Peptides (v.27, #11)

Contents continued (VII-VIII).

Three insulin–relaxin-like genes in Ciona intestinalis by Robert Piotr Olinski; Carl Dahlberg; Mike Thorndyke; Finn Hallböök (2535-2546).
The Ciona intestinalis genome harbors three insulin-like genes: INS-L1, -L2 and -L3. Conserved synteny between the Ciona-human genomes predicts that Ciona INS-Ls are orthologous to the vertebrate insulin–relaxin family, but this relation cannot be inferred from molecular phylogeny. A conserved protein core with six cysteines; typical arrangement of B-, C- and A-protein domains; pro-protein maturation mode; and putative insulin receptor-binding sites were identified in Ciona INS-L proteins. ESTs used to assemble exonic sequences of INS-Ls combined with qRT-PCR analysis provided evidence that the predicted genes are expressed in the developing and adult Ciona. Our results support that Ciona INS-L1 is orthologous to the vertebrate insulin-like/relaxin genes, INS-L2 to insulin genes and INS-L3 to IGF genes. Our analysis also implies that the insulin-like/relaxin ancestor switched receptor type from tyrosine kinase- to GPCR-type, whereas insulin–IGF subfamily retained the tyrosine kinase-type of receptor. We propose that this receptor-switch occurred after the time when urochordates branched from the common chordate lineage, but before the two genome-duplications at the root of the vertebrates.
Keywords: Ciona intestinalis; Gene duplication; IGF; Insulin; Insulin-like protein; Relaxin;

Insulin-like peptides are key regulators of metabolism, reproduction, and senescence in higher eukaryotic organisms. Here we present the identification, expression, and tissue localization of eight genes encoding insulin-like peptides (ILPs) in the yellow fever mosquito, Aedes aegypti. All eight ILPs share the conserved features of the insulin superfamily as prepropeptides consisting of contiguous signal, B, C, and A peptides. However, one of the ILPs has a truncated C peptide and a carboxy terminal extension, features consistent with insulin growth factors. Transcripts for five of the ILPs occurred predominantly in the heads (brains) of larval, pupal, and adult mosquitoes. Transcripts of two other genes, one of which was the putative insulin growth factor, were present in the head, thorax and abdomens of all stages. The final ILP was predominantly expressed in abdomen. Results from immunocytochemistry with two different ILP antisera showed cellular localizations in the nervous system and midgut that corroborated the existence of these expression patterns. Three of the ILP genes are so closely linked that only the 5′ region of the first ILP gene likely suffices as a promoter, indicating that these genes form a eukaryotic operon. The nearly identical expression pattern of these three ILPs supported this idea. Finally, the phylogenetic relationship of ILPs from three dipteran species, Ae. aegypti, the African malaria mosquito (Anopheles gambiae), and Drosophila melanogaster is presented as a step towards understanding the structural and functional diversity of insect ILPs.
Keywords: Neurosecretion; ILP; Eukaryotic operon;

Presence of immunoreactive salusin-α in human serum and urine by Kengo Sato; Takatoshi Koyama; Toru Tateno; Yukio Hirata; Masayoshi Shichiri (2561-2566).
Salusins, identified from a full-length enriched human cDNA library by bioinformatics analyses, show mitogenic, neuromodulatory and hemodynamic activities in rats. They are expressed in a wide variety of human tissues, but their precise structures and levels in human body fluids remain unknown. We developed a radioimmunoassay suitable for the detection of immunoreactive human salusin-α and characterized the molecular forms and concentrations of salusin-α in human serum and urine. The assay allowed for measurement of immunoreactive salusin-α concentrations as low as 1 fmol/tube after extraction of serum with an octyl-silica column, and the concentration required for 50% inhibition of binding was 40 fmol/tube. Cross-reactivities with salusin-β and other bioactive peptides were negligible. Salusin-α-like immunoreactivity in normal human serum and urine ranged from 11.0 to 40.4 pmol/l (mean ± S.D., 23.3 ± 8.1 pmol/l, n  = 31) and from 18.6 to 367.3 pmol/l (mean ± S.D., 156.8 ± 95.8 pmol/l), respectively. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a major immunoreactive component that coeluted with authentic salusin-α. These data indicate the presence of salusin-α in human serum and urine, thereby verifying the initially predicted processing sites for salusin-α in humans.
Keywords: Salusin-α; Radioimmunoassay; Serum; Urine; HPLC;

De novo designed cyclic cationic peptides as inhibitors of plant pathogenic bacteria by Sylvie Monroc; Esther Badosa; Lidia Feliu; Marta Planas; Emili Montesinos; Eduard Bardají (2567-2574).
Head-to-tail cyclic peptides of 4–10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of l-Phe with d-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 μM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t R), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a d-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t R. The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.
Keywords: Cyclic peptides; Antibacterial peptides; Plant pathogens; Bactericidal;

Improvement of cyclic decapeptides against plant pathogenic bacteria using a combinatorial chemistry approach by Sylvie Monroc; Esther Badosa; Emili Besalú; Marta Planas; Eduard Bardají; Emili Montesinos; Lidia Feliu (2575-2584).
Cyclic decapeptides were developed based on the previously reported peptide c(LysLeuLysLeuLysPheLysLeuLysGln). These compounds were active against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae and Xanthomonas vesicatoria. A library of 56 cyclic decapeptides was prepared and screened for antibacterial activity and eukaryotic cytotoxicity, and led to the identification of peptides with improved minimum inhibitory concentration (MIC) against P. syringae (3.1–6.2 μM) and X. vesicatoria (1.6–3.1 μM). Notably, peptides active against E. amylovora (MIC of 12.5–25 μM) were found, constituting the first report of cyclic peptides with activity towards this bacteria. A second library based on the structure c(X1X2X3X4LysPheLysLysLeuGln) with X being Lys or Leu yielded peptides with optimized activity profiles. The activity against E. amylovora was further improved (MIC of 6.2–12.5 μM) and the best peptides displayed a low eukaryotic cytotoxicity at concentrations 30–120 times higher than the MIC values. A design of experiments permitted to define rules for high antibacterial activity and low cytotoxicity, being the main rule X2  ≠ X3, and the secondary rule X4  = Lys. The best analogs can be considered as good candidates for the development of effective antibacterial agents for use in plant protection.
Keywords: Antibacterial activity; Eukaryotic cytotoxicity; Fire blight; Combinatorial chemistry; Cyclic peptides; Design of experiments;

Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice by Carlo P.J.M. Brouwer; Sylvia J.P. Bogaards; Marty Wulferink; Markwin P. Velders; Mick M. Welling (2585-2591).
The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the α-helix or β-sheet domains of the human cationic peptide ubiquicidin (UBI 1–59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1–59 and for synthetic fragments comprising amino acids 31–38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.
Keywords: Antimicrobial peptide; Synthetic fragments; MRSA; In vitro killing; Experimental infections; Microbicidal activity; Technetium; Detection of infections;

Effects of the antimicrobial peptide BMAP-27 in a mouse model of obstructive jaundice stimulated by lipopolysaccharide by Roberto Ghiselli; Oscar Cirioni; Andrea Giacometti; Federico Mocchegiani; Fiorenza Orlando; Cristina Bergnach; Barbara Skerlavaj; Carmela Silvestri; Agnese Della Vittoria; Margherita Zanetti; Marco Rocchi; Giorgio Scalise; Vittorio Saba (2592-2599).
An experimental study was designed to investigate the efficacy of BMAP-27, a compound of the cathelicidin family, in neutralizing Escherichia coli 0111:B4 lipopolysaccharide (LPS) in bile duct-ligated mice. Main outcome measures were: endotoxin and TNF-α concentrations in plasma, evidence of bacterial translocation in blood and peritoneum, and lethality. Adult male BALB/c mice were injected intraperitoneally with 2 mg/kg E. coli 0111:B4 LPS 1 week after sham operation or bile duct ligation (BDL). Six groups were studied: sham with placebo, sham with 120 mg/kg tazobactam–piperacillin (TZP), sham with 1 mg/kg BMAP-27, BDL with placebo, BDL with 120 mg/kg TZP, and BDL with 1 mg/kg BMAP-27. After LPS, TNF-α plasma levels were significantly higher in BDL mice compared to sham-operated animals. BMAP-27 achieved a significant reduction of plasma endotoxin and TNF-α concentration when compared with placebo- and TZP-treated groups. On the other hand, both TZP and BMAP-27 significantly reduced the bacterial growth compared with saline treatment. Finally, LPS induced 60% and 55% lethality in BDL placebo- and TZP-treated treated mice and no lethality in sham-operated mice, while only BMAP-27 significantly reduced the lethality to 10%. In light of its dual antimicrobial and anti-endotoxin properties, BMAP-27 could be an interesting compound to inhibit bacterial translocation and endotoxin release in obstructive jaundice.
Keywords: LPS; Lipopolysaccharide; Jaundice; BMAP-27; Bacterial translocation;

Experimental study on the efficacy of combination of α-helical antimicrobial peptides and vancomycin against Staphylococcus aureus with intermediate resistance to glycopeptides by Oscar Cirioni; Carmela Silvestri; Roberto Ghiselli; Andrea Giacometti; Fiorenza Orlando; Federico Mocchegiani; Leonardo Chiodi; Agnese Della Vittoria; Vittorio Saba; Giorgio Scalise (2600-2606).
An experimental study has been performed to compare the in vitro activity and the in vivo efficacy of magainin II and cecropin A, two α-helical antimicrobial peptides, and vancomycin against Staphylococcus aureus with intermediate resistance to glycopeptides. In vitro experiments included MIC determination, time-kill and synergy studies. For in vivo studies, a mouse model of staphylococcal sepsis has been used. Main outcome measures were: lethality, quantitative blood cultures and detection of TNF-alpha and interleukin-6 (IL-6) plasma levels. Combinations of α-helical antimicrobial peptides showed in vitro synergistic interaction. Significant increase in efficacy was also observed in vivo: combined-treated groups had significant lower bacteremia when compared to single-treated groups. Magainin II combined with vancomycin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of these combinations and provide future therapeutic alternative in infections due to glycopeptides resistant staphylococci in the coming years.
Keywords: Staphylococci; Glycopeptides; Antimicrobial peptides; Staphylococcal sepsis;

