Peptides (v.24, #4)

Structure–activity relationship of rubiscolins as δ opioid peptides by Shuzhang Yang; Soushi Sonoda; Liping Chen; Masaaki Yoshikawa (503-508).
To study the structure–activity relationship of rubiscolins (YPLDLF and YPLDL), δ opioid peptides derived from the spinach Rubisco, we substituted the amino acid residues and evaluated their activities by mouse vas deferens (MVD) and guinea pig ileum (GPI) assays as well as receptor affinity. Replacement of Leu3 with Ile and Met in rubiscolin-6 potentiated the δ opioid activity by about four times in MVD assay. Asp4 cannot be replaced by Ala, Glu or His. The original Leu5 was optimal, while substitution of Phe6 with Val potentiated its δ opioid activity by more than 10 times. The most potent derivative we obtained was YPMDLV, which was nearly 20 times more potent than rubiscolin-6 in MVD assay. The derivatives thus obtained showed higher δ receptor affinity and more potent antinociceptive activity than rubiscolins.
Keywords: Rubiscolin; Opioid peptide; Structure–activity relationship; Antinociception;

A peptide, with a molecular weight of 9 K and an N-terminal sequence identical to that of ubiquitin, was isolated from fresh fruiting bodies of the yellow mushroom Cantharellus cibarius. The peptide manifested ribonucleolytic activity toward poly A, poly C, poly G, and poly U, with the activity toward the first two polyhomoribonucleotides being higher. Maximal activity toward yeast tRNA was observed at a temperature of 70 °C and a pH of 7. The peptide was adsorbed on DEAE-cellulose, CM-Sepharose, and Q-Sepharose. The yield of the peptide was 7 mg from 1.8 kg mushroom.
Keywords: Ubiquitin; Mushroom; Ribonuclease; Cantharellus cibarius;

Pyrularia thionin (PT) is a basic 47 amino acid peptide isolated from the nuts of Pyrularia pubera. Its structure and properties have been studied in some detail. Its receptor site is a domain of membrane phosphatidyl serine (PS), where it binds with a relatively high specificity. A segment of its covalent structure, the nonapeptide Thr-Trp-Ala-Arg-Asn-Ser-Tyr-Asn-Val, designated serine nonapeptide (SNP), corresponds to amino acids 7–15 of the thionin, except for the position 12 (Ser), which substitutes for Cys, to give stability. This peptide represents what we consider to be the active site of the thionin, and it also binds to PS domains, but less tightly than thionin does. The peptide has an effect on the prothrombinase assay using the chromophore S2238 to measure the thrombin produced by the prothrombinase complex. It is shown that SNP stimulates the prothrombinase complex activity, instead of inhibiting it, as would be expected if it simply covered the PS sites on the membrane of erythrocyte ghosts, used in the prothrombinase assay. SNP appears to substitute for Va in the prothrombinase complex reaction, in a Ca2+ independent manner, being even more effective in the absence than in the presence of ghosts. In the clotting system, SNP can also substitute for Factor Va.
Keywords: Prothrombinase; Clotting process; Factor Va; Factor Xa; Thionin;

Antimicrobial peptides in the first line defence of human colon mucosa by Maria Tollin; Peter Bergman; Torgny Svenberg; Hans Jörnvall; Gudmundur Hrafn Gudmundsson; Birgitta Agerberth (523-530).
Antimicrobial peptides and proteins are effector molecules in the protection of epithelial surfaces. We have evaluated the presence of antimicrobial peptides/proteins that can participate in human colonic defence against microbes. A peptide/protein extract of normal human colon mucosa was found to be active against Gram-positive bacteria, Gram-negative bacteria, and fungi. Four polypeptides with antimicrobial activity were isolated from this material and they were identified by N-terminal amino acid sequence analysis as ubiquicidin, histone H2B, eosinophil cationic protein, and phospholipase A2 (PLA2). Using immunodetection and mass spectrometry, LL-37, HNP1–3, and HBD-1 were also identified. Combined, these results indicate that the colon mucosa is protected by a complex mixture of polypeptides, able to kill invading microbes and working in synergy as a barrier against bacterial invasion.
Keywords: Innate immunity; Histones; Ubiquicidin; Defensins; LL-37;

