Peptides (v.30, #12)

Contents (VII).

An antifungal defensin-like peptide with a molecular mass of 7.1 kDa was isolated from dried Nepalese large red beans (Phaseolus angularis). The purification protocol employed included ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The antifungal peptide was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and SP-Sepharose. The antifungal peptide inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC50 value of 1.4 and 1.8 μM, respectively. It did not inhibit HIV-1 reverse transcriptase when tested up to 200 μM. It exerted an antiproliferative action on L1210 leukemia cells and MBL2 lymphoma cells with an IC50 of 15 and 60 μM, respectively.
Keywords: Nepalese large red beans; Phaseolus angularis; Defensin-like peptide; Antiproliferative; Purification;

Differential proteomics analyses were performed on flower buds of chasmogamous (CH) and cleistogamous (CL) soybean to identify candidate genes that are associated with cleistogamy which is the production of permanently closed flowers. The proteins were extracted from flower buds of CH cv. Toyosuzu and CL cv. Karafuto-1, and separated by two-dimensional polyacrylamide gel electrophoresis. Of the 640 proteins detected on the gel, nine were differentially expressed in Toyosuzu and nine in Karafuto-1. Among these differentially expressed proteins, those associated with cleistogamy were identified using a pair of near-isogenic lines (NILs). β-Galactosidase and protein disulfide isomerase were expressed differentially between the NILs for cleistogamy. Furthermore, the mRNA expression pattern of protein disulfide isomerase corresponded to the protein level, whereas that of β-galactosidase was not consistent with the protein level. These results suggest that the protein disulfide isomerase and β-galactosidase may be useful markers for achieving a better understanding of the molecular-biological mechanisms of cleistogamy in soybean.
Keywords: Soybean; Cleistogamy; Proteomics; Floral buds; Near-isogenic lines;

A new 2S albumin from Jatropha curcas L. seeds and assessment of its allergenic properties by Fábio Menezes Maciel; Mariana Agra Laberty; Natália Deus Oliveira; Shayany Pinto Felix; Alexandra Martins dos Santos Soares; Maurício Afonso Verícimo; Olga Lima Tavares Machado (2103-2107).
Significant effort has been made world-wide to boost biofuels with the expectation of a positive contribution to renewable fuel and greenhouse gas reduction. Jatropha curcas L. has proved to be an opportunistic crop in tropical areas, particularly in unfavorable environments. For this reason, analyses of toxicity and allergy caused by its seeds and pollen are necessary. A 12 kDa, allergenic 2S albumin, denoted Jat c 1, was isolated from Physic nut (J. curcas) seeds. Jat c 1 binds IgE attached to rat mast cells, inducing histamine release. It also showed strong cross-reactivity with the major allergens from castor bean, Ric c 1 and Ric c 3.
Keywords: 2S albumin; Jatropha curcas; Jat c 1; Physic nut; Allergy;

Proteomics approach for identifying osmotic-stress-related proteins in soybean roots by Mahmoud Toorchi; Kiyoshi Yukawa; Mohammad-Zaman Nouri; Setsuko Komatsu (2108-2117).
Osmotic stress can endanger the survival of plants. To investigate the mechanisms by which plants respond to osmotic stress, protein profiles from soybean plants treated with polyethylene glycol (PEG) were monitored by a proteomics approach. Treatment with 10% aqueous PEG reduced the lengths of roots and hypocotyls of soybean seedlings. Proteins from soybean roots were separated by two-dimensional polyacrylamide gel electrophoresis, and 415 proteins were detected by Coomassie brilliant blue staining. Thirty-seven proteins changed by PEG treatment were analyzed using Edman sequencing and peptide-mass fingerprinting method and this group included proteins involved in disease/defense. Seven proteins were selected for further experiments using the results of cluster analysis and statistical analysis of the abundance change. A comparison with the effects of other abiotic stresses showed that caffeoyl-CoA-O-methyltransferase and 20S proteasome alpha subunit A were decreased and increased by abiotic stresses, respectively. Expression analyses of these transcripts were also changed by PEG treatment. Caffeoyl-CoA-O-methyltransferase and 20S proteasome alpha subunit A may control the sensitivity of several regulatory genes specific to short exposure to osmotic stress.
Keywords: Soybean; Osmotic stress; Polyethylene glycol; Proteome; Caffeoyl-CoA-O-methyltransferase; 20S proteasome alpha subunit A;

Plant aspartic proteases are of recent origin with their physiological significance in crucial processes emerging. Reports on the significance of aspartic protease inhibitors and their endogenous proteases in seeds of plants are scanty. This paper reports the purification of an aspartic protease inhibitor from the seeds of Vigna radiata, its control of the endogenous aspartic protease and their subsequent role in the early germination events. The role of the aspartic protease inhibitor and the enzyme in initial stages of germination of V. radiata has been tracked by differential timed expression and germination assays. The expression pattern revealed maximum expression of the inhibitor in the dormant seeds while the enzyme was predominant in the germinating seeds. Their expression patterns and interactions indicate their significance in initiation of germination. The expression of other classes of proteases was monitored during germination and a model predicting the events occurring during proteolysis of the storage protein in germination is hypothesized. The inhibitor was a linear, hydrophobic, pH stable and thermostable peptide with molecular weight of 1660 Da. The purified inhibitor showed a pI of 4.36 with the sequence as AEIYN KDGNK LDLYG. The inhibitor was found to be stable in a broad range of pH from 2 to 10 with an optimum of 3.0. The half-life of VrAPI at 100 °C was 30 min whereas the maximum activity was observed at 37 °C. The initial kinetic analysis of the inhibitor against the endogenous protease showed an IC50 value of 11 nM while the value of the inhibition rate constant K i was 34 × 10−9  M.
Keywords: Plant proteases; Aspartic protease; Protease inhibitors; Vigna radiata;

Peptide sequences identified by phage display are immunodominant functional motifs of Pet and Pic serine proteases secreted by Escherichia coli and Shigella flexneri by Hernández-Chiñas Ulises; Gazarian Tatiana; Gazarian Karlen; Mendoza-Hernández Guillermo; Xicohtencatl-Cortes Juan; Eslava Carlos (2127-2135).
Plasmid-encoded toxin (Pet) and protein involved in colonization (Pic), are serine protease autotransporters of Enterobacteriaceae (SPATEs) secreted by enteroaggregative Escherichia coli (EAEC), which display the GDSGSG sequence or the serine motif. Our research was directed to localize functional sites in both proteins using the phage display method. From a 12mer linear and a 7mer cysteine-constrained (C7C) libraries displayed on the M13 phage pIII protein we selected different mimotopes using IgG purified from sera of children naturally infected with EAEC producing Pet and Pic proteins, and anti-Pet and anti-Pic IgG purified from rabbits immunized with each one of these proteins. Children IgG selected a homologous group of sequences forming the consensus sequence, motif, PQPxK, and the motifs PGxI/LN and CxPDDSSxC were selected by the rabbit anti-Pet and anti-Pic IgGs, respectively. Analysis of the amino terminal region of a panel of SPATEs showed the presence in all of them of sequences matching the PGxI/LN or CxPDDSSxC motifs, and in a three-dimensional model (Modeller 9v2) designed for Pet, both these motifs were found in the globular portion of the protein, close to the protease active site GDSGSG. Antibodies induced in mice by mimotopes carrying the three aforementioned motifs were reactive with Pet, Pic, and with synthetic peptides carrying the immunogenic mimotope sequences TY PG YI N HSKA and LL P Q P P K LLLP, thus confirming that the peptide moiety of the selected phages induced the antibodies specific for the toxins. The antibodies induced in mice to the PGxI/LN and CxPDDSSxC mimotopes inhibited fodrin proteolysis and macrophage chemotaxis biological activities of Pet. Our results showed that we were able to generate, by a phage display procedure, mimotopes with sequence motifs PGxI/LN and CxPDDSSxC, and to identify them as functional motifs of the Pet, Pic and other SPATEs involved in their biological activities.
Keywords: Escherichia coli; Shigella; SPATEs; Phage display; Immunodominant epitopes; Mimotopes;

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings by Valentina Kovaleva; Ramziya Kiyamova; Rainer Cramer; Hryhoriy Krynytskyy; Ivan Gout; Valeriy Filonenko; Roman Gout (2136-2143).
A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin 1 cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins.
Keywords: Scots pine; Antimicrobial peptides; Purification; Molecular cloning; Defensins; Antifungal activity;

Antimicrobial specificity and mechanism of action of disulfide-removed linear analogs of the plant-derived Cys-rich antimicrobial peptide Ib-AMP1 by Peng Wang; Jeong-Kyu Bang; Hak Jun Kim; Jin-Kyoung Kim; Yangmee Kim; Song Yub Shin (2144-2149).
Ib-AMP1 is a 20-residue disulfide-linked β-sheet antimicrobial peptide found in the seeds of Impatiens balsamina. In order to investigate the effects of the 2 disulfide bonds on the antimicrobial specificity, to determine the mechanism of antimicrobial action of Ib-AMP1 and to develop novel cell-selective antimicrobial peptides with improved antimicrobial specificity as compared to wild-type Ib-AMP1, we synthesized a disulfide-removed linear analog of Ib-AMP1 with l-Pro, d-Pro or peptoid residues (Nala and Nlys) at the central position of the molecule. All linear analogs displayed a 3.7–4.8-fold higher antimicrobial specificity than wild-type Ib-AMP1, indicating that the disulfide bonds of Ib-AMP1 analogs are not essential for its antimicrobial specificity. Circular dichroism spectra revealed that the peptoid residues, as well as the proline at the central position of disulfide bond-removed Ib-AMP1 analogs, induce a β-turn structure in a negatively charged bacterial membrane-mimicking environment. Ib-AMP1 was not effective in depolarizing the cytoplasmic membranes of Staphylococcus aureus and showed almost no leakage of calcein from negatively charged bacterial membranes mimicking lipid vesicles. In contrast, all linear analogs caused very weak dye leakage from negatively charged vesicles, but they almost completely depolarized the membrane potential of S. aureus cells. Collectively, our results suggest that the target of Ib-AMP1 may not be the cytoplasmic membranes of bacteria but their intracellular components. All linear analogs exhibit lethality due to their ability to form small channels that permit the transit of ions or protons and not molecules as large as calcein, and not by disrupting membranes.
Keywords: Antimicrobial peptide; Ib-AMP1; Antimicrobial specificity; Disulfide-removed linear analog; Bactericidal mechanism;