The antibacterial activities of synthetic human β-defensin analogs, constrained by a single disulfide bridge and in the reduced form, have been investigated. The peptides span the carboxy-terminal region of human β-defensins (HBD-1–3), which have a majority of cationic residues present in the native defensins. The disulfide constrained peptides exhibited activity against Escherichia coli and Staphylococcus aureus whereas the reduced forms were active only against E. coli. The antibacterial activities were attenuated in the presence of increasing concentrations of NaCl and divalent cations such as Ca2+ and Mg2+. The site of action was the bacterial membrane. Peptides spanning the carboxy-terminal region of human β-defensins could be of help in understanding facets of antimicrobial activity of β-defensins such as salt sensitivity and mechanisms of bacterial membrane damage.
Keywords: Antibacterial activity; Electron microscopy; Human β-defensins; Membrane damage; Single disulfide analogs; Salt sensitivity;

The antibacterial activity of peptides without disulfide bridges, spanning the carboxy-terminal segment of arthropod defensins, has been investigated. Although all the peptides have net positive charges, they exhibited varying antibacterial potencies and spectra. Atomic force and fluorescence microscopic analyses indicate that the peptides exert their activity by permeabilizing the outer and inner membranes of Gram-negative bacteria such as Escherichia coli. It appears that the plasticity observed in the activity of mammalian defensins with respect to sequence, number of disulfide bridges or net positive charge, is also observed in insect defensins.
Keywords: Arthropod defensins; Atomic force microscopy; Carboxy-terminal analogs; Salt-sensitivity; Fluorescence microscopy;

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus by Katsuhiro Konno; Miki Hisada; Hideo Naoki; Yasuhiro Itagaki; Renato Fontana; Marisa Rangel; Joacir Stolarz Oliveira; Marcia Perez dos Santos Cabrera; João Ruggiero Neto; Izumi Hide; Yoshihiro Nakata; Tadashi Yasuhara; Terumi Nakajima (2624-2631).
A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu–Asn–Leu–Lys–Gly–Ile–Phe–Lys–Lys–Val–Ala–Ser–Leu–Leu–Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear α-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of α-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae.
Keywords: Eumenitin; Solitary wasp venom; Antimicrobial peptide; Cationic linear α-helical peptide; Amphipathic α-helix structure;

Two bradykinin-related peptides (Protopolybiakinin-I and Protopolybiakinin-II) were isolated from the venom of the social wasp Protopolybia exigua by RP-HPLC, and sequenced by Edman degradation method. Peptide sequences of Protopolybiakinin-I and Protopolybiakinin-II were DKNKKPIRVGGRRPPGFTR-OH and DKNKKPIWMAGFPGFTPIR-OH, respectively. Synthetic peptides with identical sequences to the bradykinin-related peptides and their biological functions were characterized. Protopolybiakinin-I caused less potent constriction of the isolated rat ileum muscles than bradykinin (BK). In addition, it caused degranulation of mast cells which was seven times more potent than BK. This peptide causes algesic effects due to the direct activation of B2-receptors. Protopolybiakinin-II is not an agonist of rat ileum muscle and had no algesic effects. However, Protopolybiakinin-II was found to be 10 times more potent as a mast cell degranulator than BK. The amino acid sequence of Protopolybiakinin-I is the longest among the known wasp kinins.
Keywords: Wasp toxins; Hymenoptera venom; Bradykinin-related peptides; Muscle contraction; Edman degradation chemistry; Mast cell degranulation; Inflammatory peptide;

Direct cDNA cloning of novel conotoxins of the T-superfamily from Conus textile by Sulan Luo; Dongting Zhangsun; Ben Zhang; Xin Chen; Jianchen Feng (2640-2646).
The T-superfamily is a large and diverse group of peptides, widely distributed in venom ducts of all major feeding types of Conus. These peptides are likely to be functionally diverse. A directed PCR-based approach using primers based on the conserved signal sequence was applied to investigate new conotoxins of the T-superfamily from Conus textile native to Hainan. Using RT-PCR and 3′-RACE, four novel cDNA sequences encoding precursor peptides were identified in C. textile. They share a common T-superfamily cysteine pattern (CC–CC, with two disulfide bridges). The predicted peptides are small (9–12 amino acids). TeAr193 composed of nine amino acid residues is one of the shortest T-superfamily conotoxins ever found. Patterns of sequence divergence and Cys codon usage define the major T-superfamily branches and suggest how these separate branches arose. The sequences of the signal regions exhibited highest conservation, whereas the sequences of the mature peptides were either almost identical or highly divergent; and conservation of the pro-region was intermediate between that observed in signal and toxin regions. The elucidated cDNAs of the four toxins will facilitate a better understanding of the relationship between structure and function.
Keywords: T-superfamily conotoxin; cDNA cloning; Conus textile native to Hainan;

Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels by V. Sabareesh; K. Hanumae Gowd; P. Ramasamy; S. Sudarslal; K.S. Krishnan; S.K. Sikdar; P. Balaram (2647-2654).
Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca2+ channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP D WDPWC-NH 2 and Am975, GCO D WDPWC-NH 2 (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca2+ channels. While Lo959 increases the Ca2+ current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.
Keywords: Cone snails; Contryphans; Post-translational modifications; Slow conformational interconversion; DRG neurons, Voltage-activated calcium channels;

Oxylepitoxin-1, a reversible neurotoxin from the venom of the inland taipan (Oxyuranus microlepidotus) by Carol Clarke; Sanjaya Kuruppu; Shane Reeve; A. Ian Smith; Wayne C. Hodgson (2655-2660).
This study describes the characterization of oxylepitoxin-1 (MW 6789), the first postsynaptic neurotoxin isolated from the venom of the Inland taipan (Oxyuranus microlepidotus), which is the most venomous snake in the world. Oxylepitoxin-1, purified using successive steps of size-exclusion and reverse phase-high performance liquid chromatography, produced concentration-dependent (0.3–1.0 μM) inhibition of nerve-mediated (0.1 Hz, 0.2 ms, supramaximal V) twitches of the chick biventer cervicis nerve-muscle preparation. Taipan antivenom (5 units/ml) prevented the neurotoxic activity of whole venom (10 μg/ml), but had no significant effect on oxylepitoxin-1 (1 μM). The toxin-induced inhibition of nerve-mediated twitches was significantly reversed upon washing the tissue at 5 min intervals. Oxylepitoxin-1 (30–300 nM) displayed competitive antagonism at the skeletal muscle nicotinic receptor with a pA2 value of 7.16 ± 0.28 (i.e. approximately 10-fold more potent than tubocurarine). The venom had a high level of PLA2 activity (765 ± 73 μmol/min/mg) while oxylepitoxin-1 displayed no PLA2 activity. Partial N-terminal sequencing of oxylepitoxin-1 shows high sequence identity (i.e. 93%) to postsynaptic toxins isolated from the venom of the closely related coastal taipan (Oxyuranus scutellatus scutellatus).
Keywords: Neurotoxin; Snake venom; Antivenom; Skeletal muscle; Nicotinic receptor; Chromatography;

Characterization of a novel cell penetrating peptide derived from Bag-1 protein by Dimitrios K. Niarchos; Sonia A. Perez; Michael Papamichail (2661-2669).
A highly cationic peptide (BagP), located within the normally expressed human protein Bag-1, was tested for its capacity to act as a cell penetrating peptide. BagP was found to translocate and transport high molecular weight cargos in several cell types, in varying degrees with a preference for adherent cells. The penetration phenomenon was not found to be subject to saturation for the highest amount of peptide tested (100 μM), whereas the time needed for maximum translocation to be achieved, was cell type-dependent. Finally, BagP internalization depends on its charge, cellular metabolism and cell-surface heparan sulfate proteoglycans.
Keywords: CPP; Bag-1; Protein transduction;

Identification of the first neuropeptides from the CNS of Hemiptera: CAPA peptides of the southern green stinkbug Nezara viridula (L.) by Reinhard Predel; William K. Russell; Susanne Neupert; David H. Russell; Jesus F. Esquivel; Ronald J. Nachman (2670-2677).
A direct mass spectrometric investigation of nerve homologs of the abdominal perisympathetic organs was employed to reveal the first and complete sequences of CAPA peptides from a hemipteran species, the southern green stinkbug Nezara viridula. Side-chain fragmentations allowed the assignment of internal Leu/Ile; on-plate acetylation was used to distinguish between the mass-related Lys and Gln. The following sequences were obtained: DQLFPFPRV-NH2 (CAPA-PVK-1), EQLIPFPRV-NH2 (CAPA-PVK-2), and NGSAGNGGLWFGPRL/I-NH2 (CAPA-PK). CAPA-PVKs are associated with the regulation of diuresis in insects, and identification of those native to a hemipteran will provide the experimental basis to better understand regulation of water balance in this family of insects.
Keywords: CAPA gene; Periviscerokinin; Pyrokinin; FXPRLamide; CAP2b; Perisympathetic organs; Mass spectrometry; Insect neuropeptide;

CGRP stimulates gill carbonic anhydrase activity in molluscs via a common CT/CGRP receptor by Benoit Cudennec; Marthe Rousseau; Evelyne Lopez; Martine Fouchereau-Peron (2678-2682).
The physiological significance of calcitonin gene-related peptide (CGRP) during biomineralization was investigated by assessing the effect of human CGRP on the carbonic anhydrase activity in gill membranes of the pearl oyster, Pinctada margaritifera. Salmon CT and human CGRP were able to induce a 150% increase of the basal activity. No additive effect was observed suggesting that both activities are mediated by the same receptor. The CGRP-stimulated effect was specific as demonstrated by the inhibition produced by the CGRP antagonist, hCGRP8-37. So, CGRP by its specific action on gill carbonic anhydrase controls the calcification process, an ancient role both in invertebrates and non-mammalian vertebrates.
Keywords: CGRP; CT; Gill; Carbonic anhydrase; CGRP8-37; AC187;

