urocortin-induced increase in colon temperature and attenuated the already existing elevated body temperature. Somewhat similar action was observed with urocortin 2. However, following treatment with 0.5 or 1.0 μg urocortin 2, the action was already over at 2 h, whereas 2 μg increased the colon temperature steadily, with a maximum at 4 h. Noraminophenazone blocked or diminished the action of urocortin 2. Urocortin 3 in a dose of 1 μg was the most effective in increasing the colon temperature; the maximal effect was observed at 2 h. Noraminophenazone blocked the development of urocortin 3-induced hyperthermia, or attenuated it when the hyperthermia was already present. The results demonstrated that urocortin 1, 2 or 3 caused increases in body temperature when injected into the lateral brain ventricle, though the optimal dose and the duration of hyperthermia differed for the individual urocortins. The cyclooxygenase inhibitor blocked or diminished the action of these urocortins, indicating the involvement of prostaglandins in urocortin-induced hyperthermia.
Keywords: Urocortin 1; Urocortin 2; Urocortin 3; Temperature regulation;

Effect of alpha-melanotropin hormone on serum levels of luteinizing hormone and progesterone in experimental rat autoimmune oophoritis by Victoria Berberian; Silvina Sánchez; Mariela Sánchez-Borzone; Andrés M. Attademo; Mercedes Lasaga; Maria Ester Celis (2295-2299).
We studied the effect of alpha-melanotropin hormone (α-MSH) on experimental autoimmune oophoritis (EAO), an inflammatory process induced in female rats. During proestrus, serum levels of LH and progesterone in rats with EAO were higher than those of control rats. However, administration of α-MSH to these rats decreased the levels of LH. Similarly, in the following diestrus, rats with EAO had high levels of LH but treatment with α-MSH decreased the levels to diestrus 2 control values. Treatment with α-MSH also reduced the LH levels of control rats in diestrus 2 compared to untreated controls. However, α-MSH treatment had no effect on progesterone levels of either control or rats with EAO. Thus, although α-MSH induced notable changes in levels of LH, this decrease was unable to block the illness.
Keywords: Experimental autoimmune oophoritis; Hormones; α-MSH effect; Inflammatory process; icv injections;

Melanin-concentrating hormone (MCH) reverts the behavioral effects induced by inescapable stress by Valeria P. Carlini; Helgi B. Schiöth; Susana R. de Barioglio (2300-2306).
The aim of this work was to investigate if MCH modifies the feeding and freezing responses in rats exposed to stressful stimuli. We used a basic version of contextual fear, where one group of rats were placed in a novel environment and two different groups were exposed to footshock paradigms, one of them escapable and the other one inescapable. At the end of each treatment, freezing and feeding were measured. Only the animals exposed to inescapable footshock paradigm showed significant increase in the food intake and freezing behavior in comparison to the control animals. The MCH administration (intra-hippocampal or intra-amygdaline) reverted these effects elicited by inescapable footshock. Results presented in this paper lead us to the assumption that the anxiolytic effect of the peptide is responsible for the reversion of the IS effects.
Keywords: Melanin-concentrating hormone (MCH); Escapable footshock; Inescapable footshock; Food intake; Freezing behavior;

Opposite effects of low and high doses of the gastrin-releasing peptide receptor antagonist RC-3095 on memory consolidation in the hippocampus: Possible involvement of the GABAergic system by Andrea dos Santos Dantas; Tatiana Luft; João Antônio Pêgas Henriques; Gilberto Schwartsmann; Rafael Roesler (2307-2312).
Although the gastrin-releasing peptide receptor (GRPR) has recently emerged as a system importantly involved in regulating memory formation, the role of hippocampal GRPRs in memory remains controversial. The present study examined the effects of GRPR antagonism on memory consolidation in area CA1 of the hippocampus. Male Wistar rats received bilateral infusions of the GRPR antagonist [D-Tpi6, Leu13 psi(CH2NH)-Leu14] bombesin (6-14) (RC-3095; 1, 3, or 10 μg/side) into the dorsal hippocampus immediately after inhibitory avoidance (IA) training. RC-3095 at 1 μg impaired, whereas the dose of 10 μg enhanced, 24-h IA retention. A second experiment showed that the RC-3095-induced enhancement of memory consolidation was prevented by pretraining infusion of an otherwise ineffective dose of the gamma-aminobutyric acid type A (GABAA) receptor agonist muscimol. The results indicate that high doses of GRPR antagonists can induce enhancement of memory consolidation in the hippocampus. In addition, the memory-enhancing effect of GRPR antagonists might be mediated by inhibition of GABAergic transmission.
Keywords: Bombesin-like peptides; Gastrin-releasing peptide receptor; RC-3095; Hippocampus; Memory consolidation;