A C-terminal cationic fragment derived from an arginine-rich peptide exhibits in vitro antibacterial and anti-plasmodial activities governed by its secondary structure properties by Liliana Patricia Lesmes; Magda Yenith Bohorquez; Luisa Fernanda Carreño; Manuel Elkin Patarroyo; José Manuel Lozano (2150-2160).
The differential in vitro antimicrobial activity of a 12-residue-long arginine-rich peptide derived from protamine was examined against bacterial and parasite microbes. A design of discrete peptide fragments based on the thermolysin-digestion map allowed us to propose three peptide fragments to be further assessed regarding their biological and secondary structural properties. Peptide structure allowed designing three arginine-rich fragments. All peptide fragments were assessed regarding their antimicrobial activity against Gram-positive and Gram-negative bacteria and a human malaria strain. Qualitative and quantitative assays carried out for determining all peptides’ antibacterial activity at different concentration levels included radial diffusion and a time-controlled technique. Tests demonstrated that all assessed molecules inhibited invasion of Plasmodium falciparum parasites to human red blood cells. Cytolytic activity of the parent protamine peptide was completely abolished by strategically fragmenting its aminoacid sequence. Remarkably, the cationic C-fragment exhibited stronger biological activity than its parent peptide. Interestingly, the peptide fragment denoted as 2077 displays a typical α-helix profile according to its CD spectrum. The results support proposing the protamine C-terminal fragment as a potential new antimicrobial peptide.
Keywords: Arginine-rich peptide; Antimicrobial peptide; Secondary structure; Anti-plasmodial activity; α-Helix;

The effects of various antimicrobial peptides (AMPs) on disrupting the hemagglutinating ability of cellular components of the putative oral pathogen Porphyromonas gingivalis were examined. AMP inhibition of P. gingivalis 381-induced hemagglutination using vesicles (VES) or outer membrane (OM) preparations was determined within standardized hemagglutination assays using various mammalian erythrocytes. A synthetic decapeptide (KSL-W) and its truncated peptide analogs were evaluated and compared with selected classes of AMPs derived from naturally occurring innate defense peptides. All tested AMPs were effective in disrupting P. gingivalis-induced hemagglutination among tested erythrocytes, with the exception of magainin I and the truncated KSL-W analogs. LL-37 was generally the most potent followed by histatin 5. The synthetic decapeptide (KSL-W) was found to be similar to the histatin 8 peptide in terms of inhibitory effect. In addition, co-application assays (with selected oral-related AMPs ± KSL-W) were employed to determine if co-application procedures would improve hemagglutination abrogation above that of oral-related AMPs alone. These experiments revealed that the KSL-W peptide improved hemagglutination inhibition above that of each of the oral-related peptides (histatin 5 and 8, LL-37) alone. Among mammalian erythrocytes, significant peptide-induced hemagglutination was observed for the cathelicidin class AMPs, LL-37 and indolicidin (≥25 and ≥100 μM respectively). In contrast, KSL-W did not induce erythrocyte agglutination throughout any concentration range tested (0.1–1000 μM). Our results suggest that several AMPs are effective in disrupting P. gingivalis 381-induced hemagglutination and that the co-application of a small, synthetically derived peptide may serve to augment the role of local host AMPs engaged in innate defense.
Keywords: Antimicrobial peptide; Porphyromonas gingivalis; Innate immunity; Oral bacteria; Hemagglutination;

Design of novel histone-derived antimicrobial peptides by Hoi See Tsao; Sara A. Spinella; Anna T. Lee; Donald E. Elmore (2168-2173).
Previous studies have identified several naturally occurring antimicrobial peptides derived from histone proteins. This research aimed to design novel histone-derived antimicrobial peptides (HDAPs). To this end, three novel peptides (DesHDAP1, DesHDAP2, and DesHDAP3) were designed based on a histone–DNA crystal structure and structural properties of buforin II, the best characterized naturally occurring HDAP. Molecular dynamics simulations and circular dichroism spectroscopy were used to further support the predicted structure and potential nucleic acid interactions of these three designed peptides. The antibacterial activity of the three peptides was then verified experimentally against a series of bacterial strains using a radial diffusion assay. One of these peptides is the first known fragment of histone H3 with antibacterial properties. Optical density measurements of bacterial cells exposed to the designed peptides implied that at least two of the novel peptides can induce cell death without causing significant membrane permeabilization, as observed for buforin II. The antibacterial potency of these designed HDAPs does not appear to correlate with their overall α-helical content, unlike previous observations for analogs of buforin II. However, the most potent designed peptide, DesHDAP1, shares a markedly similar circular dichroism spectrum with buforin II. These results demonstrate the potential of using histone structures as a framework for designing novel antimicrobial peptides. As well, these studies represent an important starting point for a broader characterization of properties shared by HDAPs.
Keywords: Histone-derived antimicrobial peptide; Antimicrobial peptide; Buforin II; Histone; Peptide design;

Bioavailability of β-amino acid and C-terminally derived PK/PBAN analogs by Aliza Hariton; Orna Ben-Aziz; Michael Davidovitch; Pawel Zubrzak; Ronald J. Nachman; Miriam Altstein (2174-2181).
The ability of linear β-amino acid substituted peptides (PK-βA-1: Ac-YFT[β3P]RLa; PK-βA-2: Ac-Y[β3homoF]TPRLa; PK-βA-3: Ac-Y[β3F]TPRLa; PK-βA-4: Ac-[β3F]FT[β3P]RLa) and unsubstituted analogs (Ac-YFTPRLa and YFTPRLa) of the pyrokinin(PK)/pheromone biosynthesis-activating neuropeptide (PBAN) family to penetrate the insect cuticle and exert biological activity (i.e., stimulate sex pheromone biosynthesis), was tested by topical application on Heliothis peltigera moths. The present results clearly indicate that small linear synthetic peptides can penetrate the cuticle very efficiently by contact application and activate their target organ. The time responses of the peptides applied in DDW and DMSO were tested and the activities of topically applied and injected peptides were compared. The results clearly indicate that PK-βA-4 and PK-βA-3 exhibited high bioavailability (ability to penetrate through the cuticle and exertion of bioactivity) with the latter showing longer persistence in both solvents than any other analog in the study; indicative that incorporation of a β-amino acid at the Phe2 position can enhance longevity in topical PK/PBAN analogs. PK-βA-4 was significantly more active in DMSO than in DDW, and significantly more active than the parent peptide LPK in DMSO. PK-βA-1 and PK-βA-2 exhibited negligible activity. Interestingly, Ac-YFTPRLa was highly potent in both solvents; its activity in DDW did not differ from that of PK-βA-4 and PK-βA-3, and was higher than that of LPK. Even the unacylated peptide YFTPRLa was active in both solvents, at a similar level to LPK. Topically applied PK-βA-4 and Ac-YFTPRLa exhibited significantly higher activity than the injected peptides. PK-βA-3 and YFTPRLa were equally potent in both routes of administration.
Keywords: PBAN; Topical application; Cuticular penetration; Sex pheromone biosynthesis; Bioavailability;

The Bv8 gene from Bombina orientalis: Molecular cloning, genomic organization and functional characterization of the promoter by Sara Marsango; Maria Carmela Bonaccorsi di Patti; Donatella Barra; Rossella Miele (2182-2190).
Bv8 is a secreted peptide from Bombina variegata skin glands with a molecular mass close to 8 kDa that is conserved in fish, amphibians and mammals. Bv8 has diverse regulatory roles, including an involvement in hematopoiesis and immunomodulation. Here we report the genomic organization of the gene from Bombina orientalis coding for the Bv8 homolog (Bo8). It contains three exons separated by two large introns. Several putative transcription factor binding sites have been identified in the promoter sequence. Functional analysis of this region was performed using a yeast genetic system. The results indicate that the transcription factors AP-1, NF-κB and NFAT are involved in the regulation of the expression of Bo8. Hence, amphibians are a useful model for the study of transcriptional regulation of all Bv8 homologs.
Keywords: Bombina orientalis; Prokineticin; Genomic organization; NF-κB; NFAT; One hybrid;