A novel bradykinin-like peptide from skin secretions of rufous-spotted torrent frog, Amolops loloensis by Jianguo Liang; Yaoping Han; Jianxu Li; Xueqing Xu; Huw H. Rees; Ren Lai (2683-2687).
A bradykinin-like peptide has been isolated from skin secretions of rufous-spotted torrent frog, Amolops loloensis. This bradykinin-like peptide was named amolopkinin. Its primary structure, RAPVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Amolopkinin is composed of 12 amino acid residues and is related to bradykinin composed of nine amino acid residues, identified from the skin secretions of Odorrana schmackeri. Amolopkinin was found to elicit concentration-dependent contractile effects on isolated guinea pig ileum. cDNA clones encoding the precursor of amolopkinin were isolated by screening a skin cDNA library of A. loloensis and then sequenced. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that a deficiency of an18-nucleotide fragment (TCAAGAATGATCAGACGC in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in absence of a di-basic site for trypsin-like proteinases and an unusual – APV – insertion in the N-terminal part of amolopkinin. This is the first report of a bradykinin-like peptide comprised of bradykinin with an insertion in its N-terminal part. Our results demonstrate the hypervariability of amphibian bradykinin-like peptides, as well as the diversity of antimicrobial peptides in amphibians.
Keywords: Amphibian; Bradykinin-peptide; Amolopkinin; Amolops loloensis; Skin;

Although the ancient practice of traditional Chinese medicine (TCM) utilizes predominantly herbal ingredients, many of which are now the subject of intense scientific scrutiny, significant quantities of animal tissue-derived materials are also employed. Here we have used contemporary molecular techniques to study the material known as lin wa pi, the dried skin of the Heilongjiang brown frog, Rana amurensis, that is used commonly as an ingredient of many medicines, as a general tonic and as a topical antimicrobial/wound dressing. Using a simple technology that has been developed and validated over several years, we have demonstrated that components of both the skin granular gland peptidome and transcriptome persist in this material. Interrogation of the cDNA library constructed from the dried skin by entrapment and amplification of polyadenylated mRNA, using a “shotgun” primer approach and 3′-RACE, resulted in the cloning of cDNAs encoding the precursors of five putative antimicrobial peptides. Two (ranatuerin-2AMa and ranatuerin-2AMb) were obvious homologs of a previously described frog skin peptide family, whereas the remaining three were of sufficient structural novelty to be named amurins 1–3. Mature peptides were each identified in reverse phase HPLC fractions of boiling water extracts of skin and their structures confirmed by MS/MS fragmentation sequencing. Components of traditional Chinese medicines of animal tissue origin may thus contain biologically active peptides that survive the preparation procedures and that may contribute to therapeutic efficacy.
Keywords: Amphibian; Cloning; Precursor protein; Mass spectrometry; Peptide; Antimicrobial;

A single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies by Tanja Vranac; Katrina Pretnar Hartman; Mara Popović; Anja Venturini; Eva Žerovnik; Vladka Čurin Šerbec (2695-2705).
The main step in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conformational change of the normal cellular prion protein (PrPC) into the abnormal isoform, named prion (PrPSc). Since PrP is a highly conserved protein, the production of monoclonal antibodies (mAbs) of high specificity and affinity to PrP is a difficult task. In the present study we show that it is possible to overcome the unresponsiveness of the immune system by immunizing wild-type BALB/c mice with a 13 amino acid PrP peptide from the C-terminal part of PrP, bound to the keyhole limpet hemocyanin (KLH). Immunization induced predominantly anti-PrPSc humoral immune response. Furthermore, we were able to obtain a panel of mAbs of IgG class specific for different non-self-conformations of PrP, with anti-PrPSc-specific mAbs being the most abundant.
Keywords: Monoclonal antibody; Peptide antigen; Prion protein; Epitope conformation; Immune tolerance; Anti-TSE vaccine;

The biological activity of the histidine-containing diketopiperazines cyclo(His-Ala) and cyclo(His-Gly) by F.R. Lucietto; P.J. Milne; G. Kilian; C.L. Frost; M. Van De Venter (2706-2714).
Two cyclic dipeptides, cyclo(His-Ala) and cyclo(His-Gly,) were synthesized from their linear counterparts and their structures elucidated using standard elucidation techniques. Molecular modeling and predictive NMR results indicated that the majority of energetically favourable conformers adopted a boat conformation with respect to the diketopiperazine ring. Cyclo(His-Ala), at concentrations of 100 μM inhibited the growth, in vitro, of various cancer cell lines, including HT-29, MCF-7 and HeLa carcinoma cells while cyclo(His-Gly) inhibited the growth of MCF-7 cells. While the antibacterial potential of these two compounds was limited, both cyclic dipeptides significantly inhibited the growth of C. albicans. Both compounds at a concentration of 100 μM resulted in a decrease in heart rate, coronary flow rate and left ventricular systolic pressure in the isolated rat heart. Inhibition of thrombin, amounting to a 63.3% and 36.7% reduction in the rate of fibrin formation, was noted for cyclo(His-Ala) and cyclo(His-Gly), respectively. While cyclo(His-Ala) showed no notable effects on platelet aggregation, cyclo(His-Gly) significantly inhibited both pathways tested with greatest effects on thrombin-induced platelet aggregation, yielding an IC50 of 0.0662 mM (R 2  = 0.989). The results of the anticancer and hematological studies indicate that histidine-containing diketopiperazines have potential as a novel group of cytotoxic agents with antithrombotic effects.
Keywords: Cyclo(His-Ala); Cyclo(His-Gly); Antimicrobial; Anticancer; Cardiac activity; Antithrombotic effects;

Behavioral effects of 26RFamide and related peptides by Jean-Claude do Rego; Jérôme Leprince; Nicolas Chartrel; Hubert Vaudry; Jean Costentin (2715-2721).
A novel 26-amino acid peptide possessing the Arg–Phe–NH2 motif at its C-terminal extremity has been recently characterized and named 26RFamide (26RFa). The 26RFa precursor encompasses several potential cleavage sites and thus may generate various mature peptides including an N-terminally extended form of 26RFa (termed 43RFa), two fragments of 26RFa (26RFa1–16 and 26RFa20–26), and a 9-amino acid peptide (9RFa) located in tandem in the human 26RFa precursor. In the present study, we have investigated the central effects of 26RFa and related peptides on food intake and locomotor activity in mice. We observed that i.c.v. injection of 26RFa, 43RFa, 26RFa20–26 and 9RFa stimulated food consumption while 26RFa1–16 and 26RFa8–16 had no effect. A dose-dependent stimulation of locomotor activity was observed after i.c.v. administration of 26RFa, 43RFa and 26RFa1–16, but not 26RFa20–26, 26RFa8–16 or 9RFa. These data indicate that the novel neuropeptides 26RFa and 43RFa act centrally to stimulate feeding and locomotor activities but the domains of the peptide involved in each of these responses are different suggesting that the two behavioral effects may be mediated through distinct receptors.
Keywords: RFamide–related peptide; Structure–activity relationships; Food intake; Locomotor activity; Mice;

Estrus variation in anxiolytic-like effects of intra-lateral septal infusions of the neuropeptide Y in Wistar rats in two animal models of anxiety-like behavior by Miguel Molina-Hernández; Jorge I. Olivera-Lopez; N. Patricia Tellez-Alcántara; Julían Pérez-García; M. Teresa Jaramillo (2722-2730).
Anxiolytic-like effects of intra-lateral septal infusions of the neuropeptide Y (NPY) were assessed during several estrus phases in Wistar rats tested in two animal models of anxiety-like behavior. In a conflict operant test, results showed that during late proestrus, intra-lateral septal nuclei infusions of NPY (1.0 μg/μl, P  < 0.05; 2.0 μg/μl, P  < 0.05; 2.5 μg/μl, P  < 0.05) increased the number of immediately punished responses. During metestrus–diestrus only the highest doses of NPY (2.5 μg/μl, P  < 0.05) increased the number of immediately punished reinforcers. In the elevated plus-maze test, results showed that during late proestrus, intra-lateral septal nuclei infusions of NPY (1.0 μg/μl, P  < 0.05; 2.0 μg/μl, P  < 0.05) produced anxiolytic-like actions. During metestrus–diestrus only the highest doses of NPY (2.0 μg/μl, P  < 0.05) produced anxiolytic-like actions. Neither NPY nor estrus phases significantly modified the number of closed arms entries in the elevated plus-maze test. It is concluded that anxiolytic-like effects of NPY vary within the estrus cycle in Wistar rats.
Keywords: Conflict behavior; Elevated plus-maze; Estrus cycle; Lateral septum; Neuropeptide Y;

Neuropeptide Y (NPY) Y4 receptor (Y4R) in rat brainstem has been implicated in the signaling of satiety. In this study, we investigated the effects of leptin, and refeeding-induced satiety on Y4R mRNA expression in rat brainstem. Y4R receptor-specific primers were used to amplify the mRNA obtained from hypothalamus and brainstem utilizing conventional RT-PCR and quantitative real-time RT-PCR. No PCR product for Y4R was obtained from entire hypothalamic mRNA. Real-time RT-PCR showed a significant two-fold increase in the relative quantity of Y4R mRNA in brainstem of refed rats in comparison to food deprived or ad lib fed rats. Consistently, plasma leptin level was elevated in refed rats in comparison to food deprived rats. Similarly, leptin-treated rats exhibited a significant increase in Y4R mRNA in brainstem as compared to saline-injected rats. Plasma leptin was significantly elevated in leptin-treated rats. These results suggest that refeeding stimulates the expression of Y4R gene in the brainstem and that leptin may be one of the peripheral factors involved in this anorectic signaling mechanism.
Keywords: Hindbrain; Anorexia; mRNA; Real-time RT-PCR;

Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats by Y. Oomura; N. Hori; T. Shiraishi; K. Fukunaga; H. Takeda; M. Tsuji; T. Matsumiya; M. Ishibashi; S. Aou; X.L. Li; D. Kohno; K. Uramura; H. Sougawa; T. Yada; M.J. Wayner; K. Sasaki (2738-2749).
Leptin, an adipocytokine encoded by an obesity gene and expressed in adipose tissue, affects feeding behavior, thermogenesis, and neuroendocrine status via leptin receptors distributed in the brain, especially in the hypothalamus. Leptin may also modulate the synaptic plasticity and behavioral performance related to learning and memory since: leptin receptors are found in the hippocampus, and both leptin and its receptor share structural and functional similarities with the interleukin-6 family of cytokines that modulate long-term potentiation (LTP) in the hippocampus. We therefore examined the effect of leptin on (1) behavioral performance in emotional and spatial learning tasks, (2) LTP at Schaffer collateral-CA1 synapses, (3) presynaptic and postsynaptic activities in hippocampal CA1 neurons, (4) the intracellular Ca2+ concentration ([Ca2+]i) in CA1 neurons, and (5) the activity of Ca2+/calmodulin protein kinase II (CaMK II) in the hippocampal CA1 tissue that exhibits LTP. Intravenous injection of 5 and/or 50 μg/kg, but not of 500 μg/kg leptin, facilitated behavioral performance in passive avoidance and Morris water-maze tasks. Bath application of 10−12  M leptin in slice experiments enhanced LTP and increased the presynaptic transmitter release, whereas 10−10  M leptin suppressed LTP and reduced the postsynaptic receptor sensitivity to N-methyl-d-aspartic acid. The increase in the [Ca2+]i induced by 10−10  M leptin was two times greater than that induced by 10−12  M leptin. In addition, the facilitation (10−12  M) and suppression (10−10  M) of LTP by leptin was closely associated with an increase and decrease in Ca2+-independent activity of CaMK II. Our results show that leptin not only affects hypothalamic functions (such as feeding, thermogenesis, and neuroendocrine status), but also modulates higher nervous functions, such as the behavioral performance related to learning and memory and hippocampal synaptic plasticity.
Keywords: Hippocampus; LTP; [Ca2+]i; CaMK II; Passive avoidance conditioning; Morris water maze;

Effects of glucose-dependent insulinotropic peptide on behavior by Ke-Hong Ding; Qing Zhong; Ding Xie; Huan-Xin Chen; Mary Anne Della-Fera; Roni J. Bollag; Wendy B. Bollag; Ravinder Gujral; Baolin Kang; Supriya Sridhar; Clifton Baile; Walton Curl; Carlos M. lsales (2750-2755).
Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that rises rapidly in response to nutrient ingestion. The GIP receptor is widely expressed in the brain including the brain stem, telencephalon, diencephalon, olfactory bulb, pituitary, and cerebellum. Until recently it was not clear what the endogenous ligand for this receptor was because no GIP expression had been demonstrated in the brain. GIP synthesis has now been documented in the dentate gyrus of the hippocampus. To define GIP effects on behavior we utilized a mouse model a GIP-overexpressing transgenic mouse (GIP Tg). Specifically, anxiety-related behavior, exploration, memory, and nociception were examined. Compared to age-matched adult male C57BI/6 controls GIP Tg mice displayed enhanced exploratory behavior in the open-field locomotor activity test. GIP Tg mice also demonstrated increased performance in some of the motor function tests. These data suggest that the GIP receptor plays a role in the regulation of locomotor activity and exploration. To our knowledge, this is the first report of effects of GIP on behavior.
Keywords: GIP receptor; Brain; Transgenic mice; Anxiety; Memory; Exploration;

Augmentation of insulin-stimulated ANP release through tyrosine kinase and PI 3-kinase in diabetic rats by Guang Yi Bai; Feng Lian Piao; Sun Young Kim; Kuichang Yuan; Sung Zoo Kim; Suhn Hee Kim (2756-2763).
The aim of present study was to define the effects of insulin on atrial dynamics and ANP release and its modification in diabetic rats. An isolated perfused beating atrial model was used from control and diabetic rats. Insulin was perfused with and without an inhibitor for tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Insulin increased the release of ANP and decreased atrial contractility in a dose-dependent manner. During the perfusion of 10−10  M insulin, the release of ANP abruptly increased within 8 min by approximately 40% and then decreased with time despite of continuous perfusion. In terms of increasing the dose of insulin, the time to reach the peak effect became faster and the slope to decrease became slower. In contrast, atrial contractility was gradually decreased with time. These effects were independent upon extracellular glucose. Genistein (10−5  M) or lavendustin C (10−5  M), a tyrosine kinase inhibitor, attenuated the release of ANP stimulated by insulin (10−8  M). Wortmannin (10−7  M) or LY294002 (10−5  M), a PI 3-kinase inhibitor, also attenuated insulin-stimulated ANP release. However, both inhibitors for PI 3-kinase and tyrosine kinase did not cause any significant effects on negative inotropism by insulin. Insulin-stimulated ANP release was augmented in streptozotocin-treated rat atria. The density of insulin receptor markedly increased in diabetic hearts. These results suggest that insulin stimulates the release of ANP through PI 3-kinase and tyrosine kinase, and augmentation of insulin-stimulated ANP release in diabetic rat atria may be partly due to an upregulation of insulin receptor.
Keywords: Atrial function; Insulin; Tyrosine kinase; Phosphatidylinositol 3-kinase; ANP (atrial natriuretic peptide); Diabetes mellitus; Contractility;

Adrenal expression of orexin receptor subtypes is differentially regulated in experimental streptozotocin induced type-1 diabetes by Olaf Jöhren; Julia A.F. Gremmels; Fatimunnisa Qadri; Andreas Dendorfer; Peter Dominiak (2764-2769).
Orexins (hypocretins) are involved in the regulation of energy homeostasis and sleeping behavior. Orexins were also implicated in the regulation of neuroendocrine and autonomic functions. Recent data show the expression of orexin receptors within the hypothalamic–pituitary–adrenal (HPA) axis and suggest specific actions of orexins at the pituitary and adrenal glands. To further evaluate the role of orexin in the HPA axis, we investigated the mRNA expression of prepro-orexin (PPO) and orexin receptors within the HPA axis of streptozotocin-injected (STZ) rats showing type-1 like diabetes. PPO, as well as OX1 and OX2 receptor levels were analyzed by quantitative real-time PCR (qPCR). STZ rats were characterized by decreased body weight, plasma insulin, and leptin levels and by increased plasma glucose. Hypothalamic PPO mRNA levels were significantly reduced in STZ compared to non-diabetic control rats. No differences were found in the mRNA levels of hypothalamic or pituitary OX1 and OX2 receptors between control and STZ rats. In adrenals, OX1 receptor mRNA levels were significantly elevated in STZ rats while OX2 receptors were significantly reduced. Our results imply distinct functions of adrenal orexin receptor subtypes during type-1 like diabetes.

To assess whether diabetes alters the regulatory effects of μ-opioid receptor (MOR) agonists on the cholinergic bronchoconstriction, we investigated the inhibitory effects of endomorphins (EMs) on the electrical field stimulation (EFS)-induced cholinergic bronchoconstriction in type 1 diabetic rats. At 4 weeks after the onset of diabetes, both the EFS- and exogenous acetylcholine (ACh)-induced bronchoconstriction in diabetes in vitro were greater than those in non-diabetes rats. Furthermore, endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited the response to EFS in diabetic rat isolated bronchus in a concentration- and frequency-dependent manner, which is in agreement with that in non-diabetes. However, the inhibitory effects of EMs on the EFS-induced bronchoconstriction in diabetes were significantly weaker than those in non-diabetes. Both EM1 and EM2 (1 μM) had no effect on the contractile response to exogenous ACh, indicating a prejunctional effect. Furthermore, the inhibitory effect on the EFS-induced bronchoconstriction was blocked by naloxone (10 μM). Eight weeks after the induction of diabetes, both the EFS- and exogenous ACh-induced bronchoconstrictions in diabetes were further enhanced compared to those in short-time (4 weeks) diabetic rats. Moreover, the inhibitory effects of EMs on the EFS-induced bronchoconstriction were further attenuated. These results suggest that dysfunction of presynaptic inhibitory modulation through opioid receptor by EMs may take place in the bronchus of diabetic rats.
Keywords: Type 1 diabetic rats; Endomorphin 1; Endomorphin 2; Electrical field stimulation; Cholinergic bronchoconstriction;

Endogenous β-endorphin induces thermal analgesia at the initial stages of a murine osteosarcoma by Ana Baamonde; Ana Lastra; Lucía Juárez; Olivia García-Suárez; Álvaro Meana; Agustín Hidalgo; Luis Menéndez (2778-2785).
Transient thermal, but not mechanical, hypoalgesia appears at the early stages of the development of an hyperalgesic murine osteosarcoma. This hypoalgesia is suppressed by the administration of naloxone, its peripherally acting analog naloxone methiodide, the μ- and δ-opioid receptor antagonists cyprodime and naltrindole, or the CRF receptor antagonist, α-helical CRF (9–41). When immunohistochemical assays were performed with an anti-β-endorphin antibody, whose in vivo administration suppressed the analgesia, labeled mononuclear immune cells appeared both inside and surrounding the tumoral tissue.In conclusion, the peripheral action of β-endorphin, released in response to the osteosarcoma seems responsible for the observed thermal analgesia.
Keywords: β-Endorphin; Osteosarcoma; Pain; Analgesia; Mouse; Peripheral opioid receptors;

