Peptides (v.24, #6)

New antihypertensive peptides isolated from rapeseed by Ewa D Marczak; Hachiro Usui; Hiroyuki Fujita; Yanjun Yang; Megumi Yokoo; Andrzej W Lipkowski; Masaaki Yoshikawa (791-798).
Four potent angiotensin converting enzyme (ACE) inhibitory peptides, IY, RIY, VW and VWIS, were isolated from subtilisin digest of rapeseed protein. Among them RIY and VWIS are new peptides with IC50 28 and 30 μM, respectively. All isolated peptides lowered blood pressure of spontaneously hypertensive rats (SHR) following oral administration. The maximum effect in the case of RIY was observed 4 h after administration, while maximum effect of other peptides on blood pressure occurred 2 h after administration. Furthermore, the antihypertensive effect of RIY was observed even in old rats, in which ACE inhibitors become less effective, suggesting that a different mechanism other than ACE inhibition is also involved in lowering blood pressure by this peptide. Subtilisin digest of rapeseed protein also significantly lowered blood pressure of SHR after oral administration of a single dosage 0.15 g/kg, exerting maximum antihypertensive effect 4 h after administration. This digest appears promising as a functional food, which may be useful in the prevention and treatment of hypertension.
Keywords: Angiotensin-converting enzyme inhibitory peptides; Protein digest; Rapeseed; Antihypertensive effect; Spontaneously hypertensive rat;

Purification and characterization of Luffin P1, a ribosome-inactivating peptide from the seeds of Luffa cylindrica by Feng Li; Xin-xiu Yang; Hen-chuan Xia; Rong Zeng; Wei-guo Hu; Zhen Li; Zu-chuan Zhang (799-805).
A peptide designated Luffin P1 was purified from the seeds of Luffa cylindrica. Its molecular mass was determined to be 5226.1 Da by MALDI-TOF MS analysis. The purified Luffin P1 shows a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate with IC50 of 0.88 nM. Its reaction mechanism is the same as that of the ribosome-inactivating protein trichosanthin, which is an rRNA N-glycosidase. Besides, the results of gel filtration chromatography suggested the existence of polymers of Luffin P1 and polymerization of Luffin P1 enhanced its rRNA N-glycosidase activity. Luffin P1 was the smallest peptide yet reported that has translational inhibitory activity. The cDNA and deduced amino acid sequence of Luffin P1 has also been determined.
Keywords: Luffin P1; Ribosome-inactivating protein; N-Glycosidase; Luffa cylindrica; Polymerization;

In vitro and in vivo activity of an antibacterial peptide analog against uropathogens by Mare Cudic; C.Virginia Lockatell; David E Johnson; Laszlo Otvos (807-820).
The alarming rate of bacterial resistance induction highlights the clinical need for antimicrobial agents that act by novel modes of action. Based on the activity profile, the general tissue distribution and renal clearance of peptide-based drugs, we hypothesized that our newly developed pyrrhocoricin derivative would be able to fight resistant uropathogens in vitro and in vivo. Indeed, the Pip-pyrr-MeArg dimer killed all 11 urinary tract infection-related Escherichia coli and Klebsiella pneumoniae strains we studied in the sub-low micromolar concentration range. Almost all control antibiotics, including the currently leading trimethoprim-sulfametoxazole combination for urinary tract infection, remained without considerable activity against two or more of these bacterial strains. In a mouse ascending urinary tract infection model with E. coli CFT073 as pathogen, two doses of intravenous, subcutaneous or oral treatment with the Pip-pyrr-MeArg derivative reduced the bacterial counts in the kidneys, bladder and urine to varying levels. Statistically significant elimination or reduction of bacteria compared to untreated animals was observed at dual intravenous or subcutaneous doses of 0.4 or 10 mg/kg, respectively. Serial passage of the same E. coli strain in the presence of sublethal doses of the designed peptide failed to generate resistant mutants. The Pip-pyrr-MeArg dimer showed no toxicity to COS-7 cells to the highest 500 μM concentration studied.
Keywords: Bacterial resistance; Cell toxicity; In vivo protection; Mouse model; Pyelonephritis; Pyrrhocoricin;