Characterization of a new bioactive peptide from Potamotrygon gr. orbignyi freshwater stingray venom by Katia Conceição; Juliane M. Santos; Fernanda M. Bruni; Clécio F. Klitzke; Elineide E. Marques; Márcia H. Borges; Robson L. Melo; Jorge H. Fernandez; Mônica Lopes-Ferreira (2191-2199).
Brazilian freshwater stingrays, Potamotrygon gr. orbigyni, are relatively common in the middle-western regions of Brazil, where they are considered an important public health threat. In order to identify some of their naturally occurring toxin peptides available in very low amounts, we combine analytical protocols such as reversed-phase high-performance liquid chromatography (RP-HPLC), followed by a biological microcirculatory screening and mass spectrometry analysis. Using this approach, one bioactive peptide was identified and characterized, and two analogues were synthesized. The natural peptide named Porflan has the primary structure ESIVRPPPVEAKVEETPE (MW 2006.09 Da) and has no similarity with any bioactive peptide or protein found in public data banks. Bioassay protocols characterized peptides as presenting potent activity in a microcirculatory environment. The primary sequences and bioassay results, including interactions with the membrane phospholipids, suggest that these toxins are a new class of fish toxins, directly involved in the inflammatory processes of a stingray sting.
Keywords: Potamotrygon gr. orbignyi; Venom; HPLC; Mass spectrometry; Bioactive peptide; Inflammation; Molecular dynamics; Porflan;

A novel inhibitory gonadotropin-releasing hormone-related neuropeptide in the ascidian, Ciona intestinalis by Tsuyoshi Kawada; Masato Aoyama; Iyo Okada; Tsubasa Sakai; Toshio Sekiguchi; Michio Ogasawara; Honoo Satake (2200-2205).
The gonadotropin-releasing hormone (GnRH) family peptides are most widely distributed neuropeptides and/or neurophysial hormones. GnRH is involved in diverse neuroendocrine, paracrine, autocrine, and neurotransmitter/neuromodulatory functions in the central and peripheral nervous system as well as peripheral tissues. In the present study, we show the identification of a novel GnRH-related peptide, Ci-GnRH-X, in the ascidian, Ciona intestinalis. Intriguingly, Ci-GnRH-X possesses a unique primary sequence consisting of 16 amino acids, although typical GnRH family peptides are composed of 10 amino acids. On the other hand, Ci-GnRH-X shares the GnRH consensus motifs, including the N-terminal pQHWS (‘pQ’ indicates a pyro-glutamic acid) and C-terminal Gly-amide. Reverse transcription (RT)-PCR analysis shows that the Ci-GnRH-X gene is expressed exclusively in the central nervous system. Moreover, in situ hybridization demonstrated that the Ciona GnRH-1 gene encoding Ciona GnRHs (t-GnRH-3, -5 and -6) was co-expressed with the Ci-GnRH-X gene in neurons of the cerebral ganglion. Of particular interest is that Ci-GnRH-X exhibited moderate (10–50%) inhibitory activity against t-GnRHs at their cognate receptors. Ci-GnRH-X repressed the elevation of the intracellular calcium and cAMP production by t-GnRH-6 at Ci-GnRHR-1, and cAMP production by t-GnRH-3, and t-GnRH-5 via Ci-GnRHR-3 was also inhibited by Ci-GnRH-X. In contrast, no inhibitory effect of Ci-GnRH-X at Ci-GnRHR-2 was observed. The localization and biochemical assays revealed that Ci-GnRH-X acts as an endogenous antagonist for the Ciona GnRHergic system. This is the first molecular and functional characterization of an endogenous inhibitor of GnRHs in an animal species.
Keywords: Ascidian; Ciona intestinalis; GnRH; Inhibitor; Neuropeptide;

Expression of NMS and NMU2R in the pig reproductive axis during the estrus cycle and the effect of NMS on the reproductive axis in vitro by Guihong Yang; Juan Su; Xun Li; Yuan Yao; Zhihai Lei; Xizhi Yang; Rui Kou; Yanpeng Liu (2206-2212).
Evidence has revealed that neuromedin S (NMS) and neuromedin U-receptor type-2 (NMU2R) mRNAs are expressed in the central nervous system and reproductive organs. Previous data indicated that variation of NMS and NMU2R was due to the phases of the adult rat hypothalamus estrus cycle. However, the expression and function of NMS in the pig reproductive axis remains unexplored. In this study, 16 virginal gilts were classified into four groups: proestrus, estrus, diestrus 1, and diestrus 2; the expression of NMS and NMU2R in the cyclic pig hypothalamus–pituitary–ovary axis was studied by reverse transcriptaion-polymerase chain reaction (RT-PCR), and the effect of NMS on the reproductive axis in vitro was detected by radioimmunoassay (RIA). The cloned pig NMS and NMU2R sequences were 82% and 90.2% identical to those of the corresponding human homologues, respectively. RT-PCR showed that NMS and NMU2R mRNA expression in the hypothalamus and pituitary changed with the estrus cycle, i.e., with the highest level in the proestrus group and the lowest in the estrus group. In the ovary, NMS and NMU2R expression was highest in the diestrus 2 group and the lowest in the proestrus group. In the in vitro study, different concentrations of NMS induced the release of gonadotropin-releasing hormone, luteinizing hormone, and estradiol at different levels of the reproductive axis. Taken together, the expression pattern of NMS during the estrus cycle and its role in reproductive hormones in vitro provide novel evidences of the potential roles of NMS in the regulation of pig reproduction.
Keywords: NMS; NMU2R; RT-PCR; Radioimmunoassay; Reproductive axis;

Ghrelin and peptide YY (PYY) profiles in gastrointestinal tissues and the circulation of the rat during pregnancy and lactation by Victoria J. Taylor; Michael Patterson; Mohammed A. Ghatei; Stephen R. Bloom; Catherine A. Wilson (2213-2220).
Plasma and tissue profiles of gastrointestinal hormones ghrelin and peptide YY (PYY) were investigated in different female rat reproductive states. Neither plasma nor tissue ghrelin concentrations were suppressed during pregnancy despite elevated leptin. The highest concentrations of stomach ghrelin were measured in late pregnancy. PYY concentrations in plasma, descending colon and rectum tissues were increased (P  < 0.001) throughout pregnancy and lactation. PYY peaked at day 5 of lactation in plasma, as well as descending colon and rectum tissues (proestrus vs day 5 of lactation: 25 ± 3.0 pmol/l vs 55 ± 8.0 pmol/l; 85 ± 4.5 pmol/g wwt vs 418 ± 45.0 pmol/g wwt; 23 ± 3.0 pmol/g wwt vs 78 ± 9.1 pmol/g wwt). This PYY peak was temporally associated with the luteinizing hormone peak on day 1 of lactation. Following weaning, dam adiposity and plasma leptin increased whereas ghrelin stomach peptide decreased. Relative PYY concentrations in the tissues of the gut varied in the different states suggesting regional alterations taking place in the colon. The ascending colon produced the highest concentrations in non-pregnant rats, the descending colon the highest concentrations during lactation with the pregnant rats and the dams postweaning in a transition state between. It is unclear what role the increased PYY in various tissues observed has during pregnancy and lactation as it would be expected to be reduced in these states of greatly increased appetite. PYY may have an influence on maternal dietary adaptation, intestinal hypertrophy and weight gain during pregnancy and lactation although it is still unclear precisely how it acts.
Keywords: Gastrointestinal; Ghrelin; Gut hormones; Pregnancy; Lactation; PYY; Rat;

A new role of phosphopeptides as bioactive peptides released during milk casein digestion in the young mammal: Regulation of gastric secretion by Paul Guilloteau; Véronique Romé; Luc Delaby; François Mendy; Loic Roger; Jean Alain Chayvialle (2221-2227).
The aim of this work was to study in vivo the effect of ingestion of phosphopeptides (PP) alone or associated with caseinomacropeptide (CMP) on gastric secretion and to elucidate some possible mechanisms involved. Seven calves fitted with a gastric pouch received either a diet based on whey proteins without PP and CMP (C diet) or C diet in which PP or PP + CMP was introduced at concentrations similar to that of PP or PP + CMP in cow milk (PP diet and PP + CMP diet, respectively). Gastric juice secretion was measured during successive periods throughout the day. Twenty-four calves were fitted with a catheter introduced in one external jugular vein for blood sample collections. The daily secretion of electrolytes decreased with the presence of PP or PP + CMP in the diet. During the day, peptide supplementation in the diet resulted in (1) short term (1st–2nd postprandial h), a decrease of secreted quantities of gastric juice, enzymes and electrolytes, (2) long term (7–24 h after the morning meal), a decrease of electrolyte secretions. Intervention of gastrin, CCK, somatostatin and BPP could be probable. Globally, inhibition of gastric secretions seemed more important when PP was given in association with CMP in the diet rather than alone. CMP and PP may have short and long term action respectively over the 24 h day. To our knowledge, it is the first time that phosphopeptides coming from milk casein digestion are demonstrated to inhibit gastric secretion. Therapeutic uses are suggested.
Keywords: Phosphopeptides; Gastric secretion; Acid and electrolytes; Chymosin and pepsin; Gut regulatory peptides; Calf;

We found that β-lactotensin (His-Ile-Arg-Leu), which has been isolated as an ileum-contracting peptide from chymotrypsin digest of bovine β-lactoglobulin, dose-dependently suppresses food intake after intracerebroventricular (i.c.v.) or intraperitoneal administration at a dose of 40 nmol/mouse or 100 mg/kg, respectively, in fasted mice. Orally administered β-lactotensin also suppressed food intake at 500 mg/kg. We previously reported that β-lactotensin acts as an agonist for neurotensin receptors; however, the anorexigenic activity of β-lactotensin was not inhibited by i.c.v. co-administration with SR48692 or levocabastine, an antagonist for neurotensin NT1 or NT2 receptor, respectively. On the other hand, the anorexigenic effect of β-lactotensin was blocked by i.c.v. co-administration with astressin or calcitonin gene-related peptide (CGRP)(8–37), an antagonist for corticotropin releasing factor (CRF) or CGRP, respectively. β-Lactotensin had affinity for neither CRF nor CGRP receptor. In addition, CRF-induced anorexigenic activity after i.c.v. administration was completely blocked by CGRP(8–37), while CGRP-induced anorexigenic activity was not inhibited by astressin. These results suggest that the CGRP system is activated downstream of the CRF system in food intake regulation. Taken together, β-lactotensin may suppress food intake by activating the CRF system followed by the CGRP system, independently of the neurotensin system.
Keywords: Food intake; β-Lactotensin; Neurotensin; Calcitonin gene-related peptide; Corticotropin releasing factor;

Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast-like cells by Bianca Maria Donida; Emanuela Mrak; Claudia Gravaghi; Isabella Villa; Stefania Cosentino; Elena Zacchi; Silvia Perego; Alessandro Rubinacci; Amelia Fiorilli; Guido Tettamanti; Anita Ferraretto (2233-2241).
Casein phosphopeptides (CPPs), originating by in vitro and/or in vivo casein digestion, are characterized by the ability to complex and solubilize calcium ions preventing their precipitation. Previous works demonstrated that CPPs improve calcium uptake by human differentiated intestinal tumor cell lines, are able to re-mineralize carious lesions in a dental enamel, and, as components of a diet, affect bone weight and calcium content in rats. The aim of the present study was to evaluate if CPPs can directly modulate bone cells activity and mineralization. Primary human osteoblast-like cells were established in culture from trabecular bone samples obtained from waste materials during orthopedic surgery. Commercial mixtures of bovine casein phosphopeptides were used. The CPP dependent intracellular calcium rises were monitored at the single cell level through fura2-fluorescence assays. Results show that CPPs: (i) stimulate calcium uptake by primary human osteoblast-like cells; (ii) increase the expression and activity of alkaline phosphatase, a marker of human osteoblast differentiation; (iii) affect the cell proliferation rate and the apoptotic level; (iv) enhance nodule formation by human SaOS-2. Taken together these results confirm the possibility that CPPs play a role as modulator of bone cell activity, probably sustained by their ability as calcium carriers. Although the exact mechanism by which CPPs act remains not completely clarified, they can be considered as potential anabolic factors for bone tissue engineering.
Keywords: Casein phosphopeptides; Human primary osteoblast-like cells; SaOS-2 cells; Calcium; Bone;

Islet neogenesis-associated protein-related pentadecapeptide enhances the differentiation of islet-like clusters from human pancreatic duct cells by Juan Li; Yun Wang; Xiaozhu Yu; Haiyan Chen; Ying Wu; Xiao Han; Xirong Guo; Chenyu Zhang; Qi Chen; Jiawei Chen; Tao Yang (2242-2249).
The differentiation of pancreatic ductal epithelial cells into β-cells has been considered as an alternative method for increasing the number of islets for transplantation. Critical factors have been introduced into the in vitro differentiation protocol for pancreatic duct cells in order to enhance the production of β-cells. Islet neogenesis-associated protein (INGAP) is an initiator of islet neogenesis and the peptide sequence 104–118 of INGAP has been shown to stimulate an increase in β-cell mass in animals and also found in human pathological states involving islet neogenesis. To establish a novel method for the differentiation of β-cells from human pancreatic duct cells with INGAP-related pentadecapeptide (INGAP-PP), the pancreatic duct cells were isolated, purified and expanded in vitro and differentiated using a four-step protocol that included nicotinamide, exendin-4, transforming growth factor β1 and INGAP-PP/Scrambled peptide (Scrambled-P). The production of islet-like clusters (ILCs) in the INGAP-PP group was significantly higher than that in the Scrambled-P control group after differentiation from an equal number of duct cells. The duct cells showed positive staining and expression for cytokeratin 19, pancreatic duodenal homeobox-1, nestin, and were negative for insulin and glucagon, as detected by both immunofluorescence and RT-PCR. Following differentiation the cells became insulin and glucagon positive. In addition, the ILCs from the INGAP-PP group secreted higher levels of insulin and C-peptide than the Scrambled-P group under a high glucose challenge. We conclude that INGAP peptide enhances the in vitro differentiation of pancreatic duct cells into islet-like clusters.
Keywords: Islet neogenesis; INGAP; Differentiation; Stem cells;

The structure of bioactive analogs of the N-terminal region of gastrin-17 by Jeffrey Copps; Richard F. Murphy; Sándor Lovas (2250-2262).
Gastrin-17 (G17) processing intermediates bind to non-CCK receptors which mediate growth of the colonic mucosa but also the formation and development of colonic cancers. In previous studies, we removed the C-terminal region of G17 to form G17(1–12) and considerably shorter C-terminally amidated and non-amidated analogs. Peptides as short as G17(1–4) continued to bind to a single site on DLD-1 human colonic carcinoma cells, while only the G17(1–6)-NH2 and G17(1–12) peptides retained the ability to activate the receptor and stimulate cell proliferation in vitro. In this report, we studied the structure of these analogs, using a combination of ECD and VCD spectroscopy and replica exchange molecular dynamics (REMD) simulations in water, TFE, and membrane-mimicking environments, in order to determine preferred conformations that may have importance in promoting the biological activities. Mostly random meander structures, punctuated by a β-turn at residues 1–4, were found in most peptides by REMD simulations. G17(1–3)-NH2, which cannot form a β-turn, failed to bind the non-CCK receptor, suggesting the importance of this feature for binding. Additionally, the β-turn appeared more frequently in longer sequences, possibly explaining the higher affinity of the non-CCK receptor for these peptides seen previously. Finally, C-terminally amidated peptides generally showed greater formation of turn structure than their non-amidated counterparts as shown by ECD spectra, suggesting the importance of peptide length in stabilizing turn structure in N-terminal sequences, and perhaps explaining the ability of G17(1–6)-NH2 to activate the non-CCK receptor where as the non-amidated G17(1–6) and shorter peptides do not.
Keywords: Gastrin; Gastrin-Gly; G17-Gly; Cancer; Structure; Bioactive conformation; Molecular dynamics simulation; Electronic circular dichroism; Vibrational circular dichroism;

Bioactivity of analogs of the N-terminal region of gastrin-17 by Jeffrey Copps; Shawn Ahmed; Richard F. Murphy; Sándor Lovas (2263-2267).
Gastrin-17-Gly (G17-Gly) has been shown to bind to non-CCK nanomolar and micromolar affinity sites on DLD-1 and HT-29 human colonic carcinoma cells and to stimulate cellular proliferation. However, in previous studies, we showed that C-terminal truncation of the gastrin-17 (G17) to the G17 analog G17(1–12) and then to G17(1–6)-NH2 did not remove the ability to bind to DLD-1 cells or to activate proliferation. This implies that residues and/or structural motifs required for bioactivity at these receptors rest in the N-terminal region of G17. In this work, radioligand binding studies conducted with further C-terminally truncated analogs revealed that sequences as short as G17(1–4) still bind to a single receptor with micromolar affinity. Additionally, cell proliferation assays showed that G17(1–12) stimulates proliferation of DLD-1 cells, as of HT-29 cells, but the sequences shorter than G17(1–6)-NH2, including non-amidated G17(1–6), were incapable of stimulating proliferation. These observations indicate that the tetrapeptide pGlu-Gly-Pro-Trp is the minimum N-terminal sequence for binding to the probable growth-promoting site on DLD-1 cells. Since analogs shorter than G17(1–6) are able to bind the receptor, these peptides may be of use for developing selective antagonists.
Keywords: Gastrin; G17; Gastrin-Gly; G17-Gly; Colon cancer; Minimal sequence; N-terminal sequence;

Sex-specific expression of BDNF and CART in the midbrain non-preganglionic Edinger–Westphal nucleus in the rat by Nicole M. Derks; Balázs Gaszner; Katharina Bernhardt; Eric W. Roubos; T. Kozicz (2268-2274).
In mammals, females generally appear more vulnerable to stressors than males. The non-preganglionic Edinger–Westphal nucleus (npEW) has been implicated in regulation of the stress response. Brain-derived neurotrophic factor (BDNF) and cocaine- and amphetamine-related transcript peptide (CART) are sex-specifically involved in the stress response too, and are present in the human and rat npEW. We hypothesized that male and female rats would differ in the expression of BDNF and CART in the npEW. Using immunocytochemistry and in situ hybridization we found that BDNF, CART and the estrogen receptor β (ERβ) are colocalized in the npEW. Q-RT-PCR showed no differences in CART and BDNF coding mRNAs between males and females, but quantitative immunocytochemistry revealed a 16% lower number of BDNF-immunoreactive neurons, and 19% lower CART-immunoreactivity in females compared to males. Considering the fact that Ucn1, CART and BDNF are co-expressed in the npEW with ERβ and their protein expression differs between males and females, we propose that the functioning of the npEW may contribute to the sex differences that exist in stress sensitivity.
Keywords: Sex difference; Major depression; Immunocytochemistry; In situ hybridization; Quantitative RT-PCR;

