Peptides (v.26, #11)

We report long time scale simulations of the 18-residue helical antimicrobial peptide ovispirin-1 and its analogs novispirin-G10 and novispirin-T7 in SDS micelles. The SDS micelle serves as an economical and effective model for a cellular membrane. Ovispirin, which is initially placed along a micelle diameter, diffuses out to the water–SDS interface and stabilizes to an interface-bound steady state in 16.35 ns of simulation. The final conformation, orientation, and the structure of ovispirin are in good agreement with the experimentally observed properties of the peptide in presence of lipid bilayers. The simulation succeeds in capturing subtle differences of the membrane-bound peptide structure as predicted by solid state NMR. The novispirins also undergo identical diffusion patterns and similar final conformations. Although the final interface-bound states are similar, the simulations illuminate the structural and binding properties of the mutant peptides which make them less toxic compared to ovispirin. Based on previous data and the current simulations, we propose that introduction of a bend/hinge at the center of helical antimicrobial peptides (containing a specific C-terminal motif), without disrupting the helicity of the peptides might attenuate host-cell toxicity as well as improve membrane binding properties to bacterial cellular envelopes.
Keywords: Ovispirin; SDS micelle; Molecular dynamics simulations; Antimicrobial peptides; Peptide–membrane interaction;

PTP7 is a 13-amino acid residue peptide designed from gaegurin 6, an antimicrobial peptide isolated from skin secretions of Rana rugosa. In order to examine the effect of hydrophobicity on antimicrobial activity, a series of PTP7 derivatives were constructed and analyzed the activity against bacteria and artificial membrane. We found that the mean hydrophobicity by simple summation of hydrophobicity of each constituent amino acid did not necessarily describe the hydrophobic property of antimicrobial peptides. The mean hydrophobicity did not show close correlation with the observed hydrophobicity by measuring reverse phase high performance liquid chromatography (RP HPLC) retention time. The observed hydrophobicity represented as RP HPLC retention time correlated well with the activity against artificial membrane and Gram positive bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, rather than mean hydrophobicity. However, antimicrobial activity against Gram negative bacteria, such as Escherichia coli, did not show correlation with RP HPLC retention time. These data indicate that the RP HPLC retention time should be exploited rather than the mean hydrophobicity in the analysis of the relationship between hydrophobicity and antimicrobial activity.
Keywords: RP HPLC retention time; Hydrophobicity; Antimicrobial peptide; PTP peptide; Gaegurin;

Salmonella enterica serovar Typhi (S. Typhi) is an important pathogen which infects humans exclusively and causes typhoid or enteric fever. Recently it has been discovered that type IVB pili, encoded by the S. Typhi pil operon located in the major pathogenicity island, may be important in the pathogenesis of epidemic enteric fever. To further investigate the roles of type IVB pili of S. Typhi, a 12-mer peptide (RQERSSLSKPVV), binding to the structural protein PilS of the type IVB pili of S. Typhi, was isolated with a ribosome display system. This peptide was designated as peptide R. We found that peptide R inhibited adhesion to/invasion of human monocytic THP-1 cells by piliated S. Typhi bacteria, but had no effects on nonpiliated S. Typhi bacteria. A random 12-mer peptide, of size and solubility equal to peptide R, served as a control on the specificity of peptide R. The specific interaction and binding equilibrium between the 12-mer peptide R and PilS protein was determined by isothermal titration calorimetry (ITC) and a binding constant K a determined to be between 0.4 × 105 and 2.2 × 105  L mol−1. Our findings suggest that the type IV pili-binding peptide R holds potential as an antibacterial peptide effective against S. Typhi infections, both in terms of prevention and therapeutic treatment. The data further provide insights into the understanding of the pathogenic roles of the type IVB pili of S. Typhi.
Keywords: Ribosome display; Salmonella Typhi; Type IVB pili; Binding peptide;

Diversity of wheat anti-microbial peptides by Tsezi A. Egorov; Tatyana I. Odintsova; Vitaliy A. Pukhalsky; Eugene V. Grishin (2064-2073).
From seeds of Triticum kiharae Dorof. et Migusch., 24 novel anti-microbial peptides were isolated and characterized by a combination of three-step HPLC (affinity, size-exclusion and reversed-phase) with matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and Edman degradation. Based on sequence similarity and cysteine motifs, partially sequenced peptides were assigned to 7 families: defensins, thionins, lipid-transfer proteins, hevein-like peptides, knottin-like peptides, glycine-rich peptides, and MBP-1 homologs. A novel subfamily of defensins consisting of 6 peptides and a new family of glycine-rich (8 peptides with different repeat motifs) were identified. Three 6-cysteine knottin-like peptides represented by N- and C-terminally truncated variants revealed no sequence homology to any known plant anti-microbial peptides. A new 8-cysteine hevein-like peptide and three 4-cysteine peptides homologous to MBP-1 from maize were isolated. This is the first communication on the occurrence of nearly all families of plant anti-microbial peptides in a single species.
Keywords: Triticum kiharae Dorof. et Migusch.; Wheat; Anti-microbial peptides; Glycine-rich peptides; Defensins; Thionins; Lipid-transfer proteins; Hevein-like peptides; Knottin-like peptides; MBP-1 homologs;

Short acidic peptides isolated from wheat sprout chromatin and involved in the control of cell proliferation by Isabella Calzuola; Flavio Giavarini; Paola Sassi; Leonardo De Angelis; Gian Luigi Gianfranceschi; Valeria Marsili (2074-2085).
Low molecular weight peptides were isolated from the chromatin of wheat sprouts. Following gel filtration the peptide fraction shows a sharp inhibiting activity on the growth of HeLa cancer cells. Infrared (IR) spectroscopy and mass spectrometry have been utilized to characterize the wheat sprout peptides in an attempt to recognize the peptide sequence involved in the control of cell growth. The quantitative presence of a peptide with MH+  = 572 appears proportional to the cell growth inhibition activity. This compound has been subjected to extensive mass spectrometry analysis. The automatic computational analysis of the ions of second, third and fourth generations indicate a peptide sequence, AcHis―Asp―Ser―Glu―, that binds at the C-terminal a molecule of ethanolamine. Moreover, the results show that some sequences of the wheat sprout peptide family are present in the peptide fractions isolated from several other tissues, thus supporting the hypothesis of ubiquitous regulatory peptides.
Keywords: Chromatin peptides; Cell growth; Peptide infrared spectroscopy; Peptide mass spectrometry; Peptide–ethanolamine complex;