Contribution of spinal μ1-opioid receptors and dynorphin B to the antinociception induced by Tyr-d-Arg-Phe-Sar by Hirokazu Mizoguchi; Kanenori Ito; Hiroyuki Watanabe; Chizuko Watanabe; Sou Katsuyama; Tsutomu Fujimura; Tsukasa Sakurada; Shinobu Sakurada (2786-2793).
The antinociceptive effect of Tyr-d-Arg-Phe-Sar (TAPS) at the spinal level was characterized with the mouse tail-flick test. Intrathecal (i.t.) administration of TAPS produced a dose-dependent antinociception. The antinociception induced by TAPS was completely blocked by i.t. pretreatment with the μ-opioid receptor antagonist β-funaltrexamine, the μ1-opioid receptor antagonist naloxonazine or the κ-opioid receptor antagonist nor-binaltorphimine, but not with the δ-opioid receptor antagonist naltrindole. Moreover, TAPS-induced antinociception was dose-dependently attenuated by i.t. pretreatment with an antiserum against dynorphin B, but not against dynorphin A, α-neo-endorphin, [Met5]enkephalin, or [Leu5]enkephalin. In mice lacking prodynorphin, TAPS-induced antinociception was significantly reduced compared to that in wild-type mice. These results suggest that TAPS mainly stimulates μ1-opioid receptors, which subsequently induce the release of dynorphin B, which then acts on κ-opioid receptors to produce antinociception.
Keywords: Antinociception; TAPS; Dermorphin; Dynorphin B; μ-Opioid receptor; Spinal cord;

Effect of the C-terminus of murine S100A9 protein on experimental nociception by Camila Squarzoni Dale; Rosana de Lima Pagano; Carina Cicconi Paccola; Tatiana Pinotti-Guirao; Maria Aparecida Juliano; Luiz Juliano; Renata Giorgi (2794-2802).
Calcium-binding protein S100A9 induces antinociception in mice evaluated by the writhing test. Similarly, a peptide identical to the C-terminus of murine S100A9 (mS100A9p) inhibits the hyperalgesia induced by jararhagin, a metalloprotease. Thus, we investigated the effect of mS100A9p on different models used to evaluate nociception. mS100A9p induced a dose-dependent inhibitory effect on the writhing test, and on mechanical hyperalgesia induced by carrageenan. mS100A9p inhibited thermal hyperalgesia induced by carrageenan. mS100A9p did not modify the nociceptive response in hot plate or tail-flick tests. These data demonstrate that the C-terminus of S100A9 protein interferes with control mechanisms of inflammatory pain.
Keywords: S100A9; Nociception; Hyperalgesia; Carrageenan; Inflammation;

Central administration of low doses of nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid-like orphan receptor NOP, have been shown to reduce ethanol consumption, ethanol-induced conditioned place preference and stress-induced reinstatement of alcohol-seeking behavior in alcohol preferring rats. The present study evaluated the effect of continuous (7 days) lateral brain ventricle infusions of N/OFQ (0, 0.25, 1, 4, and 8 μg/h), by means of osmotic mini-pumps, on 10% ethanol intake in Marchigian-Sardinian alcohol-preferring (msP) rats provided 2 h or 24 h access to it. N/OFQ dose-dependently increased food intake in msP rats. On the other hand, in contrast to previous studies with acute injections, continuous lateral brain ventricle infusion of high doses of N/OFQ increased ethanol consumption when the ethanol solution was available for 24 h/day or 2 h/day. The present study demonstrates that continuous activation of the opioidergic N/OFQ receptor does not blunt the reinforcing effects of ethanol. Moreover, the data suggest that continuous activation of the opioidergic N/OFQ receptor is not a suitable way to reduce alcohol abuse.
Keywords: Alcohol-preferring rats; Food intake; Ethanol intake; Nociceptin/orphanin FQ;

Lack of interaction between peripheral injection of CCK and obestatin in the regulation of gastric satiety signaling in rodents by G. Gourcerol; M. Million; D.W. Adelson; Y. Wang; L. Wang; J. Rivier; D.H. St-Pierre; Y Taché (2811-2819).
Obestatin is a new peptide for which anorexigenic effects were recently reported in mice. We investigate whether peripheral injection of obestatin or co-injection with cholecystokinin (CCK) can modulate food intake, gastric motor function (intragastric pressure and emptying) and gastric vagal afferent activity in rodents. Obestatin (30, 100 and 300 μg/kg, i.p.) did not influence cumulative food intake for the 2 h post-injection in rats or mice nor gastric emptying in rats. In rats, obestatin (300 μg/kg) did not modify CCK (1 μg/kg, i.p.)-induced significant decrease in food intake (36.6%) and gastric emptying (31.0%). Furthermore, while rats injected with CCK (0.3 μg/kg, i.v.) displayed gastric relaxation, no change in gastric intraluminal pressure was elicited by obestatin (300 μg/kg, i.v.) pre- or post-CCK administration. In in vitro rat gastric vagal afferent preparations, 20 units that had non-significant changes in basal activity after obestatin at 30 μg responded to CCK at 10 ng by a 182% increase. These data show that obestatin neither influences cumulative food intake, gastric motility or vagal afferent activity nor CCK-induced satiety signaling
Keywords: CCK; Obestatin; Food intake; Gastric emptying; Gastric relaxation; Gastric vagal afferent;

Effects of CCK-8 on independent ingestion and central c-Fos-like immunoreactivity in rats on postnatal days 10 and 11 by S. Blumberg; M. Schroeder; D. Haba; O. Malkesman; A.-M. Torregrossa; A. Weller; G.P. Smith (2820-2828).
Controls of the independent ingestion of food in the preweanling rat emerge in the second postnatal week. We investigated the effects of CCK-8 (0, 1, 5, or 10 μg/kg IP) on intake and c-Fos-like immunoreactive (CFLI) cells in hindbrain and forebrain on postnatal days 10 and 11. Five micrograms per kilogram decreased intake and increased the number of CFLI cells in four subnuclei of the nucleus tractus solitarius (NTS), in arcuate nucleus (ARC), and in central nucleus of the amygdala (CeA). Ten micrograms per kilogram decreased intake and increased CFLI in three NTS subnuclei as much as 5 μg/kg did, but was more potent than 5 μg/kg in the medial NTS subnucleus. Ten micrograms per kilogram increased CFLI in paraventricular (PVN) and supraoptic (SON) nuclei, but 5 μg/kg did not. Thus, reduction of intake by CCK-8 on days 10 and 11 is associated with increased hindbrain and forebrain CFLI.
Keywords: c-Fos; Ontogeny of food intake; Cholecystokinin; Satiety; Independent ingestion;

Food motivated behavior of melanocortin-4 receptor knockout mice under a progressive ratio schedule by C. Vaughan; M. Moore; C. Haskell-Luevano; N.E. Rowland (2829-2835).
Melanocortin-4 receptor knockout (MC4RKO) mice are hyperphagic and develop obesity under free feeding conditions. We reported previously that MC4RKO mice did not maintain hyperphagia and as a result lost weight when required to press a lever to obtain food on a fixed ratio procurement schedule. To assess the generality of this result, we tested MC4RKO mice and their heterozygous and wild type littermates using progressive ratio (PR) schedules that are believed to be sensitive indicators of motivation. Mice lived in operant chambers and obtained all of their food (20 mg pellets) via lever press responding. Food was available according to a PR schedule so that within a meal, food became progressively more costly, and we expected this would provide a stringent test of mechanisms controlling meal size. The schedule reset after either 3 or 20 min of no responding, so defining meals, and the highest ratio completed before the reset was defined as the breakpoint. The average daily number of meals was lower and mean size of meals was higher at the 20 compared with the 3 min reset condition. Mean daily food intake did not differ between the two reset criteria but did differ as a function of genotype, with MC4RKO mice eating about 25% more than heterozygous or wild type mice. Hyperphagia in the MC4RKO mice was characterized primarily by larger meals (higher breakpoints) and they emitted about twice as many responses as wild type mice. Thus, using a PR schedule, MC4RKO mice exhibit hyperphagia, and show a high level of motivation to support large meal sizes.
Keywords: Melanocortin-4 receptors; Knockout; Mice; Operant conditioning; Progressive ratio; Closed economy; Food intake;

Functional characterization of the modified melanocortin peptides responsible for ligand selectivity at the human melanocortin receptors by Min Chen; Keith E. Georgeson; Carroll M. Harmon; Carrie Haskell-Luevano; Yingkui Yang (2836-2845).
The melanocortin system plays an important role in energy homeostasis as well as skin pigmentation, steroidogenesis and exocrine gland function. In this study, we examined eight Ac-His-Phe-Arg-Trp-NH2 tetrapeptides that were modified at the Phe position and pharmacologically characterized their activities at the human MCR wild-types and their mutants. Our results indicate that at the hMC1R, all D stereochemical modified residues at the Phe position of peptides increase cAMP production in a dose-dependent manner. At the hMC3R, the DPhe peptide dose dependently increases cAMP production but all other three tetrapeptides were not. At the hMC4R, both the DPhe and DNal(1′) peptides induce cAMP production. However, both DTyr and DNal(2′) were not able to induce cAMP production. Further studies indicated that at the hMC1R M128L mutant receptor, the all D-configured tetrapeptides reduce their potencies as compared to that of hMC1R wild-type. However, at the hMC3R and hMC4R L165M and L133M mutant receptors, the DNal(2′) and DTyr tetrapeptides possess agonist activity. These findings indicate that DPhe in tetrapeptide plays an important role in ligand selectivity and specific residue TM3 of the melanocortin receptors is crucial for ligand selectivity.
Keywords: Obesity; SHU9119; MC3R; MC4R; GPCR;