Leptin receptor mRNA in rat brain astrocytes by Hung Hsuchou; Weihong Pan; Maria J. Barnes; Abba J. Kastin (2275-2280).
We recently reported that mouse astrocytes express leptin receptors (ObR), and that obesity induces upregulation of astrocytic ObR. To provide further evidence of the importance of astrocytic ObR expression, we performed double-labeling fluorescent in situ hybridization (FISH) and immunohistochemistry in the rat hypothalamus. Laser confocal microscopic image analysis showed that ObR mRNA was present in glial fibrillary acidic protein (+) cells that show distinctive astrocytic morphology as well as in neurons. In addition to the presence of ObR mRNA, ObR protein was shown in both astrocytes and neurons in the rat hypothalamus by double-labeling immunohistochemistry. In cultured rat C6 astrocytoma cells treated with different doses of lipopolysaccharide for 6 h, the mRNA for ObRa or ObRb did not show significant changes, as measured by quantitative RT-PCR. However, the protein expression of both ObRa and ObRb, determined by Western blotting, was increased after the C6 cells were treated with either lipopolysaccharide or tumor necrosis factor-α. The results indicate that astrocytic ObR expression is present in rats as well as mice, and that it probably plays a role in the neuroinflammatory response.
Keywords: Leptin; ObR; mRNA; Astrocytes; Lipopolysaccharide; TNF; Fluorescent in situ hybridization; Immunohistochemistry;

Disturbance of circulating ghrelin and obestatin in chronic heart failure patients especially in those with cachexia by Xing Xin; An-Jing Ren; Xing Zheng; Yong-Wen Qin; Xian-Xian Zhao; Wen-Jun Yuan; Zhi-Fu Guo (2281-2285).
Plasma ghrelin was elevated in chronic heart failure (CHF) patients with cachexia. Obestatin, a sibling of ghrelin, opposes several actions of ghrelin. We, therefore, investigated plasma obestatin and ghrelin levels in patients with CHF. Total plasma ghrelin and obestatin levels were measured in 65 patients with CHF (22 with cardiac cachexia) and 15 controls. Ghrelin levels were significantly higher in patients with cachexia (1237.8 ± 47.9 pg/ml) than those without cachexia (P  = 0.041) and controls (P  < 0.01). Obestatin levels correlated positively with ghrelin levels, and obestatin levels were significantly increased in patients with cachexia (282.3 ± 13.0 pg/ml) than patients without cachexia and controls (both P  < 0.01). However, the ghrelin to obestatin ratios (4.5 ± 0.2) were significantly lower in CHF patients with cachexia than controls (P  < 0.01). Ghrelin and ratio of ghrelin to obestatin were independent predictors of the development of cardiac cachexia. No association was found between ghrelin, obestatin and New York Heart Association functional class, brain natriuretic peptide. There was disturbance of circulating ghrelin and obestatin in the CHF patients especially those with cachexia, which may have a role in the pathogenesis of cardiac cachexia in CHF.
Keywords: Ghrelin; Obestatin; Heart failure; Hormones; Nutrition;

Ghrelin inhibits post-infarct myocardial remodeling and improves cardiac function through anti-inflammation effect by Cong-Xin Huang; Ming-Jie Yuan; He Huang; Gang Wu; Yu Liu; Sheng-Bo Yu; Hai-Tao Li; Tao Wang (2286-2291).
Ghrelin is a novel growth hormone-releasing peptide, which has been shown to exert beneficial cardiac effects on chronic heart failure (CHF) recently. In this study, we attempted to investigate the mechanisms for the effect of ghrelin on ventricular remodeling following acute myocardial infarction (MI). Ligation of a coronary artery was used to create an MI in rats. One week after MI, ghrelin (100 μg/kg) or saline was injected subcutaneously twice a day for 4 weeks. When compared to sham groups, ghrelin administration significantly decreased left ventricular (LV) remodeling in post-MI rats, as indicated by increased LV maximum rate of pressure, LV fractional shortening and scar thickness; and decreased LV end-diastolic pressure, LV end-systolic diameter, LV end-diastolic diameter and cardiocytocytes apoptosis. Moreover, ghrelin inhibited the inflammatory response, as shown by decreased mRNA and protein levels of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α). Subsequently, the expression of matrix metalloproteinase (MMP)-2 and MMP-9 were also inhibited by ghrelin injection. Ghrelin alleviates LV dysfunction and ventricular remodeling in post-MI rats. This suggests that the beneficial effects of ghrelin on CHF may result from an inhibition of the inflammatory response.
Keywords: Ghrelin; Myocardial infarction; Pro-inflammatory cytokines; Matrix metalloproteinase;

Ghrelin vaccination decreases plasma MCP-1 level in LDLR−/−-mice by Eija Kellokoski; Outi kummu; Raisa Serpi; Petri Lehenkari; Olavi ukkola; Y. Antero Kesäniemi; Sohvi Hörkkö (2292-2300).
Ghrelin is a novel peptide hormone having growth hormone releasing activity and many endocrine and metabolic functions. In rats and pigs, ghrelin immunizations have recently been shown to induce an antibody response against ghrelin simultaneously with a decrease in body weight gain. Our aim was to test the role of ghrelin immunization on atherosclerosis and weight gain in mice. LDLR−/−-mice (n  = 36) were immunized with ghrelin-PADRE, PADRE alone and PBS and then placed on a high fat diet for 22 weeks. Weight gain and food intake were followed throughout the study. Acylated and total ghrelin, cytokines and MCP-1 were analyzed from plasma using commercial kits. Stomach ghrelin was assessed using qRT-PCR and immunohistochemistry. Atherosclerosis was determined from aorta and cross-sections at the end of study. Mice immunized with ghrelin-PADRE developed high plasma IgG titers to ghrelin simultaneously with a significant increase in plasma acylated and total ghrelin levels. Plasma MCP-1 levels decreased in mice immunized with ghrelin-PADRE compared to mice immunized with PADRE and PBS. There were no differences in atherosclerosis determined from aorta and cross-sections as well as in body weights and food intake in LDLR−/−-mice between the different immunization groups. Our data indicates that ghrelin-PADRE vaccination induces a strong exclusive IgG response to ghrelin and increases plasma acylated and total ghrelin levels in mice. Ghrelin vaccination decreases plasma MCP-1 levels even though no effects on developing signs of atherosclerosis or weight gain in mice were observed.
Keywords: Ghrelin; MCP-1; Atherosclerosis; Immunization; Obesity; LDLR KO mouse; PADRE;

Biphasic protective effect of oxytocin on cardiac ischemia/reperfusion injury in anaesthetized rats by Fariba Houshmand; Mahdieh Faghihi; Saleh Zahediasl (2301-2308).
Oxytocin (OT) is well known for its role in reproduction. However, evidence has emerged suggesting a role in cardiovascular system. The aim of this study was to investigate the cardioprotective effect of oxytocin on ischemia/reperfusion (I/R) injury in an in vivo rat. Myocardial ischemia, was surgically induced by means of left anterior descending coronary artery occlusion for 25 min followed by reperfusion for 120 min. Infarct size was evaluated using the staining agent 2,3,5-triphenyltetrazolium chloride. Creatine kinase-MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH) levels in plasma were analyzed to assess the degree of cardiac injury. Intraperitoneal administration of OT 0.001, 0.01 and 0.1 μg significantly reduced infarct size, LDH and CK-MB levels as compared to control (I/R) group and it had a biphasic effect on the reduction of ischemia/reperfusion injury. This biphasic effect was revealed as a U-shaped curve in which efficacy was optimal between very low and very high doses. Furthermore there were no significant differences in mean arterial pressure or heart rate between the OT treatment groups and control group during I/R. Blockade of specific OT receptors by atosiban (10−6  M) abolished or attenuated the effect of OT preconditioning. The result of this study shows that OT possess a dose-dependent cardioprotective effect against ischemia/reperfusion injury and so study of OT preconditioning may provide a new target site for therapeutic exploitation.
Keywords: Oxytocin; Preconditioning; Ischemia/reperfusion; Infarct size; LDH; CK-MB;

17β-Estradiol modulates local cardiac renin-angiotensin system to prevent cardiac remodeling in the DOCA-salt model of hypertension in rats by V. Shenoy; J.L. Grobe; Y. Qi; A.J. Ferreira; R.A. Fraga-Silva; G. Collamat; E. Bruce; M.J. Katovich (2309-2315).
Ventricular remodeling can play a detrimental role in the progression of cardiovascular diseases, leading to heart failure. The current study was designed to investigate the effects of 17β-estradiol (E2) on cardiac remodeling. Cardiac fibrosis and hypertrophy were examined in deoxycorticosterone acetate (DOCA)-salt treated rats with chronic, six-week administration of two different doses of E2. Bilaterally ovariectomized (Ovex) female Sprague–Dawley rats were randomly assigned to one of the following groups: Ovex-control; Ovex-DOCA; Ovex-DOCA + low-dose E2 (1.66 μg/day); or Ovex-DOCA + high-dose E2 (2.38 μg/day). All DOCA-treated rats were uninephrectomized and drinking water was replaced by 0.15 M NaCl solution for the remainder of the study period. DOCA-salt treatment resulted in a significant increase in blood pressure, which was not altered by estrogen replacement. Histological examinations revealed marked cardiac remodeling (both ventricular hypertrophy and interstitial fibrosis) with DOCA treatment, which was attenuated in animals receiving estrogen therapy. Western blot analysis demonstrated increased cardiac levels of angiotensin converting enzyme (ACE) with DOCA treatment, which was attenuated by E2 replacement. Furthermore, increased levels of cardiac angiotensin converting enzyme 2 (ACE2) protein were observed in animals receiving high-dose E2 replacement. These findings suggest that physiologically relevant estrogen replacement therapy has blood pressure-independent cardioprotective effects, which are possibly mediated through modulation of the cardiac renin-angiotensin system.
Keywords: Estrogen replacement therapy; Cardiac remodeling; Menopause; Hypertension;