An anti-fungal peptide designated as lunatusin, with a molecular mass around 7 kDa, was purified from the seeds of Chinese lima bean (Phaseolus lunatus L.). The peptide was isolated using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. Lunatusin exerted an anti-fungal activity toward fungal species such as Fusarium oxysporum, Mycosphaerella arachidicola and Botrytis cinerea, and an antibacterial action on, Bacillus megaterium, Bacillus subtilis, Proteus vulgaris and Mycobacterium phlei. It also inhibited proliferation in the breast cancer cell line MCF-7. Lunatusin reduced the activity of HIV-1 reverse transcriptase and it also inhibited translation in a cell-free rabbit reticulocyte lysate system. Its anti-fungal activity was retained after incubation with trypsin. Lunatusin elicited a mitogenic response from mouse splenocytes.
Keywords: Lima bean; Anti-fungal peptides; HIV-1 reverse transcriptase;

Effect of amino acid substitutions on the candidacidal activity of LFampin 265–284 by Marieke I.A. van der Kraan; Christel van der Made; Kamran Nazmi; Wim van‘t Hof; Jasper Groenink; Enno C.I. Veerman; Jan G.M. Bolscher; Arie V. Nieuw Amerongen (2093-2097).
LFampin 265–284, derived from bovine lactoferrin, has broad-spectrum antimicrobial activity against the yeast Candida albicans and several Gram-positive and Gram-negative bacteria. A glycine substitution scan was used to identify residues that are important for its candidacidal activity. Each single substitution of a positively charged residue led to considerable reduction in candidacidal activity, for each residue to a different extent. Substitution within the helix-facilitating N-terminal sequence DLIW had less severe effect; substitution of Ile and Trp led to a somewhat reduced potency. No substantial effects were found on the propensity to adopt a helical structure or to bind to C. albicans cells.
Keywords: Bovine lactoferrin; Lactoferrampin; LFampin; antimicrobial peptide; Candida albicans;

Pleurostrin, an antifungal peptide from the oyster mushroom by K.T. Chu; Lixin Xia; T.B. Ng (2098-2103).
A 7 kDa peptide, with inhibitory activity on mycelial growth in the fungi Fusaerium oxysporum, Mycosphaerella arachidicola and Physalospora piricola, was isolated from fresh fruiting bodies of the oyster mushroom. The isolation procedure entailed extraction with an aqueous buffer, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 75. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel. It demonstrated an N-terminal sequence different from known antifungal proteins and peptides.
Keywords: Antifungal peptide; Oyster mushroom; Purification;

Antimicrobial and cytolytic properties of the frog skin peptide, kassinatuerin-1 and its l- and d-lysine-substituted derivatives by J. Michael Conlon; Bency Abraham; Sehamuddin Galadari; Floyd C. Knoop; Agnes Sonnevend; Tibor Pál (2104-2110).
Kassinatuerin-1, a 21-amino-acid C-terminally α-amidated peptide first isolated from the skin of the African frog Kassina senegalensis, adopts an amphipathic α-helical conformation in a membrane-mimetic solvent (50% trifluoroethanol) and shows broad-spectrum antimicrobial activity. However, its therapeutic potential is limited by its relatively high cytolytic activity against mammalian cells. The antimicrobial and cytolytic properties of a peptide are determined by an interaction between cationicity, hydrophobicity, α-helicity and amphipathicity. Replacement of the C-terminal α-amide group in kassinatuerin-1 by carboxylic acid decreased both cationicity and α-helicity, resulting in an analog with decreased potency against Escherichia coli (4-fold) and Staphylococcus aureus (16-fold). Low cytolytic activities against human erythrocytes (LD50 > 400 μM) and L929 fibroblasts (LD50 = 105 μM) were also observed. Increasing cationicity, while maintaining amphipathic α-helical character, by progressively substituting Gly7, Ser18, and Asp19 on the hydrophilic face of the α-helix with l-lysine, increased antimicrobial potency against S. aureus and Candida albicans (up to 4-fold) but also increased hemolytic and cytolytic activities. In contrast, analogs with d-lysine at positions 7, 18 and 19 retained activity against Gram-negative bacteria but displayed reduced hemolytic and cytolytic activities. For example, the carboxylic acid derivative of [d-Lys7, d-Lys18, d-Lys19]kassinatuerin-1 was active (minimum inhibitory concentration (MIC) = 6–12.5 μM) against a range of strongly antibiotic-resistant strains of E. coli but showed no detectable hemolytic activity at 400 μM and was 4-fold less cytolyic than kassinatuerin-1. However, the reduction in α-helicity produced by the d-amino acid substitutions resulted in analogs with reduced potencies against Gram-positive bacteria and against C. albicans.
Keywords: Antimicrobial peptide; Structure-activity; Kassinatuerin-1; Cytolysis; Hemolysis;

In vitro activity of amphibian peptides alone and in combination with antimicrobial agents against multidrug-resistant pathogens isolated from surgical wound infection by Andrea Giacometti; Oscar Cirioni; Wojciech Kamysz; Carmela Silvestri; Alberto Licci; Alessandro Riva; Jerzy Łukasiak; Giorgio Scalise (2111-2116).
The in vitro activities of three amphibian peptides magainin II amide, citropin 1.1 and temporin A alone and in combination with eight clinically used antimicrobial agents (imipenem, ceftazidime, clarithromycin, vancomycin, amikacin, polymyxin E, ciprofloxacin and linezolid) were investigated against several multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus strains isolated from surgical wound infections. Antimicrobial activities were measured by MIC, MBC and time-kill studies. P. aeruginosa strains were more susceptible to magainin II amide and less susceptible to temporin A. S. aureus isolates were highly susceptible to temporin A and citropin 1.1. The combination studies showed synergy between citropin 1.1 and clarithromycin. Magainin II amide and temporin A showed synergism with imipenem and ceftazidime. Finally, all peptides showed synergistic effects with polymyxin E.These results provide evidence for the potential use of these antimicrobial peptides in the topical or systemic treatment of surgical wound infections.
Keywords: Amphibian peptides; Antibiotics; Wound infection; Resistance; Susceptibility;

Isolation and structural characterization of novel Rugosin A-like insulinotropic peptide from the skin secretions of Rana saharica frog by Lamin Marenah; Peter R. Flatt; David F. Orr; Chris Shaw; Yasser H.A. Abdel-Wahab (2117-2123).
Skin secretions of Rana saharica were evaluated for the isolation and characterization of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11cells, fractions 36–43, 46–54 and 57–63 showed the significant 2–8-fold increase in insulin-releasing activity (P  < 0.001) compared with 5.6 mM of glucose alone. A pool of fractions 36–43 was subsequently rechromatographed to 28 homogenous peaks out of which 7 were capable of subsequent 1.5–3-fold increase in insulin release (P  < 0.001). Structural analysis of the non-toxic peptides with greatest insulin-releasing activity was performed by mass spectrometry and Edman degradation. Mass spectrometry analysis of two peaks indicated the molecular masses of 1892.6 and 2930.8 Da. The sequence of the 1892.6-Da peptide was determined as KGAAKGLLEVASCKLSKSC, which has 68% homology with Rugosin A originally isolated from the skin secretion of Rana rugosa. A partial N-terminal sequence was determined for the 2930.8-Da peptide as AVITGACERDVQCGGGTCCAVSLI…. These data indicate that the skin secretions of Rana saharica frogs contain novel peptides with insulin-releasing activity.
Keywords: Rana saharica; Insulin secretion; Peptides;