In the blood coagulation cascade, heparin activates human plasma antithrombin III (hAT III), resulting in the inhibition of factor Xa. This polysaccharide also exhibits hemorrhagic tendency mediated by the inhibition of thrombin in heparinotherapy. Therefore, attention has focused on the development of low molecular weight heparins (LMW-heparins) that inhibit factor Xa but not thrombin. In this investigation, we examined the biochemical and physicochemical properties of hAT III-derived heparin-binding peptides (HBPs). Of all the tested HBPs, hAT III (123–139) exhibited the highest affinity with heparin and showed an inhibitory effect on the heparin-induced enhancement of hAT III activity toward factor Xa, indicating that hAT III (123–139) specifically interacts with the active region in heparin. We prepared a synthetic hAT III (123–139)-coupled affinity chromatography system, and demonstrated that this novel affinity chromatography is useful for fractionation of highly active moieties in LMW-heparins.
Keywords: Antithrombin III; Heparin; Factor Xa; Heparin-binding peptide;

Solution structure of a peptide derived from the β subunit of LFA-1 by Zhang Shuxing; Wong S. Ying; Teruna J. Siahaan; Seetharama D.S. Jois (827-835).
Cell-adhesion molecules are critical for immune response. It is well known that the inhibition of adhesion is very effective in immunotherapy and that the peptides derived from leukocyte function associated antigen (LFA-1) and intercellular adhesion molecule (ICAM-1) modulate cell-adhesion interaction. The three-dimensional structure of a cyclic peptide, Cyclo(1,12)Pen1-Asp2-Leu3-Ser4-Tyr5-Ser6-Leu7-Asp8-Asp9-Leu10-Arg11-Cys12 (cLBEL) derived from the β subunit of LFA-1 which is known to modulate homotypic T-cell-adhesion process has been studied using NMR, CD and molecular dynamics (MD) simulation. The peptide exhibits two possible conformations in solution. Structure I has a conformation with two consecutive β-turns involving residues Tyr5-Ser6-Leu7-Asp8 and Asp9-Leu10-Arg11-Cys12. Structure II has a β-turn at Tyr5-Ser6-Leu7-Asp8 and forms a β-hairpin type of conformation.
Keywords: Leukocyte function associated antigen-1 (LFA-1); MIDAS domain; Peptide conformation; T-cell adhesion; β-Turn;

The structure–activity relations of [His7]-corazonin were studied using two different bioassay systems; i.e. inhibitory effect on spinning rate in the silkworm, Bombyx mori, and darkening response in albino nymphs of the migratory locust, Locusta migratoria. Deletion of the N-terminus, shortening of the peptide and single amino acid substitutions reduced activity in a similar manner except for the minimum effective dose in the two insects. The results also revealed that the residues at position 1, 3 and 5 were particularly important for biological activity. Despite the different physiological affects, the two insect species exhibited similar structure–activity relationships, suggesting that they might have similar receptor systems.
Keywords: [His7]-corazonin; Corazonin derivatives; Structure–activity; Locusts; Locusta migratoria; Silkworm; Bombyx mori; Spinning; Dark color;

A cDNA, encoding a crustacean hyperglycemic hormone (cHH) of the South African spiny lobster, Jasus lalandii has been cloned. The cDNA consists of 1773 bp with an open reading frame of 399 bp that encodes a preprohormone of 133 amino acid residues. The preprohormone consists of a 25 amino acid hydrophobic signal peptide, a 32 amino acid cHH precursor-related peptide (CPRP) and the cHH sequence of 72 amino acid residues. The cHH sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by Gly-Lys, where Gly serves as an amidation site. The deduced amino acid sequence of the CPRP is in complete agreement with a peptide previously elucidated from sinus glands of J. lalandii, code-named CPRP 2 and the sequence of the cHH peptide matches that of the minor cHH isoform of J. lalandii, i.e. crustacean hyperglycemic hormone-II (cHH-II), which was also previously obtained by peptide sequencing. In situ hybridization on eyestalks revealed strong cHH-II mRNA expression in a subset of neurosecretory cells of the X-organ.
Keywords: Crustacean hyperglycemic hormone; cHH; Spiny lobster; Molecular cloning; Jasus lalandii; Preprohormone; In situ hybridization;