Electroacupuncture suppresses expression of gastric ghrelin and hypothalamic NPY in chronic food restricted rats by Nan Tian; Fei Wang; De-Run Tian; Yuan Zou; Shi-Wei Wang; Li-Li Guan; Yu-Shun Shi; Jaw-Kang Chang; Jun Yang; Ji-Sheng Han (2313-2320).
Electroacupuncture (EA) has been reported to reduce body weight in overweight subjects in clinical practice, as well as in rats and mice with diet-induced obesity. In the present study, this effect of EA was tested in lean rats subjected to long-term food restriction (FR, food was offered only 1 h/day). Two hertz EA administered once every other day produced a further reduction in body weight in FR rats. Exploration of the mechanism involved revealed significant downregulation of the orexigenic peptides: ghrelin in the stomach, and neuropeptide Y (NPY) but not Agouti-related peptide (AgRP) in the hypothalamus, which was in line with the reduction in food intake in rats receiving EA stimulation as compared with those receiving restraint only. Uncoupling protein 3 (UCP3), involved in accelerating energy expenditure, was not significantly altered. These results suggest that the EA-induced body weight reduction was due mainly to a decrease in food intake rather than an increase in energy expenditure. A reduction in the orexigenic peptides ghrelin and NPY may be involved in the underlying mechanism.
Keywords: Electroacupuncture; Ghrelin; Neuropeptide Y; Agouti-related peptide; Uncoupling protein 3;

Regulation of food intake by acyl and des-acyl ghrelins in the goldfish by Kouhei Matsuda; Tohru Miura; Hiroyuki Kaiya; Keisuke Maruyama; Sei-Ichi Shimakura; Minoru Uchiyama; Kenji Kangawa; Seiji Shioda (2321-2325).
Our recent research has indicated that intracerebroventricular (ICV) and intraperitoneal (IP) administration of n-octanoic acid-modified ghrelin (acyl ghrelin) stimulates food intake and locomotor activity in the goldfish. The manner in which peripherally administered acyl ghrelin regulates food intake, however, remains unclear. In contrast to acyl ghrelin, non-acylated ghrelin (des-acyl ghrelin) does not exert an orexigenic action or induce hypermotility. To this extent, the biological role of des-acyl ghrelin in fish is unknown. Given the possible involvement of afferent pathways in mediating the effects of acyl ghrelin, as is known to occur in rodents, we examined the effect of capsaicin, a neurotoxin which destroys primary sensory (vagal and splanchnic) afferents, on the orexigenic activity induced by IP-injected acyl ghrelin. Pretreatment with IP-injected capsaicin (0.16 μmol/g body weight (BW)) cancelled the orexigenic action of IP-injected acyl ghrelin (8 pmol/g BW), although IP-injected capsaicin alone did not affect food intake. The effect of des-acyl ghrelin on the orexigenic action of acyl ghrelin in the goldfish was also investigated. The ICV and IP injection of des-acyl ghrelin at doses 3–10 times higher than that of acyl ghrelin suppressed the orexigenic action of ICV- and IP-injected acyl ghrelin (doses of 1 and 8 pmol/g BW). In contrast, injection of des-acyl ghrelin alone did not show any inhibitory effect on food intake. These results suggest that, as is seen in rodents, circulating acyl ghrelin derived from peripheral tissues acts via primary sensory afferent pathways on feeding centers in the brain. The results also show that des-acyl ghrelin inhibits acyl ghrelin-induced orexigenic activity in goldfish.
Keywords: Goldfish; Acyl ghrelin; Des-acyl ghrelin; Capsaicin; Feeding behavior;

Sex difference in body weight gain and leptin signaling in hypocretin/orexin deficient mouse models by Nobuhiro Fujiki; Yasushi Yoshida; Shengwen Zhang; Takeshi Sakurai; Masashi Yanagisawa; Seiji Nishino (2326-2331).
Recent studies in human and animal models of narcolepsy have suggested that obesity in narcolepsy may be due to deficiency of hypocretin signaling, and is also under the influence of environmental factors and the genetic background. In the current study, using two hypocretin/orexin deficient narcoleptic mouse models (i.e. preproorexin knockout (KO) and orexin/ataxin-3 transgenic (TG) mice) with cross-sectional assessments, we have further analyzed factors affecting obesity. We found that both KO and TG narcoleptic mice with mixed genetic backgrounds (N4–5, 93.75–96.88% genetic composition of C57BL/6) tended to be heavier than wild type (WT) mice of 100–200 days old. The body weight of heterozygous mice was intermediate between those of KO and WT mice. Obesity was more prominent in females in both KO and TG narcoleptic mice and was associated with higher serum leptin levels, suggesting a partial leptin resistance. Obesity is less prominent in the congenic TG narcoleptic mice, but is still evident in females. Our results confirmed that hypocretin/orexin ligand deficiency is one of the critical factors for the obese tendency in narcolepsy. However, multiple factors are also likely to affect this phenotype, and a sex difference specific alteration of leptin–hypocretin signaling may be involved.
Keywords: Narcolepsy; Obesity; Hypocretin; Orexin; Leptin; Sex difference;