We used a combinatorial chemical approach to identify novel agonists for the endogenous melanocortin receptor expressed in Xenopus laevis melanophores. A random one-bead one-compound hexapeptide library was screened to detect new molecules able to induce pigment dispersion in melanophores. Our approach led to the discovery of seven related novel peptides able to stimulate pigment dispersion with EC50 in the range of 0.1–10 μM. Their action was inhibited by the amphibian melanocortin receptor antagonist dWRL. These novel peptides share no significant sequence homology with known melanocortins. This study may aid in the understanding of the chemical interaction between the melanocortin receptors and their ligands.
Keywords: MSH; Melanocortin; G-protein coupled receptor; Melanophore; Peptide; Screening;

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis by Patrick Bulau; Atsuro Okuno; Elke Thome; Tina Schmitz; Jasna Peter-Katalinic; Rainer Keller (2129-2136).
The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue (∼99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.
Keywords: Molt-inhibiting hormone; Crustacean; Sinus gland; Neuropeptide; Mass spectrometry;

Crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs) are produced during the proteolytic processing of CHH preprohormones. Currently, the physiological roles played by CPRPs are unknown. Due to their large size, direct mass spectrometric sequencing of intact CPRPs is difficult. Here, we describe a novel strategy for sequencing Cancer productus CPRPs directly from a tissue extract using nanoflow liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. Four novel CPRPs were characterized with the aid of MS/MS de novo sequencing of 27 truncated CPRP peptides. Extensive modifications (methionine oxidation and carboxy-terminal methylation) were identified in both the full-length and truncated peptides. To investigate the origin of the modifications and truncations, a full-length CPRP was synthesized and subjected to the same storage and extraction protocols used for the characterization of the native peptides. Here, some methionine oxidation was seen, however, no methylation or truncation was evident suggesting much of the chemical complexity seen in the native CPRPs is unlikely due to a sample preparation artifact. Collectively, our study represents the most complete characterization of CPRPs to date and provides a foundation for future investigation of CPRP function in C. productus.
Keywords: Capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry; Neuropeptide; Peptide sequencing; Sinus gland; Crustacean hyperglycemic hormone precursor-related peptide (CPRP); Cancer productus;

Mass spectrometric assignment of Leu/Ile in neuropeptides from single neurohemal organ preparations of insects by Ronald J. Nachman; William K. Russell; Geoffrey M. Coast; David H. Russell; Reinhard Predel (2151-2156).
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF–TOF) tandem mass spectrometry has been applied for the first time on an insect/arthropod target, focusing on PVK/CAP2b neuropeptides in the housefly Musca domestica and flesh fly Neobellieria bullata. The peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The confirmation of side-chain fragments which allows assignment of Leu/Ile even from samples as small as neurohemal organs will greatly accelerate the identification of novel neuropeptides that are implicated in the regulation of critical physiological processes in insects. The unnatural Ile analog is 4.5 times more active than the native Leu sequence in a housefly Malpighian tubule fluid secretion assay, which reinforces the caveat that potency values in a biological assay cannot be relied upon to predict the native sequence.
Keywords: MALDI-TOF/TOF mass spectrometry; Insect; Neuropeptide; Periviscerokinin; CAP2b; Musca domestica; Neobellieria bullata; Peptidomics;

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista by Bibiana M. Souza; Maria A. Mendes; Lucilene D. Santos; Maurício R. Marques; Lilian M.M. César; Roberta N.A. Almeida; Fernando C. Pagnocca; Katsuhiro Konno; Mario S. Palma (2157-2164).
Keywords: Polybia paulista; Hymenoptera insect; Polycationic peptide; Wasp venom; Mastoparans; Chemotactic peptides;

The phosphorylation of phospholipase C-gamma1, Raf-1, MEK, and ERK1/2 induced by a conserved retroviral peptide by Tian xue Fan; Noorbibi K. Day; Voravich Luangwedchakarn; Yenhui Chang; Susumu Ikehara; Danica L. Lerner; Soichi Haraguchi (2165-2174).
A synthetic 17-amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins has been found to exhibit suppressive properties for numerous immune functions. It has been shown that CKS-17 causes an imbalance of human types 1 and 2 cytokines and inhibition of the immune responses of lymphocytes, monocytes, and macrophages. CKS-17 induced increased intracellular levels of cAMP, which plays an important role in regulation of cytokine biosynthesis. In this study, using a Jurkat T-cell line and Western blot analysis, CKS-17 induced phosphorylation of PLC-gamma1, Raf-1, MEK and ERK1/2. Using a PLC selective inhibitor U73122 or PLC-gamma1-deficient Jurkat cell line, phosphorylation induced by CKS-17 of ERK1/2, PLC-gamma1, or Raf-1, respectively, were undetectable or significantly reduced. Reintroduction of PLC-gamma1 into the PLC-gamma1-deficient Jurkat cells restored the phosphorylation of ERK1/2 and PLC-gamma1 induced by CKS-17. Further, pretreatment of Jurkat cells with PKC inhibitors blocks the phosphorylation of Raf-1, MEK, and ERK1/2 induced by CKS-17. These results indicate that CKS-17 induces the PLC-gamma1-PKC-Raf-1-MEK-ERK1/2 signaling pathway.
Keywords: CKS-17; Synthetic peptide; Retroviral transmembrane protein; Signal transduction; Phospholipase C-gamma1; Raf-1; ERK1/2; Jurkat T-cell line; Human;