Melanocortin-4 receptor-mediated inhibition of apoptosis in immortalized hypothalamic neurons via mitogen-activated protein kinase by Biaoxin Chai; Ji-Yao Li; Weizhen Zhang; Erika Newman; John Ammori; Michael W. Mulholland (2846-2857).
The melanocortin-4 receptor (MC4R) is a seven transmembrane member of the melanocortin receptor family. The GT1-1 cell line exhibits endogenous expression of MC4R. In this study, GT1-1 cells were used to study MC4R signaling pathways and to examine the effects of melanocortin receptor agonist NDP-MSH on apoptosis. MC4R mRNA expression was demonstrated by RT-PCR. Functional melanocortin receptor expression was implied by specific binding of NDP-MSH and cAMP production. NDP-MSH-stimulated GnRH release in a dose-dependent manner. Serum deprivation-induced apoptosis in GT1-1 cells, and the NDP-MSH inhibited this effect. The melanocortin receptor antagonist SHU9119 blocked the antiapoptotic actions of NDP-MSH, and the MAP kinase inhibitor PD98059 significantly attenuated the antiapoptotic effect. NDP-MSH-stimulated ERK1/2 phosphorylation in a dose-dependent manner. ERK1/2 phosphorylation could be abolished by SHU9119. In GT1-1 cells, melanocortin receptor activation causes ERK1/2 phosphorylation. In these cells, MC4R activation is also associated with antiapoptotic effects.
Keywords: Melanocortin-4 receptor; Apoptosis; GT1-1 cells; NDP-MSH; Mitogen-activated protein kinase;

β-Amyloid (Aβ) is a hydrophobic peptide that drives the pathogenesis of Alzheimer's disease (AD) due to its aberrant aggregation. Inhibition of Aβ aggregation process is one of the most promising strategies for therapeutic intervention in AD. Here, we demonstrate that sterically stabilized (PEGylated) phospholipid nanomicelles (SSM) are effective in mitigating Aβ-42 aggregation using several deterministic techniques such as (1) Turbidimetry (2) Congo red binding (3) Thioflavine-T binding (4) Laser light scattering and (5) Electron Microscopy. α-Helicity of Aβ-42 is significantly augmented in the presence of SSM as demonstrated by circular dichroism (p  < 0.05). Cytotoxicity studies, employing human neuroblastoma SHSY-5Y cells, established that PEGylated phospholipid associated peptide demonstrated significantly lower neurotoxicity compared to lipid untreated Aβ-42 (p  < 0.05). Collectively, our results establish that PEGylated phospholipids abrogate transformation of Aβ-42 to amyloidogenic β-sheeted form and impart neuroprotection in vitro. This study provides a foundation for designing nanoconstructs of PEGylated phospholipid nanomicelles in conjunction with a therapeutic agent for multitargeting the different pathophysiologies associated with AD.
Keywords: β-Amyloid aggregation; Alzheimer's disease; DSPE-PEG2000; PEGylated phospholipid; Nanomicelles; SHSY-5Y cells; Nanomedicine;

Vasoactive intestinal peptide (VIP) was known to provide neuroprotection. Three VIP receptors have been cloned: VPAC1, VPAC2 and PAC1. A specific splice variant of PAC1 in the third cytoplasmatic loop, hop2, was implicated in VIP-related neuroprotection. We aimed to clone the hop2 splice variant, examine its affinity to VIP and investigate whether it mediates the VIP-related neuroprotective activity. The PAC1 cDNA was cloned from rat cerebral astrocytes. Using genetic manipulation the hop2 splice variant was obtained, then inserted into an expression vector and transfected into COS-7 cells that were used for binding assays. Results showed that VIP bound the cloned hop2 splice variant. Stearyl-neurotensin6–11 VIP7–28 (SNH), an antagonist for VIP, was also found to bind hop2. In addition, VIP protected COS-7 cells expressing hop2 from oxidative stress. Parallel assays demonstrated that VIP increased cAMP accumulation in COS-7 cells expressing hop2. These results support the hypothesis that hop2 mediates the cytoprotective effects attributed to VIP.
Keywords: Vasoactive intestinal peptide (VIP); Pituitary adenylate cyclase-activating polypeptide (PACAP); Neuroprotection; Splice variant; Stearyl-neurotensin6–11 VIP7–28 (SNH);

Urinary oxytocin as a non-invasive biomarker for neurohypophyseal hormone secretion by Anthony B. Polito; David L. Goldstein; Lylian Sanchez; David R. Cool; Mariana Morris (2877-2884).
The objective was to characterize the urinary oxytocin (OT) system with the goal of using it as a biomarker for neurohypophyseal peptide secretion. We studied urinary OT secretion in mice under three conditions: (1) in OT gene deletion mice (OT −/−) which lack the ability to produce the peptide; (2) after arterial vascular infusion of OT and (3) after physiological stimulation with consumption of 2% sodium chloride. OT was measured by radioimmunoassay (RIA) and Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectroscopy (SELDI TOF MS). In OT −/− mice (n  = 25), urinary OT levels were not detectable, while in OT +/+ mice (n  = 23) levels were 250.2 ± 35.3 pg/ml. To evaluate blood/urine transfer, mice with chronic carotid arterial catheters were infused with saline or OT (5 or 20 pmol/min). Peak urine OT levels were 89 ± 11.5 and 844 ± 181 ng/ml in the low and high OT groups, respectively. Proteomic evaluation showed MS peaks, corresponding to OT (∼1009 Da) and a related peptide (∼1030 Da) with highest levels in mice infused with OT. Salt loading (5 days of 2% NaCl as drinking water) increased plasma osmolality (3.3%), increased plasma and urinary vasopressin (AVP), but caused no changes in OT. Thus, using non-invasive urine samples, we document that urinary OT and AVP can be used to monitor changes in peptide secretion. Urinary OT and AVP, as well as other urinary peptides, may provide a viable biomarker for peptide secretion and may be useful in clinical studies.
Keywords: Sodium chloride; Vasopressin; Hypothalamus; Genetic models; Kidney; Osmotic control; Mouse;

Neural interaction between galanin-like peptide (GALP)- and luteinizing hormone-releasing hormone (LHRH)-containing neurons by Fumiko Takenoya; Jian-Lian Guan; Masakatsu Kato; Yasuo Sakuma; Yuri Kintaka; Yoshitaka Kitamura; Shinji Kitamura; Hiromi Okuda; Masao Takeuchi; Haruaki Kageyama; Seiji Shioda (2885-2893).
Galanin-like peptide (GALP), commonly known as an appetite-regulating peptide, has been shown to increase plasma luteinizing hormone (LH) through luteinizing hormone-releasing hormone (LHRH). This led us to investigate, using both light and electron microscopy, whether GALP-containing neurons in the rat brain make direct inputs to LHRH-containing neurons. As LHRH-containing neurons are very difficult to demonstrate immunohistochemically with LHRH antiserum without colchicine treatment, we used a transgenic rat in which LHRH tagged with enhanced green fluorescence protein facilitated the precise detection of LHRH-producing neuronal cell bodies and processes. This is the first study to report on synaptic inputs to LHRH-containing neurons at the ultrastructural level using this transgenic model. We also used immunohistochemistry to investigate the neuronal interaction between GALP- and LHRH-containing neurons. The experiments revealed that GALP-containing nerve terminals lie in close apposition with LHRH-containing cell bodies and processes in the medial preoptic area and the bed nucleus of the stria terminalis. At the ultrastructural level, the GALP-positive nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts with the EGFP-positive neurons in these areas. These results strongly suggest that GALP-containing neurons provide direct input to LHRH-containing neurons and that GALP plays a crucial role in the regulation of LH secretion via LHRH.
Keywords: Galanin-like peptide (GALP); Luteinizing hormone-releasing hormone (LHRH); Rat; Hypothalamus; Electron microscopy; Immunostaining;

The extracellular domain (ECD) of the follicle stimulating hormone receptor (FSHR) has been shown to be a major determinant of hormone selectivity. The N-terminal 9–30 region, the sequence of which is unique to FSHR, has been extensively studied earlier and has been proposed to be an FSHR neutralizing epitope. In this study antipeptide antibodies specific to the peptide 9–30 were generated and used for identifying a specific immunodominant region within it. Overlapping peptides corresponding to the regions 9–19, 15–25 and 20–30 were synthesized. The ability of the antipeptide antibodies to 9–30 of FSHR to bind to different peptides was checked. The results indicated that the antibodies mainly recognized the peptide 20–30 and not the other two overlapping peptides. Further, the effect of the peptide 20–30 on the binding of radiolabeled FSH to its receptor was monitored. This peptide showed FSH-binding inhibitory activity with an IC50 value of 0.598 × 10−4  M and was more effective than the peptide 9–30 itself. Binding kinetics revealed that the observed effect of the peptide 20–30 is due to mixed type of inhibitory mechanism. This is the smallest peptide from the rat FSHR sequence having ability to inhibit FSH binding to its receptor by more than 90%.
Keywords: Antipeptide antibodies; FSH binding inhibitor; Radioreceptor assay;

Treatment with valproate (Valp) facilitates the synthesis of TRH-like peptides (pGlu-X-Pro-NH2) in rat brain where “X” can be any amino acid residue. Because high levels of TRH-like peptides occur in the pancreas and pGlu-Glu-Pro-NH2 (Glu-TRH) has been shown to be a fertilization promoting peptide, we hypothesized that these peptides mediate some of the metabolic and reproductive side effects of Valp. Male WKY rats were treated with Valp acutely (AC), chronically (CHR) or chronically followed by a 2 day withdrawal (WD). AC, CHR and WD treatments significantly altered TRH and/or TRH-like peptide levels in pancreas and reproductive tissues. Glu-TRH was the predominant TRH-like peptide in epididymis, consistent with its fertilization promoting activity. Glu-TRH levels in the epididymis increased 3-fold with AC Valp. Phe-TRH, the most abundant TRH-like peptide in the pancreas, increased 4-fold with AC Valp. Phe-TRH inhibits both basal and TRH-stimulated insulin release. Large dense core vesicles (LDCV's) contain a copper-dependent enzyme responsible for the post-translational processing of precursors of TRH and TRH-like peptides. Copper (500 μM) increased the in vitro C-terminal amidation of TRH-like peptides by 8- and 4-fold during 24 °C incubation of homogenates of pancreas and testis, respectively. Valp (7 μM) accelerated 3-fold the processing of TRH and TRH-like peptide precursors in pancreatic LDCV's incubated at 24 °C. We conclude that copper, an essential cofactor for TRH and TRH-like peptide biosynthesis that is chelated by Valp, mediates some of the metabolic and reproductive effects of Valp treatment via acceleration of intravesicular synthesis and altered release of these peptides.
Keywords: Prostate; Epididymis; Testis; RIA; HPLC;