Cloning of maximakinin precursor cDNAs from Chinese toad, Bombina maxima, venom by Tianbao Chen; Anthony J. Bjourson; Stephen McClean; David F. Orr; Edmund J. O’Kane; Pingfan Rao; Chris Shaw (853-861).
Using a novel technique that we have developed for cloning of amphibian skin secretion peptide cDNAs from lyophilized samples, we report here that maximakinin (DLPKINRKGP-bradykinin) is encoded by two different cDNAs, named BMK-1 and BMK-2, containing either four tandem repeat sequences or a single copy. The open reading frames of both precursor cDNAs were found to be 152 and 116 amino acid residues, respectively. These data provide evidence that the structural diversity of peptides in amphibian skin secretions arising from molecular evolutionary events, can be mediated by parallel diversity in encoding mRNAs that in itself may reflect serial gene duplications.
Keywords: Amphibian; Venom; Bradykinin; cDNA; 3′ and 5′ RACE;

Bradykinins and their cDNA from piebald odorous frog, Odorrana schmackeri, skin by Long Li; Anthony J Bjourson; Jianyuan He; Guangxian Cai; Pingfan Rao; Chris Shaw (863-872).
Bradykinin, (des-Arg9)-bradykinin and bradykinyl-VAPAS, were identified in the skin secretion of the piebald odorous frog, Odorrana schmackeri. Using 3′- and 5′-RACE reactions, bradykinin precursor cDNA was cloned and found to contain an open-reading frame of 311 amino acid residues. The preprobradykinin was found to consist of a putative signal peptide of approximately 20 amino acid residues, followed by seven tandem repeat coding domains of 43–44 amino acids. Bradykinin and its C-terminal extended molecular form were encoded on this single precursor and could be generated by differential post-translational processing.
Keywords: Amphibian; Peptides; Cloning; Kininogen; Molecular evolution;

The structural diversity of polypeptides in amphibian skin secretion probably reflects different roles in dermal regulation or in defense against predators. Here we report the structures of two novel trypsin inhibitor analogs, BOTI and BVTI, from the dermal venom of the toads, Bombina orientalis and Bombina variegata. Cloning of their respective precursors was achieved from lyophilized venom cDNA libraries for the first time. Amino acid alignment revealed that both deduced peptides, consisting of 60 amino acid residues, including 10 cysteines and the reactive center motif, -CDKKC-, can be affirmed as structural homologs of the trypsin inhibitor from Bombina bombina skin.
Keywords: Amphibian; Mass spectrometry; Trypsin inhibitor; cDNA cloning;

Angiotensin II reduces serotonin release in the rat subfornical organ area by Junichi Tanaka; Katsuhide Kariya; Masahiko Nomura (881-887).
In the present study we used intracerebral microdialysis techniques to examine whether angiotensin II (ANG II) modulates the release of serotonin (5-hydroxytryptamine, 5-HT) in the subfornical organ (SFO) in freely moving rats. Extracellular concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the region of the SFO were significantly decreased by microinjection of ANG II (10 pmol, 50 nl), but not by vehicle, into the dialysis site. No significant changes in the 5-HT and 5-HIAA levels caused by ANG II were observed in the sites away from the SFO. Water ingestion significantly enhanced the amount of the decrease in the 5-HT and 5-HIAA concentrations in the SFO area elicited by the ANG II injection. These results show that ANG II may reduce the release of 5-HT in the SFO area, and imply that the 5-HT receptor mechanism in the SFO area may participate in the elicitation of the drinking behavior to ANG II.
Keywords: Subfornical organ; Angiotensin II; 5-Hydroxytryptamine; 5-Hydroxyindoleacetic acid; Drinking;