Stronger anti-HIV-1 activity of C-peptide derived from HIV-189.6 gp41 C-terminal heptad repeated sequence by Jeong Kon Seo; Hee Kyung Kim; Tae Young Lee; Kyung-Soo Hahm; Kil Lyong Kim; Myung Kyu Lee (2175-2181).
C34-LAI containing amino acids 118 to 151 of the HIV-1LAI gp41 ectodomain exhibits potent anti-HIV-1 activity. However, the N-terminal halves of C34 peptides vary more according to the HIV-1 strain than the C-terminal halves. Therefore, an analysis was conducted on the anti-HIV-1 activities of the C34 peptides derived from various HIV-1 strains. C34-89.6 exhibited the strongest anti-HIV-1 activity among the C34 peptides tested. Interestingly, its N-terminal half was more acidic than those of the other C34 peptides, whereas its C-terminal half was more basic. Since the C-peptides derived from the HIV-1LAI strain are used extensively, the anti-HIV-1 activities of these peptides were compared between the HIV-1 strains 89.6 and LAI. When using chimeric peptides, it was found that the C-terminal basic region of C34-89.6 was more critical than its N-terminal basic region. The anti-HIV-1 activity of T20-89.6 and C28-89.6 was also stronger than that of T20-LAI and C28-LAI, respectively. The anti-HIV-1 activity of C28-89.6 was weakened when the C-terminal basic residues were changed to the corresponding residues of C28-LAI. However, no conformational differences were found among the C28 peptides. Accordingly, these results imply that introducing the C-terminal basic residues of the HIV-189.6 C-peptide may be useful for developing potent anti-HIV-1 drugs.
Keywords: Anti-HIV-1 assay; Anti-HIV-1 peptide; HIV-189.6 strain; C-peptide of HIV-1 gp41;

Aminopeptidase N/CD13 targeting fluorescent probes: Synthesis and application to tumor cell imaging by Zhouen Zhang; Hiroshi Harada; Kazuhito Tanabe; Hiroshi Hatta; Masahiro Hiraoka; Sei-ichi Nishimoto (2182-2187).
A family of fluorescein–peptide conjugates (CNP1–3) for aminopeptidase N (APN/CD13) targeting fluorescent probes were designed and synthesized. Among the three conjugates, CNP1 bearing tumor-homing cyclic peptide CNGRC, could selectively label APN/CD13 over-expressing on the surface of tumor cells of HT-1080, as identified by means of fluorescent microscopic cell imaging. CNP1 was shown to be a promising fluorescent probe applicable to tumor-targeting molecular imaging.
Keywords: Aminopeptidase N (APN/CD13); Tumor-homing peptide; CNGRC; Fluorescent probe; Molecular imaging;

A proline-rich polypeptide complex (PRP) with immunoregulatory and procognitive activities shows beneficial effects in Alzheimer's disease (AD). The mechanism of action of PRP in AD is not yet clarified. Here, we present results of the effect of PRP on Vitamin D3-induced phenotypic (CD11b and CD14) and functional (phagocytic) differentiation/maturation of monocytes/macrophages using the premonocytic HL-60 cell line as a model. This cell line can be induced to differentiate into monocyte/macrophage cells by incubation with Vitamin D3. However, when Vitamin D3 was applied together with PRP, a 30–40% inhibition of the expression of the differentiation markers and an over-60% inhibition of phagocytic ability were observed. When PRP was administered to the cells after treatment with Vitamin D3, no attenuation of the differentiation/maturation process of the HL-60 cells was observed. This indicates that PRP affects the early stages of differentiation/maturation of these cells. Our results, therefore, suggest that PRP, which affects the differentiation/maturation processes of cells of monocyte/macrophage lineage, may regulate in this way the inflammatory processes in which these cells participate.
Keywords: Proline-rich polypeptide complex (PRP); HL-60 cells; Vitamin D3; Differentiation/maturation; Phagocytosis;

Rapid presymptomatic detection of PrPSc via conformationally responsive palindromic PrP peptides by A. Grosset; K. Moskowitz; C. Nelsen; T. Pan; E. Davidson; C.S. Orser (2193-2200).
Structurally unique, synthetic prion peptides provide the basis of a simple assay to serve as both a detection and signal amplification system that distinguishes the normal prion protein, PrPC, from the misfolded prion protein, PrPSc, that is associated with the occurrence of transmissible spongiform encephalopathies (TSE). Proof-of-principle has been shown on brain samples from an experimental scrapie hamster model. The assay demonstrates very sensitive detection of PrPSc in animal brain tissue with potential application for early presymptomatic detection in animal screening. Furthermore, the sensitivity of the assay could enable blood tests for this TSE disease as well as other amyloid and/or misfolded protein diseases.
Keywords: Prion protein; Presymptomatic detection; Hamster; Scrapie;

Insulin-like growth factor (IGF) induced proliferation of human lung fibroblasts is enhanced by neurotensin by Richard C. Scarpa; Robert E. Carraway; David E. Cochrane (2201-2210).
Fibroblasts are key cells in tissue repair and important contributors to the inflammatory response. Insulin-like growth factors (IGFs) have been shown to participate in growth, in immune responses and in tissue repair where they stimulate cell growth. Neurotensin (NT) has been suggested to participate in inflammation and in tissue repair and is an autocrine or paracrine growth factor for several cancer cell types. Here we show that IGF-induced proliferation of fibroblasts is enhanced by NT in a concentration and type 1 NT-receptor dependent manner. This action of NT was blocked by inhibitors of phospholipase C and protein kinase C but not by inhibitors of phosphoinositide-3-kinase. An inhibitor of MEK 1/2 significantly reduced the proliferative effects of the IGFs but NT's ability to enhance IGF-induced proliferation was not effected. The ability of NT to enhance IGF-induced proliferation did not involve an autocrine factor. These results suggest that interactions between NT and the IGFs may contribute to the regulation of fibroblasts in for example, inflamed or injured tissues.
Keywords: Insulin-like growth factors; Neurotensin; Human lung fibroblasts; Cell proliferation;

Urotensin II: Evidence for cardiac, hepatic and renal production by Christopher J. Charles; Miriam T. Rademaker; A. Mark Richards; Timothy G. Yandle (2211-2214).
Although urotensin II (UII) has been reported to circulate in human plasma and be raised in cardiovascular disorders, little, if any, information is available regarding the source of plasma UII. Accordingly, we have performed trans-organ arteriovenous sampling for measurement of UII concentration in anesthetized sheep. Plasma UII levels were measured in the low picomolar range in normal sheep and arterial plasma levels rose steadily with increasing time of anesthesia. Significant arteriovenous gradients were observed across the heart (36%), liver (40%) and kidney (44%). This is the first report to identify the heart, liver and kidney as sources of UII in the circulation.
Keywords: Heart; Liver; Kidney; Heart failure; Arteriovenous gradient; Urotensin II;