Effect of estrogen on neprilysin expression in uterus and kidney of Sprague–Dawley normotensive and heterozygous (mRen2)27-transgenic hypertensive rats by L.A.A. Neves; M.C. Chappell; C.M. Ferrario; P.E. Gallagher; D. Ganten; K.B. Brosnihan (2912-2918).
The present study was designed to determine whether estrogen modulates the angiotensin processing enzymes in membrane homogenates obtained from uterus and kidney cortex and medulla of Sprague–Dawley (SD) and heterozygous (mRen2)27-transgenic hypertensive (Tg(+)) female rats treated with or without 17β-estradiol (E2). We evaluated estrogen's influence on neprilysin (NEP), an endopeptidase that forms angiotensin-(1–7) [Ang-(1–7)] and on aminopeptidase (AMP), which degrades Ang-(1–7). Renal tissue from normotensive and hypertensive male rats was also evaluated. E2 up-regulated NEP mRNA in the uterus of both SD and Tg(+) and this was associated with increased NEP activity in the uterus of SD (0.31 ± 0.03 nmol/min/mg versus 0.18 ± 0.04 nmol/min/mg of protein, p  < 0.05) and Tg(+) (0.26 ± 0.04 nmol/min/mg versus 0.13 ± 0.02 nmol/min/mg of protein, p  < 0.05) female). E2 had no significant effect on NEP activity in cortex and medulla of hypertensive and normotensive female. In female animals, cortical NEP activity is two-fold higher than medullary; in males there is a four-fold higher cortical NEP activity as compared to medulla. In male animals, medullary NEP was significantly lower than females with or without E2 treatment; no gender specific effect was found in cortex. E2 treatment also caused a two-fold increase in AMP activity in the uterus and 1.6-fold decrease in kidney cortex of SD and Tg(+) female (p  < 0.05). Our studies indicate that NEP may be a primary candidate for increased Ang-(1–7) processing in the uterus with estrogen treatment; kidney NEP, on the other hand, showed no modulation by estrogen, suggesting that down regulation of other processing enzymes, like AMP and ACE, may come into play in the kidney with estrogen replacement. In addition, these studies showed that there is tissue-specific regulation of NEP with estrogen treatment that is strain independent.
Keywords: Hypertension; Renin-angiotensin system; Estrogen replacement; Peptidases;

Urotensin-II receptor blockade with SB-611812 attenuates cardiac remodeling in experimental ischemic heart disease by Nicolas Bousette; Julia Pottinger; Wisam Ramli; Eliot H. Ohlstein; Dashyant Dhanak; Stephen A. Douglas; Adel Giaid (2919-2926).
It is now well established that urotensin-II (UII) levels are increased in several cardiovascular diseases. We previously demonstrated that UII and the UII receptor (UT) protein levels are significantly increased in the hearts of both humans and rats with congestive heart failure (CHF). We have also recently demonstrated that UII blockade, with a selective UII antagonist, improves heart function in a rat model of ischemic CHF. Here, we evaluated the attenuation of cardiac remodeling associated with UII antagonism in the same rat model of ischemic CHF. Animals were administered a specific UT receptor antagonist, SB-611812 (30 mg/kg/day, gavage), or vehicle 30 min prior to coronary artery ligation followed by daily treatment for 8 weeks. Myocardial interstitial fibrosis was analyzed by Masson's trichrome and picrosirius red staining. RT-PCR analysis was utilized for mRNA expression studies. We used Western blotting to assess levels of collagen types I and III. Mitogenic activity of UII on cultured neonatal cardiac fibroblasts was also evaluated. Following coronary ligation, SB-611812 significantly attenuated both myocardial and endocardial interstitial fibrosis, and reduced collagen type I:III ratio (P  < 0.01). UII induced proliferation of cardiac fibroblasts and this mitogenic effect was significantly inhibited with 1 μM of SB-611218 (P  < 0.05). We demonstrate here that selective blockade of UT reduces diastolic dysfunction by decreasing myocardial fibrosis post-coronary ligation in vivo, and inhibits UII-mediated fibroblast proliferation in vitro.
Keywords: Rat; Myocardial infarction; Fibrosis; Collagen; Urotensin-II;

Parathyroid hormone-related peptide (PTHrP) is known to be a strong vasorelaxant peptide. The mechanisms by which PTHrP reduces the coronary resistance of the rat heart have not been worked out but seem to be independent of the classical PTH/PTHrP receptor-mediated, cAMP-dependent effect. In this study we hypothesized that PTHrP reduces the coronary resistance of the rat heart via endothelial cell hyperpolarization. Isolated microvascular endothelial cells from rat heart were incubated with PTHrP(1–36), and changes in the membrane potential were recorded via DiBAC fluorescence. Cells exposed to PTHrP showed a hyperpolarization of approximately 7 mV. In the isolated Langendorff preparation, PTHrP-dependent vasodilatation of l-nitro-arginine-exposed hearts was abolished under depolarizing conditions (high potassium). Denudation of the endothelial cell layer significantly impaired the vasodilatory effect of PTHrP. In the presence of H89 (a cAMP/protein kinase A pathway antagonist) and indomethacin (a cyclooxygenase inhibitor), PTHrP dilated the vessels. In conclusion, PTHrP exerted a nitric oxide-independent vasodilatory effect that depends on endothelial cell hyperpolarization.
Keywords: EDHF; Vasodilation; PTHrP; Rat;

Adrenomedullin antagonizes angiotensin II-stimulated proliferation of human aortic smooth muscle cells by Fabio Rossi; Cora Bertone; Silvia Petricca; Vittorio Santiemma (2935-2941).
The vasodilating peptide adrenomedullin has been reported to regulate vascular tone as well as proliferation and differentiation of various cell types in an autocrine/paracrine manner. Conflicting data have been reported on the adrenomedullin (AM) effect on vascular smooth muscle cell proliferation, a process involved in the progression of vascular remodeling and atherosclerotic lesion. In this paper we investigate the effect of AM on proliferation of human aorta smooth muscle cell (HASMC). AM showed a potent dose-dependent inhibiting effect on angiotensin II (AngII) induced-proliferation and a stimulatory effect on proliferation of quiescent cells. The cAMP/PKA pathway was involved in the AM inhibitory effect of AngII-induced proliferation in HASMC. PI3K/Akt and ERK pathways were involved in the proliferative effect exerted by AM per se. Our results suggest that AM plays a role in the regulation of HASMC growth antagonizing the AngII effect and may be involved in conditions of altered regulation of the blood vessels.
Keywords: Adrenomedullin; Angiotensin II; HASMC; Proliferation;

Tissue-specific modulation of angiotensin-converting enzyme (ACE) in hyperthyroidism by M.S. Carneiro-Ramos; V.B. Silva; R.A.S. Santos; M.L.M. Barreto-Chaves (2942-2949).
We have previously demonstrated the interaction between the RAS and thyroid hormones (TH). The present study was designed to determine the role of TH in the local regulation of ACE activity and expression in different tissues. Adult male Wistar rats were randomized into three groups: T4-25 and T4-100 (0.025 and 0.100 mg/kg of body weight/day of l-thyroxine for 14 days, respectively) and control. Hemodynamic parameters as well as cardiac and renal hypertrophy were evaluated. ACE activity and mRNA levels were determined by Fluorimetric and Northern blot assays, respectively. Both doses increased SBP and HR, as well as inducing cardiac and renal hypertrophy. Pulmonary and serum ACE levels were comparable among the groups. Both doses promoted increased renal ACE activity and expression but surprisingly ACE was diminished in the heart in both hyperthyroid groups. This change was mediated by a tissue-specific transcription mechanism.
Keywords: Angiotensin-converting enzyme; Hyperthyroidism; Local renin-angiotensin system;

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin by Yike Yu; Jianen Hu; Yuji Miyaguchi; Xuefang Bai; Yuguang Du; Bingcheng Lin (2950-2956).
Animal blood is potentially an untapped source of drugs and value-added food production. More than 400 million pigs are slaughtered each year but porcine blood is usually discarded in China. This study describes the isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from porcine hemoglobin. The most active hydrolysate was obtained from the peptic digestion of porcine hemoglobin. After the purification of ACE-inhibitory peptides with Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) on C18 column, two active fractions were obtained. They were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). They were LGFPTTKTYFPHF and VVYPWT, corresponding to the 34–46 fragment of the α chain and the 34–39 fragment of the β chain of porcine hemoglobin, with IC50 values of 4.92 and 6.02 μM, respectively. They were the first found from porcine hemoglobin; in particular, LGFPTTKTYFPHF was a novel ACE-inhibitory peptide. In addition, the purified ACE inhibitors both competitively inhibited ACE, and maintained inhibitory activity even after incubation with gastrointestinal proteases. This suggests that these peptides might have a potential antihypertensive effect.
Keywords: Angiotensin I-converting enzyme; Inhibitory activity; Peptide; Porcine hemoglobin;