Gene expression of (pro)renin receptor is upregulated in hearts and kidneys of rats with congestive heart failure by Takuo Hirose; Nobuyoshi Mori; Kazuhito Totsune; Ryo Morimoto; Takahiro Maejima; Takuya Kawamura; Hirohito Metoki; Kei Asayama; Masahiro Kikuya; Takayoshi Ohkubo; Masahiro Kohzuki; Kazuhiro Takahashi; Yutaka Imai (2316-2322).
Recent studies have revealed that (pro)renin receptor ((P)RR), a newly identified member of the renin–angiotensin system, was associated with organ damage in the kidney. However, there has been little information for (P)RR in hearts. To investigate the regulation of (P)RR in heart failure, we examined the expression of (P)RR in hearts and kidneys of rats with congestive heart failure (CHF) due to coronary ligation by quantitative RT-PCR and immunohistochemistry. Significantly increased levels of (P)RR mRNA were found in the atrium, right ventricle, non-infarcted part of left ventricle, infarcted part of left ventricle and kidney of CHF rats, when compared with sham operated rats (about 1.6-fold, 1.4-fold, 1.6-fold, 1.7-fold and 1.5-fold, respectively). Expression levels of mRNAs encoding renin and angiotensinogen in these heart and kidney tissues were also increased in the CHF rats. Immunohistochemistry showed positive (P)RR immunostaining in the myocardium, the renal tubular cells, and vascular smooth muscle and endothelial cells in the heart and the kidney. The renal tubular cells were more intensely immunostained in CHF rats than in sham operated rats. These findings suggest that the expression of (P)RR is increased in the hearts and kidneys of rats with heart failure, and that (P)RR may contribute to heart failure.
Keywords: Renin–angiotensin system; Organ damage; RT-PCR; Immunohistochemistry;

Vasoactive intestinal peptide (VIP), a 28 amino acid peptide, has been shown to inhibit proliferation of vascular smooth muscle cells. In previous studies VIP and VIP analogs have been used to study the effects of the peptide on vascular smooth muscle cell function. In this study an adenovirus encoding the VIP gene was used to investigate the mechanism of the antiproliferative action of VIP in vascular smooth muscle cells. Primary cultures of aortic and pulmonary artery smooth muscle cells from male Sprague–Dawley rats were transfected with varying concentrations of serotype 5 adenovirus encoding human VIP (Ad5CMVhVIP). Transfection efficiency and subsequently VIP gene expression were confirmed by western blot analysis and immunohistochemistry. In this study a decrease in vascular smooth muscle cell proliferation at vector concentrations of 150, 300 and 600 MOI (multiplicity of infection) was observed. In addition, there was increased production of cAMP in pulmonary artery and aortic smooth muscle cells transfected with VIP. Treatment of cells with a PKA inhibitor (Rp-8-BrcAMPs) restored proliferation to about 80% of control whereas treatment with the PKG inhibitor Rp-8-BrcGMPs had no significant effect suggesting the involvement of the PKA pathway in the antiproliferative actions of VIP.
Keywords: Pulmonary hypertension; Vascular smooth muscle cells; Vasodilators; cAMP;

Peptide corresponding to the C terminus of tissue factor pathway inhibitor inhibits mesangial cell proliferation and activation in vivo by Wang Liang; Juan Cheng; Rui Liu; Ji-ping Wang; Jin-gui Mu; Qing-hua Wang; Hui-jun Wang; Duan Ma (2330-2336).
Mesangial cells (MsCs) are one of the resident cell types in the glomerulus and are important with respect to its function and structure. The activation and proliferation of MsCs occur in several types of glomerulonephritis, particularly proliferative glomerulonephritis, producing a series of protein factors and matrix components that impair the normal structure and function of the glomerulus. To inhibit proliferation or induction of apoptosis is considered to be one mechanism that can be used to treat these diseases. In previous studies, we found that the tissue factor pathway inhibitor (TFPI) induces the apoptosis of cultured rat MsCs. Here, we expressed a series of TFPI fragments as fusion proteins to maltose binding protein (MBP-TFPI162–188, MBP-TFPI187–241, MBP-TFPI240–276, MBP-TFPI162–241, MBP-TFPI187–276 and MBP-TFPI162–276) and applied them to cultured rat mesangial cells. The C terminus of TFPI, a peptide corresponding to residues 240–276 of TFPI, was confirmed to induce apoptosis of MsCs in vitro. To observe the effect of this peptide on MsCs in vivo, we performed intramuscular gene transfer treatment on a rat model of proliferative glomerulonephritis with a plasmid containing the gene for the C terminus of TFPI. This revealed that the C terminus of TFPI exhibited suppressive effects on the activation and proliferation of MsCs and, thereby, improved renal function. Our data indicate that the C terminus of TFPI could be used in the treatment of proliferative glomerulonephritis.
Keywords: Anti-proliferation; Apoptosis; C terminus; Mesangial cell; Proliferative glomerulonephritis; TFPI;

cANF causes endothelial cell hyperpolarization by activation of chloride channels by Aaron Simon; Gong Xin Liu; Gideon Koren; Gaurav Choudhary (2337-2342).
Objectives: Natriuretic peptides bind with natriuretic peptide receptor (NPR)-C, which can alter cellular function through its interaction with the Gi protein complex. NPR-C has been found to mediate the activation of K+ channels and non-selective cation channels in vascular smooth muscle and cardiac fibroblast cells, respectively. However, the electrophysiological effect of NPR-C activation on endothelial cells (EC) has not been previously examined. In this study we sought to elucidate the effect of cANF(4-23), a selective NPR-C ligand, on EC membrane potential (E m). Methods/results: Changes in EC E m was measured through non-invasive fluorescence imaging. EC were preincubated in the potentiometric dye, DiBAC4(3) and subsequently exposed to cANF(4-23), in the presence of selective inhibitors of ion-channels or second messengers. NPR-C expression in rat lung microvascular endothelial cells was assessed by RT-PCR. cANF(4-23) induced a sustained decrease in EC cellular fluorescence, indicating endothelial cell hyperpolarization. The cANF-induced hyperpolarization could not be attenuated by TEA, barium, ouabain or by the reduction of extracellular Ca2+. Further, the cANF-induced hyperpolarization was insensitive to inhibition of Gi and protein kinase G (PKG), downstream messengers of NPRs. However, the Cl channel inhibitors, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, niflumic acid, and hypertonic saline attenuated the cANF-induced hyperpolarization. Perforated patch clamp recordings confirmed the cANF-induced current was carried by Cl and could be inhibited by niflumic acid. RT-PCR confirmed expression of NPR-C in vascular smooth muscle cells but not in EC. Conclusions: cANF causes hyperpolarization that is most likely mediated via activation of Cl channels by a PKG and Gi independent mechanism.
Keywords: Natriuretic peptide; Natriuretic peptide receptor-C; Endothelial cell; Ion channel; Membrane potential;

Skeletal contributions to plasma CNP forms: Evidence from regional sampling in growing lambs by Timothy C.R. Prickett; Chris J. Charles; Timothy G. Yandle; A. Mark Richards; Eric A. Espiner (2343-2347).
Unlike the cardiac circulating hormones, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP) appears to be largely tissue-based and circulates at concentrations considered insufficient to affect organ function. Consistent with CNP's crucial role in regulating skeletal growth, serial studies in juveniles show that both plasma CNP and aminoterminal proCNP (NTproCNP) are highly correlated with growth velocity raising the possibility that skeletal tissues contribute to circulating concentrations of CNP forms during the growing period. Hypothesizing that venous blood draining from bone dense regions is relatively enriched in CNP, we have performed trans-organ regional blood sampling for measurement of CNP forms in 4-week-old lambs and compared the findings to simultaneous levels of ANP and BNP. Because bone growth and CNP synthesis are inhibited by glucocorticoids, identical studies were also undertaken in lambs pretreated with dexamethasone. Highly significant positive arterio-venous gradients of CNP were found across the head, heart, leg and foot. Dexamethasone significantly reduced the CNP arterio-venous gradient across the head and leg but not heart, liver or kidney. In contrast, there was no evidence of tissue secretion of ANP or BNP except across the heart, and no effect on these gradients from dexamethasone. These findings of CNP enrichment in samples from bone dense regions in growing lambs, and their selective reduction by dexamethasone, provide in vivo evidence linking plasma and skeletal tissue concentrations of CNP and further support the use of plasma CNP forms as markers of bone growth.
Keywords: C-type natriuretic peptide; Growth; Bone; Glucocorticoids;

Involvement of orexin-A on micturition reflex in normal and cyclophosphamide-induced cystitis bladder in rat by Mizuki Kobayashi; Masayoshi Nomura; Hiroaki Fujihara; Hitoshi Suzuki; Hiroki Otsubo; Hisae Nishii; Naohiro Fujimoto; Tetsuro Matsumoto; Yoichi Ueta (2348-2356).
The purpose of the present study was to investigate the effect of orexin-A in the spinal cord on bladder function in normal rats and cyclophosphamide (CYP)-induced cystitis rat models. The effects of intrathecal (i.t.) injection of orexin-A (0.01, 0.1 and 1.0 nmol) on bladder function were examined during continuous infusion cystometrogram (CMG) in urethane anesthetized normal and CYP-induced cystitis rats. The effects of i.t. injection of selective orexin-1 receptor (OXR1) antagonist SB334867 (10 nmol) on orexin-A-induced bladder overactivity in normal rats and SB334867 (10 and 30 nmol) on changes in bladder function in normal and CYP-induced cystitis rats were investigated. The effects of intravenous (i.v.) injection of orexin-A (0.3 and 1.0 nmol) on micturition reflex were also investigated in normal rats. I.t. injection of orexin-A (0.1 and 1.0 nmol) significantly decreased the intercontraction intervals (ICI) in normal and CYP-induced cystitis rats. I.t. injection of SB334867 (10 nmol) significantly increased the ICI of orexin-A induced overactive bladder in normal rats and i.t. injection of SB334867 (30 nmol) also increased the ICI in normal rat bladder. However, in CYP-injected cystitis rat models, i.t. injection of SB334867 did not change the bladder function. I.v. injection of orexin-A failed to affect the bladder function in normal rats. Orexin mRNA levels in the lateral hypothalamus were significantly decreased in CYP-induced cystitis rats. These results indicate that orexin-A in the spinal cord activates micturition reflex via OXR1 in normal rats. In addition, OXR1 antagonist did not have any effect on micturition reflex in CYP-induced cystitis rats.
Keywords: Bladder; Micturition reflex; Orexin-A; Orexin-1 receptor; Spinal cord;