Systemic administration of lipopolysaccharide upregulates angiotensin II expression in rat renal tubules: Immunohistochemical and ELISA studies by Mika Fukada; Shinsuke Kato; Michio Miyoshi; Kenichi Yamaguchi; Toshiaki Imoto; Tatsuo Watanabe (2215-2221).
We investigated whether angiotensin II (AII) peptide is induced in the rat kidney under endotoxemic conditions. Immunohistochemistry revealed strong AII-like immunoreactivity in the renal tubules of rats given high-dose lipopolysaccharide (LPS; 1000 μg/kg) intraperitoneally (i.p.). AII-like immunoreactivity in renal tubules was slight at 1 h after the LPS injection, but marked at 3 h. There were few signals in the kidney in saline-injected control rats. When injected at 0.1, 10, or 1000 μg/kg i.p., LPS-induced a dose-related increase in AII-like immunoreactivity in renal tubules that was unaffected by treatment with the prostaglandin-synthesis blocker indomethacin. ELISA measurement of the AII concentration in the whole kidney supported the above findings. These results suggest that systemically administered LPS induces AII peptide expression in renal tubules by a prostaglandin-independent mechanism.
Keywords: Angiotensin II; Lipopolysaccharide; Kidney; Immunohistochemistry; ELISA;

Adrenomedullin release in the rat mesenteric resistance artery by Shinji Akiyama; Narumi Hobara; Naomi Maruo; Seiichi Hashida; Kazuo Kitamura; Tanenao Eto; Hiromu Kawasaki (2222-2230).
Adrenomedullin (AM) is a potent vasodilator peptide whose major source is the vascular wall. In the present study, the mechanism of release of AM was investigated in the rat mesenteric resistance artery. The isolated mesenteric vascular bed was perfused with Krebs solution at a constant flow rate (5 ml/min) and AM in the perfusate was measured by a highly sensitive enzyme immunoassay (Immunoenzymometric assay; IEMA) method. In preparations without endothelium, spontaneous release of AM was detected in the perfusate (68.7 ± 5.8 fmol/ml, n  = 45). Periarterial nerve stimulation (PNS, 4 and 8 Hz) caused 11.4 ± 3.9% (4 Hz) and 9.1 ± 3.5% (8 Hz) decreases in the spontaneous release of AM. Removal of Ca2+ from the medium did not affect the spontaneous AM release, but abolished the PNS-induced inhibition of spontaneous AM release. Perfusion of 10 nM calcitonin gene-related peptide (CGRP) or 0.1 μM capsaicin (inducer of CGRP release) inhibited significantly the spontaneous AM release. PNS (8 Hz)-induced inhibition of spontaneous AM release was antagonized by CGRP(8–37) (CGRP receptor antagonist). These results suggest that AM is mainly released from vascular smooth muscle cells of the rat mesenteric artery and endogenous or exogenous CGRP inhibits AM release.
Keywords: Adrenomedullin; Calcitonin gene-related peptide (CGRP); Adrenomedullin receptor; CGRP receptor; Neurotransmission of CGRPergic nerves; Rat mesenteric resistance artery;

The purpose of this study is to elucidate the mechanism of action and site of action of calcitonin gene-related peptide (CGRP) and its effects on calcium concentrations in two types of cardiomyocytes, neonatal and adult, by employing real-time fluorescence imaging. CGRP caused an increase in intramyocytic calcium with adult cells, but a decrease with neonates. Treatment of adult myocytes with ouabain and ryanodine yielded results suggesting that CGRP action is not at the ryanodine receptor (RyR) and does not involve Na+  + K+ ATPase. Furthermore, in neonatal cardiomyocytes CGRP caused a reduction in intramyocytic calcium levels, and challenges with ryanodine and ouabain gave results supporting the hypothesis that CGRP acts at the sarcolemmal L-type calcium channel. Employing real-time fluorescence measurements in cultured, dedifferentiated adult cardiomyocytes, which are known to express a fetal phenotype and exhibit neonatal-like calcium transients, our acquisitions demonstrated a major reduction in intracellular calcium levels. Finally, our collaborative studies in human myocardium using fluorescence deconvolution microscopy revealed that CGRP localization was found in a pattern similar to that of the sarcolemmal L-type calcium channel.
Keywords: Calcitonin gene-related peptide; Calcium; Myocytes; Fluorescence imaging;

The newly isolated peptide, urocortin (UCN), is a member of the corticotropin-releasing factor (CRF)-related peptides that has been found to have potent cardiovascular protective effects. In order to investigate the effect of UCN on the viability of adult rat vascular smooth muscle cells (VSMC) and the relevant mechanisms, we exposed the VSMC to UCN to observe the change in cell viability using MTT assay and intracellular calcium concentration using confocal laser scanning microscope methods. Our results showed that UCN (10−7  M) inhibited the viability of VSMC by about 26% (P  < 0.05, compared to control). The effect was concentration-dependent, but it was not dependent on the affecting time. Glybenclamide (Gly, 10−5  M), the ATP-sensitive potassium channel (KATP channel) blocker, and astressin (10−6  M), a competitive antagonist of CRF receptors, had no influence on this inhibition. Bay K8644 (10−6  M), a special L-type calcium channel activator, increased the viability of VSMC. Pre-treatment of the cells with UCN diminished the effect of Bay K8644 (n  = 6, P  < 0.05). UCN was also observed to reduce the intracellular Ca2+ increase induced by KCl and Bay K8644. There was no significant difference in nitrite accumulation between UCN groups and the control. In conclusion, UCN reduced the viability of VSMC through L-type calcium channels. These interesting results might suggest that UCN may be a new vasoactive agent involved in hindering vascular remodeling in combination with previous reports about UCN's hypotensive effects.
Keywords: Urocortin; Calcium channel; Vascular smooth muscle cells; Cell viability; Vascular remodeling;

Doxapram increases corticotropin-releasing factor immunoreactivity and mRNA expression in the rat central nucleus of the amygdala by Song-hyen Choi; Sung-Jin Kim; Sang-Ha Park; Bo-Hyun Moon; Eunju Do; Boe-Gwun Chun; Min-Soo Lee; Kyung-Ho Shin (2246-2251).
Doxapram causes panic anxiety in humans. To determine whether doxapram alters corticotropin-releasing factor (CRF) expression in the central nucleus of the amygdala (CeA), paraventricular nucleus of hypothalamus (PVN), or bed nucleus of the stria terminalis (BNST), we used immunohistochemistry to measure CRF peptide in these brain areas after doxapram injection. Doxapram injection significantly increased CRF-like immunoreactivity (CRF-IR) within the CeA, but not in the BNST or PVN, and this increase was significant 2 h after injection. In addition, doxapram significantly increased CRF mRNA expression within the CeA, and this was most prominent 30 min after injection. These results suggest that doxapram selectively increases CRF expression within the CeA, and that this is mediated by increased CRF gene transcription. This increase in CRF-IR within the CeA might explain the doxapram-induced anxiety reaction.
Keywords: Amygdala; Corticotropin-releasing factor; Anxiety; Doxapram;