We have tested the hypothesis that sustained leptin action in the hypothalamus alone can engender and maintain euglycemia in wild type mice and in two monogenic diabetic models, the insulin-deficient nonobese Akita mice and the hyperinsulinemic leptin-deficient obese, ob/ob mice. A single intracerebroventricular injection of recombinant adeno-associated virus vector encoding leptin (rAAV-lep) enhanced leptin transgene expression in the hypothalamus without any evidence of leptin leakage to the peripheral circulation, and promptly reinstated euglycemia that persisted along with severe insulinopenia in all three genotypes through the 7-week period of observation. A comparative evaluation of known etiologic factors of hyperglycemia showed that this long-term benefit on glucose homeostasis was not due to diminished energy consumption, weight and adiposity, but was conferred by at least two mechanisms operating simultaneously, enhanced glucose metabolism to meet the demand for the rAAV-lep induced increased non-shivering thermogenesis mediated by brown adipose tissue and insulin hypersensitivity. These findings endorse the hypothesis that increased leptin action locally in the hypothalamus can impose euglycemia independent of pancreatic insulin, and central leptin reinforcement may serve as a newer adjunct therapy to treat type 1 and type 2 diabetes.
Keywords: Gene therapy; Leptin; Hypothalamus; Euglycemia;

Long-term administration of PACAP receptor antagonist, PACAP(6-27), impairs glucose tolerance and insulin sensitivity in obese diabetic ob/ob mice by Brian D. Green; Nigel Irwin; Roslyn S. Cassidy; Victor A. Gault; Peter R. Flatt (2343-2349).
Pituitary adenylate cyclase-activating peptide (PACAP) is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the PACAP receptor antagonist, PACAP(6-27) to evaluate the role of endogenous PACAP in genetic obesity-related diabetes and related metabolic abnormalities using ob/ob mice. Acute in vivo antagonistic potency of PACAP(6-27) was confirmed in ob/ob mice by blockade of the insulin-releasing action but not hyperglycaemia. In longer-term studies, ob/ob mice were given once daily injections of PACAP(6-27) or vehicle for 14 days. Feeding activity, body weight, basal plasma glucose and plasma insulin concentrations were not significantly affected by chronic PACAP(6-27) treatment. However, PACAP(6-27) treatment impaired glucose tolerance, insulin sensitivity and the glycaemic response to feeding. Plasma glucagon and lipids were unchanged. These observations indicate a role of endogenous PACAP for normal glucose homeostasis, but indicate a minor involvement in the regulation of insulin secretion in ob/ob mice.
Keywords: PACAP; Diabetes; Insulin; Glucose; Antagonist;

Apidaecins (apidaecin-type peptides) refer to a series of small, proline-rich (Pro-rich), 18- to 20-residue peptides produced by insects. They are the largest group of Pro-rich antimicrobial peptides (AMPs) known to date. Structurally, apidaecins consist of two regions, the conserved (constant) region, responsible for the general antibacterial capacity, and the variable region, responsible for the antibacterial spectrum. The small, gene-encoded and unmodified apidaecins are predominantly active against many Gram-negative bacteria by special antibacterial mechanisms. The mechanism of action by which apidaecins kill bacteria involves an initial non-specific binding of the peptides to an outer membrane (OM) component. This binding is followed by invasion of the periplasmic space, and by a specific and essentially irreversible combination with a receptor/docking molecule that may be a component of a permease-type transporter system on inner membrane (IM). In the final step, the peptide is translocated into the interior of the cell where it meets its ultimate target. Evidence that apidaecins are non-toxic for human and animal cells is a prerequisite for using them as novel antibiotic drugs. This review presents the biodiversity, structure–function relationships, and mechanism of action of apidaecins.
Keywords: Apidaecins; Biodiversity; Structure–function relationships; Antibacterial activity; Action mechanism;