Multifunctional role of VIP in prostate cancer progression in a xenograft model: Suppression by curcumin and COX-2 inhibitor NS-398 by Ana B. Fernández-Martínez; Ana M. Bajo; Ana Valdehita; M. Isabel Arenas; Manuel Sánchez-Chapado; María J. Carmena; Juan C. Prieto (2357-2364).
We used an in vivo model of human experimental prostate cancer in order to shed a new light on the effects of vasoactive intestinal peptide (VIP) on tumor growth as well as its pro-metastatic potential in this disease. We used nude mice subcutaneously injected with prostate cancer androgen-independent PC3 cells for 30 days. The regulatory role of VIP on cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) expression as well as on matrix metalloproteinase-2 and 9 (MMP-2 and 9) activities was examined. A selective COX-2 inhibitor, NS-398, and curcumin were used to block VIP effects. Xenografts of VIP-treated PC3 prostate cancer cells in nude mice gave tumors that grew significantly faster than those in the untreated group. It is conceivably a result of both the trophic effect of VIP on prostate cancer cells and the proangiogenic action of the neuropeptide in the growing tumor. We show the overexpression at mRNA and/or protein levels of VIP, its main receptor VPAC1, the major angiogenic factor VEGF, and the pro-inflammatory enzyme COX-2 as well as the increased activity of MMP-2 and 9 in tumors derived from VIP-treated PC3 cells as compared with control group. The overexpression of the above biomarkers was suppressed in tumors derived from VIP-treated PC3 cells that had been previously incubated with curcumin or NS-398. Thus, the potential therapeutic role of curcumin and selective COX-2 inhibitors in combination with available VIP antagonists should be considered in prostate cancer therapy as supported by their inhibitory activities on tumor cell growth.
Keywords: VIP; Curcumin; COX-2; Matrix metalloproteinases; Prostate cancer;

Epinecidin-1 peptide induces apoptosis which enhances antitumor effects in human leukemia U937 cells by Jyh-Yih Chen; Wei-Ju Lin; Jen-Leih Wu; Guor Mour Her; Cho-Fat Hui (2365-2373).
Epinecidin-1 is an antimicrobial peptide present in the grouper (Epinephelus coioides). In this study, the antitumor activity of a synthetic epinecidin-1 peptide was tested. The in vitro results showed that epinecidin-1 inhibited the proliferation of human leukemia U937 cells and increased the ADP/ATP ratio after 24 h of treatment. The DNA fragmentation assay, flow cytometric assay, and caspases-3, -8, and -9 assays indicated that epinecidin-1 could induce apoptosis in U937 cells. Real-time RT-PCR results showed regular increases in tumor necrosis factor (TNF)-α after treatment with 4 μg/ml epinecidin-1 from 4 to 24 h; interleukin (IL)-10, interferon (INF)-r, p53, IL-15, and IL-6 increased after treatment with 2 μg/ml epinecidin-1 for 4–12 h. These results suggest that the epinecidn-1 inhibited U937 cells, induced apoptosis in response to cytokine production, and may have pleiotropic effects on different cells.
Keywords: Epinecidin-1; Tumor cell inhibition; Lytic peptide;

Inhibiting the inhibitors: Retro-inverso Smac peptides by Julia Hossbach; Elke Michalsky; Peter Henklein; Marten Jaeger; Peter T. Daniel; Robert Preissner (2374-2379).
Resistance against apoptosis-inducing anti-cancer drugs remains a severe problem in therapy. One reason is the overexpression of inhibitors of apoptosis proteins (IAPs), a group of proteins responsible for the prevention of apoptosis induction by inactivation of initiator caspases. The natural inhibitor of the IAPs is the protein Smac, which impedes the binding to the caspases. Although Smac is a potent inhibitor, Smac peptides are not very stable in vivo and thus not applicable in therapy. Bioinformatical methods were applied to design Smac-derived peptides to break the therapy resistance in IAP high-expressing tumor cells. The exchange of amino acids in the Smac peptides AVPI and AVPF against unnatural amino acids leads to an improvement of the apoptosis sensitivity. The variety of Smac peptides was filtered by computational docking. Moreover, Smac-derived peptides with sufficient binding to the IAPs were tested in IAP-expressing Hodgkin Lymphoma cell lines.
Keywords: Apoptosis; Smac; Inhibitor of apoptosis proteins; Computational docking;

Novel peptide mimetic small molecules of the HAV motif in N-cadherin inhibit N-cadherin-mediated neurite outgrowth and cell adhesion by Susan M. Burden-Gulley; Theresa J. Gates; Sonya E.L. Craig; Sara F. Lou; Samantha A. Oblander; Scott Howell; Mukur Gupta; Susann M. Brady-Kalnay (2380-2387).
The cell adhesion molecule, N-cadherin, stabilizes cell–cell junctions and promotes cellular migration during tissue morphogenesis in development. N-cadherin is also implicated in mediating tumor progression and metastasis in cancer. Therefore, developing antagonists of N-cadherin adhesion may be of therapeutic value in cancer treatment. The amino acid sequence HAV in the extracellular domain of N-cadherin is required for N-cadherin-mediated adhesion and migration. A cyclic peptide, ADH-1, derived from the N-cadherin HAV site is an effective antagonist of N-cadherin-mediated processes and is now in clinical trials for cancer chemotherapy. Because it is a peptide, ADH-1 has certain limitations as a drug, namely its metabolic instability and lack of oral delivery. Adherex set out to identify small molecule antagonists of N-cadherin, which would be more amenable to therapeutic use. Using three-dimensional computational screening, Adherex identified a set of small molecules as potential antagonists with sufficient structural similarity to the HAV region of N-cadherin. We tested the ability of these small molecules to interfere with two N-cadherin-dependent processes: neurite outgrowth (axonal migration) and N-cadherin-dependent cell adhesion. We identified 21 N-cadherin antagonists of varying potency. More importantly, our studies demonstrate that these compounds are significantly more potent than ADH-1 at perturbing N-cadherin-mediated processes. The IC50 of ADH-1 is 2.33 mM while the IC50 of the small molecules ranges from 4.5 to 30 μM. Given the efficacy of ADH-1 for treating cancer, these small molecule antagonists will be highly effective in treatment of cancer metastasis and conditions of aberrant neurite outgrowth, such as neuropathic pain.
Keywords: N-cadherin; Neurite outgrowth; Adhesion; Peptidomimetic; Small molecules;

Inflammation is part of the host defense mechanism against harmful matters and injury; however, aberrant inflammation is associated to the development of chronic diseases such as cancer. Lunasin is a novel peptide that demonstrates potential anticancer activity against mammalian cancer cell lines and may play a role in inflammation. The objective of this study was to determine the mechanism of action by which lunasin and lunasin-like peptides exert their anti-inflammatory properties using RAW 264.7 macrophage cell line as an in vitro model. We purified three peptides (5, 8, and 14 kDa) from defatted soybean flour with a positive immunoreactivity towards lunasin mouse monoclonal antibody. Treatment with these peptides (10–50 μM) resulted in the inhibition of pro-inflammatory markers in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. The 5 kDa peptide inhibited most potently pro-inflammatory markers including interleukin-6 production (IC50  = 2 μM), interleukin-1β production (IC50  = 13 μM), nuclear factor-kappa B (NF-κB) transactivation (IC50  = 21 μM), cyclooxygenase-2 expression (IC50  = 25 μM), nitric oxide production (IC50  = 28 μM), inducible nitric oxide synthase expression (IC50  = 37 μM), prostaglandin E2 production (IC50  = 41 μM), p65 nuclear translocation (IC50  = 48 μM) and p50 nuclear translocation (IC50  = 77 μM). In conclusion, lunasin and lunasin-like peptides purified from defatted soybean flour inhibited inflammation in LPS-induced RAW 264.7 macrophage by suppressing NF-κB pathway.
Keywords: Lunasin; Lunasin-like peptides; Soybean; Inflammation; NF-κB;