Corticotropin-releasing factor from the rat brain measured by protein immunoblot by Edward G. Meloni; Alexandra V. Jackson; Bruce M. Cohen; William A. Carlezon (2252-2256).
The ability to measure changes in brain levels of corticotropin-releasing factor (CRF) is an important step toward understanding the role of this neuropeptide in mood states. Here, we report for the first time that the protein (Western) immunoblot assay can be used to detect and quantify CRF (4.7 kDa) from the rat brain. Intracerebroventricular (ICV) injections of the neuronal transport-inhibitor colchicine (0, 7.5, 15 and 75 μg) produced a dose-dependent increase in CRF levels within the paraventricular nucleus (PVN) of the hypothalamus with a concomitant and dose-dependent decrease in CRF levels within the median eminence (ME). These data provide a positive validation for the use of the immunoblot assay to detect treatment-induced changes in brain CRF levels.
Keywords: Corticotropin-releasing factor (CRF); CRH; Paraventricular nucleus (PVN); Median eminence (ME); Immunoblot; Western blot; Colchicine; HPA axis;

A distinctive feature of galanin expression is that it is extensively increased by neuronal injury, estrogens, Alzheimer's disease and during development. Since stroke is amongst the clinically most important causes of neuronal injury we studied the tissue concentrations of galanin in a rat stroke model and the possibility of modulating this effect with estrogen. Transient focal middle cerebral artery ischemia was induced in rats that 2 weeks earlier underwent ovariectomy and received 1.5 mg 17β-estradiol slow-release or placebo pellets. The concentrations of galanin and neuropeptide Y were measured after observation periods of 3, 7 and 14 days in extracts of punch biopsies from both the lesioned and the contra lateral control hemisphere. The galanin levels were not changed in any of the brain regions studied except in the hippocampus where they were lower in the ischemic hemisphere in both the estrogen- and placebo-treated animals compared to the corresponding contra lateral intact hemisphere (p  = 0.015). Estrogen treatment up-regulated galanin concentrations in both the ventral and dorsal hippocampus (p  = 0.003). The effects on the galanin concentrations were similar after all observation periods: 3, 7 and 14 days (p  = 0.144). No significant changes were observed in the concentration of neuropeptide Y in response to the lesions. The ischemic lesions were markedly larger in the estrogen-treated animals observed after 3 days compared to the corresponding control group. In the estrogen group the lesion was largest at bregma and the slice 2 mm anterior to the bregma, 82% and 435% larger than in the control group (p  < 0.001). A similar, but much less pronounced (not statistically significant) difference was seen in the groups observed after 7 and 14 days. Earlier studies of lesions in the peripheral and central nervous systems have generally shown an up-regulation of galanin markers in response to but at a distance from the injury. Our results indicate that galanin is not involved in the response of the ischemic penumbra itself to stroke, whereas it may participate in the reactions of the neural stem-cell rich hippocampus to stroke.
Keywords: Galanin; Neuropeptide Y; Neuropeptides; Stroke; Cerebral ischemia; Estrogen; Neuroprotection; Middle cerebral artery occlusion; Rat;

PVN galanin increases fat storage and promotes obesity by causing muscle to utilize carbohydrate more than fat by R. Yun; J.T. Dourmashkin; J. Hill; E.C. Gayles; S.K. Fried; S.F. Leibowitz (2265-2273).
To understand the function of the feeding-stimulatory peptide, galanin (GAL), in eating and body weight regulation, the present experiments tested the effects of both acute and chronic injections of this peptide into the paraventricular nucleus (PVN) of rats. With food absent during the test, acute injection of GAL (300 pmol/0.3 μl) significantly increased phosphofructokinase activity in muscle, suggesting enhanced capacity to metabolize carbohydrate, and reduced circulating glucose levels. It also decreased β-hydroxyacyl-CoA dehydrogenase activity in muscle, indicating reduced fat oxidation, while increasing circulating non-esterified fatty acids (NEFA) and lipoprotein lipase activity in adipose tissue (aLPL). Chronic PVN injections of GAL (300 pmol/0.3 μl/injection) versus saline over 7–10 days significantly stimulated daily caloric intake and increased the weight of four dissected fat depots by 30–40%. These effects, accompanied by elevated levels of leptin, triglycerides, NEFA and aLPL activity, were evident only in rats on a diet with at least 35% fat. Thus, by favoring carbohydrate over fat metabolism in muscle and reversing hyperglycemia, PVN GAL may have a function in counteracting the metabolic disturbances induced by a high-fat diet. As a consequence of these actions, GAL can promote the partitioning of lipids away from oxidation in muscle towards storage in adipose tissue.
Keywords: Galanin; Paraventricular nucleus; Carbohydrate metabolism; Dietary fat; Eating behavior; Obesity;

Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens by Amy M. Naleid; Martha K. Grace; David E. Cummings; Allen S. Levine (2274-2279).
Ghrelin, a powerful orexigenic peptide released from the gut, stimulates feeding when injected centrally and has thus far been implicated in regulation of metabolic, rather than hedonic, feeding behavior. Although ghrelin's effects are partially mediated at the hypothalamic arcuate nucleus, via activation of neurons that co-express neuropeptide Y and agouti-related protein (NPY/Agrp neurons), the ghrelin receptor is expressed also in other brain sites. One of these is the ventral tegmental area (VTA), a primary node of the mesolimbic reward pathway, which sends dopaminergic projections to the nucleus accumbens (Acb), among other sites. We injected saline or three doses of ghrelin (0, 0.003, 0.03, or 0.3 nmol) into the VTA or Acb of rats. We found a robust feeding response with VTA injection of ghrelin, and a more moderate response with Acb injection. Because opioids modulate feeding in the VTA and Acb, we hypothesized that ghrelin's effects in one site were dependent on opioid signaling in the opposite site. The general opioid antagonist, naltrexone (NTX), injected into the Acb did not affect feeding elicited by ghrelin injection into the VTA, and NTX in the VTA did not affect feeding elicited by ghrelin injected into the Acb. These results suggest interaction of a metabolic factor with the reward system in feeding behavior, indicating that hedonic responses can be modulated by homeostatic factors.
Keywords: Ghrelin; Reward; Opioid; Food intake; Microinjection; Rat;