Hemorphin and hemorphin-like peptides isolated from dog pancreas and sheep brain are able to potentiate bradykinin activity in vivo by Danielle Ianzer; Katsuhiro Konno; Carlos Henrique Xavier; Reto Stöcklin; Robson Augusto S. Santos; Antônio Carlos Martins de Camargo; Daniel Carvalho Pimenta (2957-2966).
Hemorphins are biologically active peptides, derived from hemoglobin, which presents a number of physiological activities. Proteolytic generation of these peptides is not fully understood; however, among their roles, is to provoke reduction on blood pressure. In this work, this particular biological effect was chosen as the monitor for the selection of mammalian vasoactive peptides. By combining high-performance liquid chromatography and mass spectrometry, including ‘de novo’ sequencing, several hemorphin-like peptides were identified presenting bradykinin potentiating activity. Moreover, taking LVV-hemorphin-7 as model compound, we evaluated its biological effect on blood pressure of anaesthetized rats. By summarizing all the results, it is possible to present the hemorphins as a family of proteolytically generated peptides that are able to potentiate bradykinin activity in vivo.
Keywords: Hemorphin; Hemoglobin; Bradykinin potentiating peptide; Bradykinin; Mass spectrometry; De novo sequencing;

Effect of novel selective non-peptide kinin B1 receptor antagonists on mouse pleurisy induced by carrageenan by Robson Costa; Elizabeth S. Fernandes; Octávio Menezes-de-Lima; Maria M. Campos; João B. Calixto (2967-2975).
Two novel selective non-peptide kinin B1 receptor antagonists, the benzodiazepine antagonist and SSR240612, were evaluated in carrageenan-induced mouse pleurisy. The peptide R-715 (0.5 mg/kg, i.p.) and the non-peptide benzodiazepine (3 mg/kg, i.p.) antagonists significantly decreased cellular migration (predominantly neutrophils), without altering plasma exudation. SSR240612 (1 mg/kg, i.p.) diminished total cells and neutrophils, besides exudation. Oral administration of SSR240612 (10 mg/kg) also reduced total cell and neutrophil counts. Only the benzodiazepine antagonist inhibited the lung myeloperoxidase activity. No tested antagonist significantly altered the lung and pleural TNFα and IL-1β production. We provide interesting evidence on the anti-inflammatory in vivo effects of non-peptide B1 receptor antagonists.
Keywords: B1 receptor; Peptide antagonists; Non-peptide antagonists; Carrageenan; Pleurisy; Mice;

Substance P and neurokinin-1 immunoreactivities in the neural circadian system of the Alaskan northern red-backed vole, Clethrionomys rutilus by Rayna E. Samuels; Ronald J. Tavernier; Marina R. Castillo; Abel Bult-Ito; Hugh D. Piggins (2976-2992).
The suprachiasmatic nucleus (SCN) of the hypothalamus houses the main mammalian circadian clock. This clock is reset by light–dark cues and stimuli that evoke arousal. Photic information is relayed directly to the SCN via the retinohypothalamic tract (RHT) and indirectly via the geniculohypothalamic tract, which originates from retinally innervated cells of the thalamic intergeniculate leaflet (IGL). In addition, pathways from the dorsal and median raphe (DR and MR) convey arousal state information to the IGL and SCN, respectively. The SCN regulates many physiological events in the body via a network of efferent connections to areas of the brain such as the habenula (Hb) in the epithalamus, subparaventricular zone (SPVZ) of the hypothalamus and locus coeruleus of the brainstem—areas of the brain associated with arousal and behavioral activation.Substance P (SP) and the neurokinin-1 (NK-1) receptor are present in the rat SCN and IGL, and SP acting via the NK-1 receptor alters SCN neuronal activity and resets the circadian clock in this species. However, the distribution and role of SP and NK-1 in the circadian system of other rodent species are largely unknown. Here we use immunohistochemical techniques to map the novel distribution of SP and NK-1 in the hypothalamus, thalamus and brainstem of the Alaskan northern red-backed vole, Clethrionomys rutilus, a species of rodent currently being used in circadian biology research. Interestingly, the pattern of immunoreactivity for SP in the red-backed vole SCN was very different from that seen in many other nocturnal and diurnal rodents
Keywords: Tachykinin; Suprachiasmatic; Intergeniculate leaflet; Raphe nuclei; Habenula; Subparafascicular; Interpeduncular; Rodent;

Renal hyporesponsiveness to brain natriuretic peptide: Both generation and renal activity of cGMP are decreased in patients with pulmonary hypertension by Anne Charloux; Ari Chaouat; François Piquard; Gabrielle Brandenberger; Emmanuel Weitzenblum; Bernard Geny (2993-2999).
We examined the mechanisms of renal resistance to atrial and brain natriuretic peptides (ANP and BNP) in pulmonary hypertension (PH). Compared to eight controls, nine PH patients showed a reduced ability to excrete an acute sodium load despite increased circulating ANP, BNP and cyclic guanosine monophosphate (cGMP), their second messenger. Patients’ reduced urinary cGMP/BNP and natriuresis/urinary cGMP ratios demonstrated impaired generation of and reduced renal response to cGMP, respectively. Therefore, PH patients hyporesponsiveness to cardiac natriuretic peptides is likely located both upstream and downstream cGMP generation. Natriuretic peptide signalling pathway disruptions might be accessible to therapy.
Keywords: Natriuresis; Natriuretic peptides; cGMP; Pulmonary hypertension; Renal function;

Quantification of human angiotensinogen by a novel sandwich ELISA by Yuki Suzaki; Yuri Ozawa; Hiroyuki Kobori (3000-3002).
The urinary angiotensinogen excretion rates show a clear relationship to kidney angiotensin II content, suggesting that urinary angiotensinogen may serve as an index of angiotensin II-dependent hypertensive rats. However, simple and accurate methods to measure human angiotensinogen are unavailable at this time. We have developed two antibodies and a sensitive and specific quantification ELISA system for human angiotensinogen to be applicable to human subjects. The ELISA is able to detect human angiotensinogen at range of 0.01–1 μg/well (R 2  = 0.9945) using standard ELISA plates. This ELISA will be a useful tool to investigate the relationship between urinary angiotensinogen excretion rates and reactivity to antihypertensive drugs in hypertensive human subjects.

Hypoxia increases endothelin-1 mRNA expression but not immunoreactive endothelin in the medium of T98G glioblastoma cells under cytokine treatment by Yan Zhang; Yin Li; Kazuhito Totsune; Kumi Kikuchi; Osamu Murakami; Shigeki Shibahara; Kazuhiro Takahashi (3003-3006).
Endothelin-1 (ET-1) levels in the culture medium were considered to reflect the transcription of the ET-1 gene and the subsequent secretion of ET-1 from cultured cells. It has not been clarified how different ET-1 mRNA expression levels and immunoreactive (IR)-ET levels in the culture medium are in the cell culture system. We studied ET-1 mRNA expression levels and IR-ET levels in the medium of T98G glioblastoma cells treated with cytokines. T98G glioblastoma cells were cultured with cytokines (interferon-γ 100 U/ml, tumor necrosis factor-α 20 ng/ml and interleukin-1β 10 ng/ml) under normoxia or hypoxia (1% O2). Northern blot analysis showed that ET-1 mRNA expression levels were increased by tumor necrosis factor-α alone or a combination of tumor necrosis factor-α and interleukin-1β, or three cytokines, and the increase was further enhanced under hypoxia. Particularly, relative expression levels of ET-1 mRNA were significantly higher under hypoxia than in normoxia in the treatment with a combination of three cytokines. IR-ET levels in the medium were increased by treatment with tumor necrosis factor-α, interleukin-1β or a combination of tumor necrosis factor-α and interleukin-1β, or three cytokines. In contrast to the mRNA expression levels, IR-ET levels in the medium of T98G cells treated with a combination of three cytokines were rather decreased under hypoxia compared with those in normoxia. These findings indicate that hypoxia induces ET-1 mRNA expression in the treatment of three cytokines, but IR-ET levels in the medium do not reflect this induction in T98G glioblastoma cells.

Tachykinins and the control of prolactin secretion by Luciano Debeljuk; Mercedes Lasaga (3007-3019).
Tachykinins are present in the pituitary gland and in brain areas involved in the control of the secretion of pituitary hormones. Tachykinins have been demonstrated to stimulate prolactin release acting directly on the anterior pituitary gland. These peptides have also been revealed to be able to act at the hypothalamic level, interacting with neurotransmitters and neuropeptides that have the potential to affect prolactin secretion. Tachykinins seem to act by stimulating or inhibiting the release of the factors that affect prolactin secretion. Among them, tachykinins have been demonstrated to stimulate oxytocin and vasopressin release, which in turn results in prolactin release. Tachykinins also potentiated the response to vasoactive intestinal peptide (VIP) and reinforced the action of glutamate, which in turn result in prolactin release. They have also been shown to interact with serotonin, a neurotransmitter involved in the control of prolactin secretion. In addition, tachykinins have been shown to inhibit GABA release, a neurotransmitter with prolactin-release inhibiting effect. This inhibition may result in an increased prolactin secretion by removal of the GABA inhibition. On the other hand, tachykinins have also been shown to stimulate dopamine release by the hypothalamus, an action that results in an inhibition of prolactin release. Dopamine is a well known inhibitor of prolactin secretion. In conclusion, although tachykinins have been shown to have a predominantly stimulatory effect on prolactin secretion, especially at the pituitary level, under some circumstances they may also exert an inhibitory influence on prolactin release, by stimulating dopamine release at the hypothalamic level.
Keywords: Tachykinins; Prolactin; Neurotransmitters; Hypothalamus; Anterior pituitary;

Carrier-mediated delivery of peptidic drugs for cancer therapy by Crispin R. Dass; Peter F.M. Choong (3020-3028).
Protein and peptide drugs are used for treatment of a variety of ailments. However, their wider use has been hindered by issues such as poor bioavailability in vivo and the cost involved in producing these drugs. This review discusses the various carrier-mediated methods used for delivery of peptide and protein drugs, with emphasis on liposomal and microspherical drug delivery systems. A brief look at the types of peptidic drugs currently in use clinically, and a brief discourse on several novel ideas for better protein delivery systems for cancer therapy is included.
Keywords: Peptide; Protein; Drug delivery; Cancer; Liposome; Microparticle;