Synthesis, radiolabeling and biological evaluation of a neutral tripeptide and its derivatives for potential nuclear medicine applications by Susmita Chandra; Kakali De; Santanu Ganguly; Bharat Sarkar; Mridula Misra (2399-2408).
Peptides are important regulators of growth and cellular functions not only in normal tissue but also in tumors. So they are becoming radioligands of increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. So development of new peptide radiopharmaceuticals is becoming one of the most important areas in nuclear medicine research. A small tripeptide derivative NH2PhePheCys was synthesized by Fmoc solid phase peptide synthesis using an automated synthesizer. The oxidized form, i.e. NH2PhePheCysCysPhePheNH2, was also prepared by iodine oxidation method from NH2PhePheCys(ACM). The ligands were analyzed by HPLC and mass spectroscopy. They were radiolabeled with 99mTc using SnCl2. In vitro analytical studies and biological characterizations were performed using the peptide radiopharmaceuticals. Images taken under gamma camera showed very high uptake in the liver, lung and spleen. Significant uptake was also observed in bone marrow and brain for 99mTc-NH2PhePheCys. Metabolites were produced in vivo when the radiopharmaceuticals were injected intravenously and were identified from rat brain and liver homogenate studies. Clearance through kidney did not show any evidence of breaking of the labeled compounds and formation of free 99mTc. Radiopharmaceuticals prepared using tripeptide and hexapeptide ligands were transported into the brain through blood brain barrier depending on the size and sequence characteristics. Using this property of peptide new derivatives can be prepared to develop 99mTc radiopharmaceuticals for imaging normal brain tissues as well as for diagnosing various brain disorders.
Keywords: Peptide; 99mTc-labeled peptides; Brain imaging;

In vitro binding and in vivo biodistribution studies of the neuroprotective peptide humanin using [125I]humanin derivatives by Alexandra Evangelou; Christos Zikos; Dimitra Benaki; Maria Pelecanou; Penelope Bouziotis; Minas Papadopoulos; Lenka Borovickova; Iva Vesela; Tomas Elbert; Gabriela Kunešová; Ioannis Pirmettis; Maria Paravatou-Petsotas; Jirina Slaninová; Evangelia Livaniou (2409-2417).
Humanin (HN) and HN-derivatives are a family of peptides first reported in the last decade with potent in vitro and in vivo neuroprotective activity, which is mediated through a not completely elucidated mechanism. Recently, our group has evaluated the effect of various HN-derivatives on the 3-quinuclidinyl benzilate (QNB)-induced impairment of spatial orientation and memory in rats, by employing the T-maze test. In the present work four new, tyrosine containing HN-derivatives were synthesized (Y-PAGASRLLLTGEIDLP, peptide I; Y-PAGASRLLLLTGEIDLP, peptide II; Y-SALLRSIPAPAGASRLLLTGEIDLP, peptide III; Y-SALLRSIPAPAGASRLLLLTGEIDLP, peptide IV). The neuroprotective action of these peptides was evaluated in the T-maze test and the most active among them (peptides I and III) was radiolabeled with 125I. The pure monoradioiodinated peptides were used in: (i) in vitro binding studies with various neuronal cell lines and with brain and stomach membranes from rats and mice and (ii) in vivo biodistribution studies in rats and mice. Moreover, the metabolic stability of the above radiolabeled peptides was studied. Under the experimental conditions used, our data do not confirm the existence of specific binding sites for HN on the neuronal tissue. Nevertheless, they are setting the basis for further relevant studies aiming at the clarification of the mode of the neuroprotective action of HN-peptides.
Keywords: Neuroprotective peptides; Humanin derivatives; [125I]Radiolabeling; In vitro cell binding; In vivo biodistribution; Metabolic stability;

The C-terminal amidated analogue of the substance P (SP) fragment SP1–7 attenuates the expression of naloxone-precipitated withdrawal in morphine dependent rats by Qin Zhou; Anna Carlsson; Milad Botros; Rebecca Fransson; Anja Sandström; Torsten Gordh; Mathias Hallberg; Fred Nyberg (2418-2422).
We previously demonstrated that intracerebroventricular (i.c.v.) administration of the substance P (SP) aminoterminal fragment SP1–7 attenuates the expression of morphine withdrawal in the male rat. In this study we have used a synthetic analogue of this peptide, i.e. the SP1–7 amide showing higher binding potency than the native heptapeptide, in a similar experimental set-up. Thus, Wistar male rats were made tolerant to morphine by daily injections of the opiate during 8 days. Following peptide administration (i.c.v.) and a subsequent naloxone challenge a variety of physical syndromes of withdrawal were recorded. We observed that the SP1–7 amide potently and dose-dependently reduced several signs of reaction to morphine withdrawal. Interestingly, the effect of the peptide amide was significantly attenuated by the addition of the sigma agonist (+)-SKF-10047. We conclude that the SP1–7 amide mimics the effect of the native SP fragment and that the mechanisms for its action involve a sigma receptor site.
Keywords: Morphine; Opiate; Substance P (SP); SP1–7 amide; Rat; Withdrawal; Sigma receptor;

The peptides galanin (GAL) and orexin (OX) share common features with the opioid enkephalin (ENK) in their relationship to ingestive behavior, stimulating consumption of a fat-rich diet and ethanol when injected into the hypothalamus. Since receptors for GAL and OX are dense in areas where ENK-expressing neurons are concentrated, these non-opioid peptides may exert their effects, in part, through the stimulation of endogenous ENK. This study was conducted to determine whether injection of GAL or OX affects the expression of ENK in hypothalamic and mesolimbic nuclei involved in consummatory behavior. Rats were injected with GAL (1 μg), OX-A (1 μg), or saline vehicle just dorsal to the hypothalamic paraventricular nucleus (PVN). They were sacrificed 1 h later for analysis of ENK mRNA levels in the PVN, ventral tegmental area (VTA), central nucleus of the amygdala (CeA), and nucleus accumbens (NAc). Both GAL and OX had similar effects, significantly increasing ENK mRNA expression in each of these areas, except for the NAc. This enhanced ENK expression in the PVN, VTA and CeA was demonstrated with real-time quantitative polymerase chain reaction and confirmed in separate groups using radiolabeled and digoxigenin-labeled in situ hybridization. These findings demonstrate that the non-opioid peptides, GAL or OX, which have similar effects on consummatory behavior, are also similar in their effect on endogenous ENK. In light of published findings showing an opioid antagonist to block GAL- and OX-induced feeding, these results provide additional evidence that ENK is involved in mediating the common behavioral effects of these peptides.
Keywords: Orexin; Galanin; Enkephalin; Rat; mRNA;

Endogenous opiates and behavior: 2008 by Richard J. Bodnar (2432-2479).
This paper is the 31st consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2008 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17).
Keywords: Mu-opioid receptor; Delta-opioid receptor; Kappa-opioid receptor; ORL-1 receptor; Enkephalin; Endorphin; Endomorphin; Nociceptin; Opiate agonists; Opaite antagonists;

NAPVSIPQ (NAP) is a small, active fragment of activity-dependent neuroprotective protein that has neuroprotective and memory enhancing properties at very low concentrations. Previous research demonstrated that 1–2 weeks of treatment provided memory enhancing effects in normal middle-aged and cholinergically lesioned rats. Improvement in cognitive performance was shown in 12-month-old C57Bl6/J mice after 10 days of oral treatment with D-NAP and D-SALLRSIPA. Additionally, NAP-related cognitive benefits on spatial memory were observed in a 3 × Tg Alzheimer mouse model after 6 months of chronic administration at a moderate stage of disease. In this study, the potential memory enhancing effect of NAP was investigated using the APP23 transgenic mouse model for Alzheimer's disease. Twelve-month-old male heterozygous APP23 mice and their wild-type control littermates were intraperitoneally injected with 0.3 μg NAP/g body weight or with saline vehicle for 22 consecutive days. Cognitive performance training in the Morris Water Maze (MWM) started on day 8 of treatment. The internal validity of our study was demonstrated by the fact that the APP23 mice performed significantly worse in the MWM than wild-type animals. Treatment with NAP, however, did not exert any significant effects on MWM performance. Although we failed to show significant memory enhancing effects in this study, NAP might be a promising peptide for disease-modifying therapy in neurodegenerative disease, but short-term effects are probably not to be expected. Also, most likely, treatment should start in an early stage, i.e. before full-blown pathology is eminent, and the necessary treatment period should enclose several months.

Neuromedin U-induced anorexigenic action is mediated by the corticotropin-releasing hormone receptor-signaling pathway in goldfish by Keisuke Maruyama; Kohei Wada; Kotaro Ishiguro; Sei-Ichi Shimakura; Tatsuya Wakasugi; Minoru Uchiyama; Seiji Shioda; Kouhei Matsuda (2483-2486).
Our recent research has indicated that neuromedin U (NMU) orthologs exist in goldfish, and that NMU consisting of 21 amino acid residues (NMU-21) can potently inhibit food intake in goldfish, as is the case in rodents. However, the anorexigenic pathway of NMU-21 has not yet been clarified in this species. Corticotropin-releasing hormone (CRH), CRH-related peptides and α-melanocyte-stimulating hormone (α-MSH), which exert potent anorexigenic effects, are important mediators involved in feeding regulation in fish. We examined whether CRH or α-MSH mediates NMU-21-induced anorexigenic action in goldfish. We first investigated the effect of intracerebroventricular (ICV) administration of NMU-21 at 100 pmol/g body weight (BW), which is enough to suppress food intake, on expression levels of mRNA for CRH and proopiomelanocortin (POMC) in the hypothalamus. ICV-injected NMU-21 induced a significant increase in the expression level of CRH mRNA, but not that of POMC mRNA. We also examined the effects of ICV administration of the CRH 1/2 receptor antagonist, α-helical CRH(9–41), and the melanocortin 4 receptor antagonist, HS024, on the anorexigenic action of ICV-injected NMU-21. The anorexigenic effect of NMU-21 was blocked by treatment with α-helical CRH(9–41) at 400 pmol/g BW, but not HS024 at 200 pmol/g BW. These results suggest that the anorexigenic action of NMU-21 is mediated by the CRH 1 or 2 receptor-signaling pathway in goldfish.