Stimulation of neurogenesis in rat nucleus of the solitary tract by ghrelin by Weizhen Zhang; Yuexuan Hu; Theodore R. Lin; Yongyi Fan; Michael W. Mulholland (2280-2288).
Ghrelin, a gastric hormone, regulates growth hormone secretion and energy homeostasis. The present study shows that ghrelin promotes neural proliferation in vivo and in vitro in the rat nucleus of the solitary tract (NTS). Systemic administration of ghrelin significantly increased 5-bromo-2′-deoxyuridine (BrdU) incorporation in the NTS in adult rats with cervical vagotomy. Cultured NTS neurons contain immature precursor cells as shown by expression of Hu protein. Exposure of cultured NTS neurons to ghrelin significantly increased the percentage of BrdU incorporation into cells in both dose- and time-dependent manners. Co-localization of Hu immunoreactivity with BrdU labeling was demonstrated by double fluorescent staining, suggesting that cells labeled with BrdU are neuronal cells. Ghrelin receptor mRNA was detected in tissues from the NTS. The mitotic effect of ghrelin was abolished by treatment of cultured NTS neurons with ghrelin receptor antagonists: d-Lys-3-GHRP-6 and [d-Arg1, d-Phe-5, d-Trp-7, 9, Leu-11] substance P. Diltiazem, a l-type calcium channel blocker, significantly attenuated ghrelin-mediated increments in BrdU incorporation. Ghrelin acts directly on NTS neurons to stimulate neurogenesis.
Keywords: Brain stem; Growth hormone secretagogue receptor; Cell proliferation; Calcium channel blocker; Rat;

Evidence of involvement of leptin and IL-6 peptides in the action of interferon-beta in secondary progressive multiple sclerosis by Francesco Angelucci; Massimiliano Mirabella; Marcella Caggiula; Giovanni Frisullo; Katia Patanella; Cristina Sancricca; Viviana Nociti; Pietro Attilio Tonali; Anna Paola Batocchi (2289-2293).
Leptin is a peptide hormone which acts on cells of immune system by influencing the production of cytokines. Serum leptin levels and cytokine production by peripheral blood mononuclear cells (PBMC) were measured in 18 secondary progressive multiple sclerosis (SPMS) patients under IFN-beta-1b treatment. There were no overall effects on leptin, interleukin-6 (IL-6), IL-10 and IL-12 p40 after 2, 6 and 12 months of treatment. However, leptin and IL-6 decreased after 6 and 12 months of treatment in 12 patients who did not show progression of disability. Thus, our pilot data show that the beneficial effect of IFN-beta on some SPMS patients might be associated with the reduced levels of leptin and reduced IL-6 production by PBMC.
Keywords: Leptin; IL-6; Cytokines; Secondary progressive multiple sclerosis; Interferon-beta;

We investigated the effect of melanocortin 4 receptor (MC4) antagonists on food intake in mice. Food intake during the light phase was significantly increased by ICV administration of mixed MC3/MC4 antagonists (AgRP and SHU9119) or MC4 selective antagonist peptide [(Cyclo (1–5)[Suc–d–Nal–Arg–Trp–Lys]NH2] (MBP10) and the small molecule antagonists THP and NBI-30. Both mixed and selective antagonists significantly reversed anorexia induced by ICV administration of the MC4 agonist (c (1–6) HfRWK-NH2) and the cytokine IL-1β. These findings provide pharmacological evidence that the MC4 receptor mediates the effects of melanocortin agonists and antagonists on food intake in mice, and support the idea that selective small molecule MC4 antagonists may be useful as therapeutics for cachexia.
Keywords: Feeding; Anorexia; Cytokine; Cachexia;

Isolation and characterization of antagonist and agonist peptides to the human melanocortin 1 receptor by Stéphane Bonetto; Isabelle Carlavan; Daniel Baty (2302-2313).
We identified a large number of peptide mimotopes of the adrenocorticotropic hormone (ACTH) and the α-melanocyte stimulating hormone (α-MSH) to analyze better the structure–function relationships of these hormones with the human MC1 receptor (hMC1R). We have investigated the use of phage-display technology to isolate specific peptides of this receptor by using three monoclonal anti-ACTH antibodies (mAbs). A library of 108 phage-peptides displaying randomized decapeptides was constructed and used to select phage-peptides that bind to mAbs. Forty-five phage-peptides have been isolated and from their amino acid sequences, we have identified two consensus sequences, EXFRWGKPA and WGXPVGKP, corresponding to the regions 5–13 and 9–16 of ACTH, respectively. A biological assay on cells expressing the hMC1-R was developed to determine the capacity of phage-peptides to stimulate the receptor. Only two phage-peptides showed detectable activity. Thirty-one peptides were synthesized to analyze their biological effect. We identified two weak agonists, EC50  = 16 and 11 μM, two strong agonists, EC50  = 25 and 14 nM and a partial antagonist, IC50  = 36 μM. This work confirmed the modulator agonist role of the regions 11–12 of α-MSH and ACTH, and the importance of the methionine residue at position 4 for the stimulation of the hMC1-R. We also identified analogues of the regions 8–17 of ACTH that exhibited a weak activator effect, and of one analogue of the N-terminal regions 1–9 of ACTH and α-MSH having a partial antagonist effect. These results may be useful in the development of potential agonists or antagonists of the hMC1R.
Keywords: Melanocortin; α-MSH; ACTH; Agoniste; Antagoniste; hMC1R; Phage-display; Mimotope;

The brain melanocortin system mediates downstream effects of hypothalamic leptin and insulin signaling. Yet, there have been few studies of chronic intracerebroventricular (i.c.v.) melanocortin receptor (MCR) agonist or antagonist infusion. Although there is evidence of interaction between melanocortin and dopamine (DA) systems, effects of chronic MCR ligand infusion on behavioral sensitivity to non-ingestive reward stimuli have not been investigated. The objective of this study was to investigate effects of chronic i.c.v. infusion of the MCR agonist, MTII, and the MCR antagonist, SHU9119, on food intake, body weight, and sensitivity to rewarding lateral hypothalamic electrical stimulation (LHSS) and the reward-potentiating (i.e., threshold-lowering) effect of d-amphetamine. The MCR antagonist, SHU9119 (0.02 μg/h) produced sustained hyperphagia and weight gain during the 12-day infusion period, followed by compensatory hypophagia and an arrest of body weight gain during the 24-day post-infusion period. At no point during the experiment was sensitivity to LHSS or d-amphetamine (0.25 mg/kg, i.p.) altered. The MCR agonist, MTII (0.02 μg/h) produced a brief hypophagia (3 days) followed by a return to control levels of daily intake, but with body weight remaining at a reduced level throughout the 12-day infusion period. This was followed by compensatory hyperphagia and weight gain during the 24-day post-infusion period. There was no change in sensitivity to non-ingestive reward stimuli during the infusion of MTII. However, sensitivity to d-amphetamine was increased during the 24-day post-infusion period. It therefore seems that changes in ingestive behavior that occur during chronic MCR ligand infusion may not affect the response to non-ingestive reward stimuli. However, it is possible that the drive to re-feed and restore body weight following MCR agonist treatment includes neuroadaptations that enhance the incentive effects of drug stimuli.
Keywords: Melanocortin; MTII; SHU9119; Amphetamine; Reward; Self-stimulation;