Ventricular cardiotrophin-1 activation precedes BNP in experimental heart failure by Michihisa Jougasaki; Hanna Leskinen; Amy M. Larsen; Andreas Luchner; Alessandro Cataliotti; Issei Tachibana; John C. Burnett (889-892).
Both cardiotrophin-1 (CT-1) and B-type or brain natriuretic peptide (BNP) are activated by cardiomyocyte stretch, and gene expression of CT-1 and BNP are augmented in the heart in experimental and human congestive heart failure (CHF). The goal of this study was to define cardiac gene expression of CT-1 and BNP by Northern blot analysis in normal (n=5), early left ventricular dysfunction (ELVD, n=5) and overt CHF dogs (n=5), in which ventricular function is progressively decreased. CT-1 mRNA was detected in both atria and ventricles in normal dogs. Ventricular CT-1 mRNA production increased in ELVD, and it further increased in overt CHF. Ventricular BNP mRNA remained below or at the limit of detection in normal and ELVD models, and it markedly increased in overt CHF. This study reports differential regulation of gene expression of CT-1 and BNP in the heart during the progression of CHF, and demonstrates that ventricular CT-1 gene activation precedes ventricular BNP gene activation.
Keywords: BNP; Cardiotrophin-1; Congestive heart failure; Cytokines; Northern blot analysis;

Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue by M.Olga Garcı́a-Fernández; Rosa M. Solano; Marı́a J. Carmena; Rebeca Busto; Guillermo Bodega; Antonio Ruı́z-Villaespesa; Juan C. Prieto; Manuel Sánchez-Chapado (893-902).
Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC1/VPAC2 receptors whereas PACAP binds with higher affinity than VIP to PAC1 receptor. Here we systematically studied the presence and distribution of functional PAC1, VPAC1 and VPAC2 receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC1 (various isoforms due to alternative splicing), VPAC1 and VPAC2 receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC1, VPAC1 and VPAC2 receptors in human prostate as well as its maintenance after malignant transformation.
Keywords: PACAP; VIP; PAC1; VPAC1 and VPAC2 receptors; Prostate cancer;

Central effects of selective NK1 and NK3 tachykinin receptor agonists on two models of experimentally-induced colitis in rats by Giovanna Improta; Francesco Carpino; Vincenzo Petrozza; Antonio Guglietta; Alessandra Tabacco; Maria Broccardo (903-911).
Peripheral tachykinins (TKs) are believed to play a role in the pathogenesis of inflammatory bowel diseases (IBD). In this study we investigated changes induced by central administration of two natural TK receptor agonists, NK1 (PG-SPI) and NK3 (PG-KII), on trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulphate (DSS)-induced experimental colitis in rats. Colitis was induced by instilling a single intracolonic dose of TNBS 50 mg kg−1 (0.5 ml in 50% ethanol) or by oral administration of 5% DSS for 7 days. Each group of rats was intracerebroventricularly injected daily with PG-SPI and PG-KII (0.5, 5, and 50 μg kg−1). On day 3, TNBS-treated animals were killed and the severity of gut inflammation was evaluated by measuring myeloperoxidase (MPO) activity, interleukin-1β (IL-1β) production and by scoring macroscopic and histologic colonic damage. DSS-treated animals were checked daily for the length of survival and for stool consistency and faecal blood. In the TNBS group, PG-SPI and PG-KII increased scores for the severity of colonic damage, stimulated the production of IL-1β and increased granulocyte infiltration into the colon (MPO activity). In the DSS group, PG-SPI and PG-KII decreased the percentage of surviving animals, and increased the number of rats that developed loose stools and blood in the faeces and the MPO activity. These results indicate that centrally injected NK1 and NK3 tachykinin receptor agonists play a proinflammatory role in experimentally-induced colitis in rats.
Keywords: Experimental colitis; Natural NK1 and NK3 receptor agonists; Central nervous system; Rats;

Ghrelin-induced feeding is dependent on nitric oxide by F.Spencer Gaskin; Susan A. Farr; William A. Banks; Vijaya B. Kumar; John E. Morley (913-918).
Ghrelin is a newly discovered gastric peptide, which has orexigenic effects. Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor and stimulates growth hormone and gastrointestinal motility. We have previously shown that nitric oxide (NO) plays an important role as a mediator of feeding induced by a variety of neuropeptides. This raises the question of whether ghrelin’s effects are NO dependent. Here, we first determined that intracerebroventricular administration of 100 ng of ghrelin significantly increased food intake in satiated mice. We next examined the effects of N ω-nitro-l-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, on ghrelin-induced increase in food intake. A subthreshold dose (12.5 mg/kg; SC) of L-NAME significantly blocked the ghrelin-induced increase in food intake. Ghrelin administration increased the levels of nitric oxide synthase in the hypothalamus. This supports the hypothesis that nitric oxide is a central regulator of food consumption.
Keywords: Ghrelin; L-NAME; Mouse; Food intake; Nitric oxide; Orexigenic peptides; Hypothalamus;

Hypothalamic paraventricular injections of ghrelin: effect on feeding and c-Fos immunoreactivity by Pawel K Olszewski; Martha K Grace; Charles J Billington; Allen S Levine (919-923).
The paraventricular hypothalamic nucleus (PVN) appears to integrate orexigenic properties of a novel peptide, ghrelin. Thus, we examined central mechanisms underlying feeding generated by intra-PVN ghrelin. We established that 0.03 nmol of PVN-injected ghrelin was the lowest dose increasing food consumption and it induced c-Fos immunoreactivity (a marker of neuronal activation) in the PVN itself, as well as in other feeding-related brain areas, including the hypothalamic arcuate and dorsomedial nuclei, central nucleus of the amygdala, and nucleus of the solitary tract. We conclude that the PVN, as part of larger central circuitry, mediates orexigenic properties of ghrelin.
Keywords: Food intake; Ghrelin; Immunohistochemistry; c-Fos; Brain stem; Amygdala; Hypothalamus; Injection;

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus by Qing-Ping Wang; Jian-Lian Guan; Takashi Matsuoka; Yumi Hirayana; Seiji Shioda (925-930).
The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.
Keywords: Ultrastructure; Synapse; Sleep; Pain; Immunostain; Electron microscopy;

Effect of bradykinin analogues on the B1 receptor of rat ileum by Gabriele Vietinghoff; Enzo Hilscher; Inge Paegelow; Siegmund Reissmann (931-935).
The longitudinal muscle of isolated rat ileum is a sensitive bioassay suitable for testing compounds with antagonistic effects on the B1 receptor. Bradykinin analogues with replacement of proline by alkyl-substituted phenylalanine at position 7 are effective on this receptor as entire molecules and have a stronger antagonistic effect than on the B2 receptor. A corresponding desArg9-compound has a specific effect on the B1 receptor and a very high antagonistic potency. [LNMPhe2]bradykinin as a compound without any replacement at position 7 or 8 shows antagonistic activity as well.
Keywords: Bradykinin; DesArg9-bradykinin; B1 receptor; B2 receptor; Antagonists; Smooth muscle contraction;

Relation of β-casomorphin to apnea in sudden infant death syndrome by Zhongjie Sun; Zhonge Zhang; Xiuqing Wang; Robert Cade; Zaher Elmir; Melvin Fregly (937-943).
Sudden infant death syndrome (SIDS) is the most common cause of death in infants and its pathogenesis is complex and multifactorial. The aim of this review is to summarize recent novel findings regarding the possible association of β-casomorphin (β-CM) to apnea in SIDS, which has not been widely appreciated by pediatricians and scientists. β-CM is an exogenous bioactive peptide derived from casein, a major protein in milk and milk products, which has opioid activity. Mechanistically, circulation of this peptide into the infant’s immature central nervous system might inhibit the respiratory center in the brainstem leading to apnea and death. This paper will review the possible relationship between β-CM and SIDS in the context of passage of β-CM through the gastrointestinal tract and the blood–brain barrier (BBB), permeability of the BBB to peptides in infants, and characterization of the casomorphin system in the brain.
Keywords: β-Casomorphin; Sudden infant death syndrome; Blood–brain barrier; Peptides; Exogenous peptides; Peptidase; Opioid; Opioid receptor; Respiratory center; Apnea;