Cholecystokinin (CCK) and serotonin (5-HT) systems have been shown to cooperate interdependently in control of food intake. To assess mechanisms by which CCK and 5-HT systems interact in control of food intake we examined: (1) participation of CCK-1 and 5-HT3 receptors in 5-HT-induced suppression of sucrose intake; (2) the interaction between CCK and 5-HT in suppression of food intake; (3) the role of CCK-1 and 5-HT3 receptors in mediating this interaction. Intraperitoneal administration of 5-HT (0.25, 0.5 and 1.0 mg/kg) significantly reduced intake compared to control in a dose responsive fashion (r 2  = 0.989). Suppression of food intake by 5-HT was significantly attenuated by prior treatment with the 5-HT3 receptor antagonist ondansetron at each 5-HT dose tested (P  < 0.05), while blockade of CCK-1 receptors by lorglumide had no effect on 5-HT-induced suppression of intake. Administration of CCK-8 (0.5 μg/kg) or 5-HT (0.5 mg/kg) alone significantly reduced sucrose intake by 22.9 and 22.2% respectively, compared to control (P  < 0.0001). Co-administration of CCK and 5-HT resulted in a synergistic suppression of intake leading to an overall 48.4% reduction in sucrose intake compared to saline (P  < 0.0001). Concomitant CCK-1 and 5-HT3 receptor blockade by lorglumide and ondansetron respectively, resulted in a complete reversal of the combined CCK and 5-HT-induced suppression of intake. Independent administration of lorglumide or ondansetron did not alter intake compared to control. These studies provide evidence that 5-HT causes suppression in food intake by acting at 5-HT3, not CCK-1 receptors. Furthermore, CCK and 5-HT interact to produce an enhanced suppression of food intake, an effect mediated through concomitant activation of CCK-1 and 5-HT3 receptors.
Keywords: Satiety; Gastric; Synergistic;

An orexin-1 receptor antagonist decreases food intake whereas orexin-A selectively induces hyperphagia to a high-fat diet. In the present study, we evaluated the effect of an orexin antagonist in two strains of rats that differ in their sensitivity to becoming obese while eating a high-fat diet. Male Osborne-Mendel (OM) and S5B/Pl (S5B) rats were treated acutely with an orexin-1 receptor antagonist (SB-334867), after adaptation to either a high-fat (56% fat energy) diet or a low-fat (10% fat energy) diet that were equicaloric for protein (24% energy). Ad libitum fed rats were injected intraperitoneally with SB-334867 at doses of 3, 10 or 30 mg/kg, or vehicle at the beginning of the dark cycle, and food intake and body weight were measured. Hypothalamic prepro-orexin and orexin-1 receptor mRNA expression were analyzed in OM and S5B rats fed at a high-fat or low-fat diet for two weeks. SB-334867 significantly decreased food intake in both strains of rats eating the high-fat diet but only in the OM rats eating the low fat diet. The effect was greatest at 12 and 24 h. Body weight was also reduced in OM rats 1 d after injection of SB-334867 but not in the S5B rats. Prepro-orexin and orexin-1 receptor expression levels did not differ between strains or diets. These experiments demonstrate that an orexin antagonist (SB-334867) reduces food intake and has a greater effect in a rat strain that is susceptible to dietary-induced obesity, than in a resistant strain.
Keywords: Dietary-induced obesity; Orexin; SB-334867; OM; S5B; Rat;

CSF hypocretin-1/orexin-A concentrations in patients with subarachnoid hemorrhage (SAH) by K. Dohi; B. Ripley; N. Fujiki; H. Ohtaki; S. Shioda; T. Aruga; S. Nishino (2339-2343).
The aim of this study was to examine the role of the hypothalamic hypocretin/orexin system in complications of delayed ischemic neuronal deficit (DIND) resulting from symptomatic vasospasm in patients with aneurysmal subarachnoid hemorrhage (SAH). CSF hypocretin-1/orexin-A levels were measured in 15 SAH patients. DIND complications occurred in seven patients with symptomatic vasospasm. Hypocretin-1/orexin-A levels were low in SAH patients during the 10 days following the SAH event. CSF hypocretin-1/orexin-A levels were lower in patients with DIND complications than in those who did not develop DIND. A significant transient decline in CSF hypocretin-1/orexin-A levels was also observed at the onset of DIND in all patients with symptomatic vasospasm. The reduced hypocretin/orexin production observed in SAH patients may reflect reduced brain function due to the decrease in cerebral blood flow. These results, taken together with recent experimental findings in rats that indicate hypocretin receptor 1 (orexin 1 receptor) mRNA and protein are elevated following middle cerebral artery occlusion, suggest that a reduction in hypocretin/orexin production in SAH and DIND patients is associated with alterations in brain hypocretin/orexin signaling in response to ischemia.
Keywords: Hypocretin/orexin; Subarachnoid hemorrhage (SAH); Human; Vasospasms; Cerebral blood flow (CBF);

Pituitary adenylate cyclase activating polypeptide plays a role in olfactory memory formation in chicken by Rita Józsa; Tibor Hollósy; Andrea Tamás; Gábor Tóth; István Lengvári; Dóra Reglődi (2344-2350).
PACAP plays an important role during development of the nervous system and is also involved in memory processing. The aim of the present study was to investigate the function of PACAP in chicken embryonic olfactory memory formation by blocking PACAP at a sensitive period in ovo. Chicken were exposed daily to strawberry scent in ovo from embryonic day 15. Control eggs were treated only with saline, while other eggs received a single injection of the PACAP antagonist PACAP6-38 at day 15. The consumption of scented and unscented water was measured daily after hatching. Animals exposed to strawberry scent in ovo showed no preference. However, chickens exposed to PACAP6-38, showed a clear preference for plain water, similarly to unexposed chicken. Our present study points to PACAP's possible importance in embryonic olfactory memory formation.
Keywords: PACAP; Chicken; Olfactory memory; Embryonic;

Immunocytochemical distribution of NK-1 and NK-3 tachykinin receptors in isolated pancreatic acini of guinea pigs and rats by Maria Broccardo; M. Teresa Ciotti; Giorgio Linari; Simona Agostini; Carla Petrella; Giusy Amadoro; Cinzia Severini; Giovanna Improta (2351-2354).
In this study, we investigated the immunocytochemical distribution of NK-1 and NK-3 tachykinin receptors in guinea pig and rat isolated pancreatic acini. In dispersed acinar cells from guinea pig, immunofluorescence staining detected similar densities of NK-1 and NK-3 receptors; conversely, rat acinar cells expressed NK-1 receptors more strongly than NK-3 receptors. In line with previous functional studies, these immunocytochemical findings suggest that guinea pig NK-1 and NK-3 receptors and rat NK-1 receptors alone play a direct stimulatory role in the basal pancreatic acinar amylase release.
Keywords: Immunofluorescence; Tachykinin receptors; Pancreatic acini; Guinea pig; Rat;