Two new peptides to improve post-operative gastric ileus in dog by L. Trudel; M. Bouin; C. Tomasetto; P. Eberling; S. St-Pierre; P. Bannon; M.C. L’Heureux; P. Poitras (531-534).
Peptides can influence gastrointestinal motility, and from data obtained earlier in rats, we hypothesized that MTL-RP/Ghrelin, as well as CGRP receptor antagonist 8–37, could improve gastric post-operative ileus in dog. Dogs submitted to laparotomy were perfused with or saline or CGRP 8–37 or MTL-RP/Ghrelin on days 1–4 post-operatively while gastric emptying was estimated by measuring the postprandial increase in plasma acetaminophen ingested with a meal. As expected, in saline-treated animals the gastric emptying function was impaired post-operatively. The total amount of acetaminophen emptied (AUC over 150 min) on post-operative days 1–4 reached respectively 31±5%, 65±8%, 60±8% and 62±8% of the normal emptying capacity. CGRP antagonist increased the total emptying of acetaminophen to 52±5% on day 1, 95±2% on day 2 and 103±3% (P<0.05) on day 3. The delayed emptying of acetaminophen seen post-operatively in saline-treated animals could be completely reversed by MTL-RP/Ghrelin (P<0.01) whether it was given at 100 μg/kg on day 2 (102±7% of the normal emptying capacity), 4 μg/kg on day 3 (106±7%) or 20 μg/kg on day 4 (132±8%). As found earlier in rodents, CGRP receptor antagonist 8–37 as well as MTL-RP/Ghrelin are potent prokinetics to improve post-operative gastric ileus in dog.
Keywords: CGRP; Calcitonin gene-related peptide; MTL-RP; Motilin-related peptide; Ghrelin; Gastrointestinal hormones; Gastrointestinal motility; Regulatory peptides;

Effect of long-term CCK blockade on the pancreatic acinar cell renewal in rats with acute pancreatitis by Ana M de la Mano; Sara Sevillano; Manuel A Manso; Isabel de Dios (535-541).
This study determines the effect of 7-day pretreatment with L364,718 (a potent cholecystokinin (CCK) receptor antagonist) on pancreatic cell turnover during the course of acute pancreatitis (AP) induced in the rat by bile-pancreatic duct obstruction (BPDO). Cell cycle distribution and apoptosis were analyzed by flow cytometry using propidium iodide (PI) and Annexin V staining. Besides altering the pancreatic redox status, long-term CCK blockade inhibited the normal proliferation of acinar cells as indicated by the significant increase in G0/G1-phase cells and the decrease in G2/M-cells found in control rats treated with L364,718 for 7 days. A progressive depletion in pancreatic GSH was found from 3 to 24 h after BPDO with similar values in L364,718-pretreated and non-treated rats, which led to a maximum peak in malondialdehyde (MDA) levels 6 h after BPDO. However, plasma amylase activity and ascites volume indicated higher severity of AP in L364,718-pretreated rats. CCK blockade enhanced the alterations that appear in cell cycle distribution of acinar cells during AP demonstrated by the significantly higher increase in G0/G1-cells and decrease in S-cells found in L364,718-treated rats 48 h after BPDO. Our results indicate that the renewal of acinar cells deleted by apoptosis 48 h after BPDO worsens if CCK is blocked before inducing AP.
Keywords: Acute pancreatitis; Apoptosis; CCK blockade; Cell cycle; Acinar cells;

Biologic activity of the tachykinins on lamb and sheep gallbladder by Paolo Tucci; Paola Bolle; Cinzia Severini; Pacifico Valeri (543-551).
In this study, we examined the activity of the tachykinins (TKs) on lamb and sheep isolated gallbladder and whether the TKs are involved in the capsaicin-induced activity in these tissues. Substance P (SP) and physalaemin (PHYS) contracted lamb gallbladder, PHYS-induced striking tachyphylaxis. This tissue was nearly insensitive to neurokinin A (NKA), neurokinin B (NKB), septide, and capsaicin. As in lamb tissues, SP and PHYS both contracted sheep gallbladder although PHYS induced no tachyphylaxis. At doses that had no effect on lamb tissue, NKA, NKB, septide, and capsaicin contracted sheep gallbladder. Our findings indicate that TK receptors differ in adult and young ovine gallbladder. The activity of PHYS on lamb gallbladder could depend on the existence of an unusual binding site, carrying one or more residues critical for the N-terminal sequence present in PHYS but not in SP.
Keywords: Ovine gallbladder; Tachykinins; Substance P; Physalaemin;

Endothelin receptor antagonists restore morphine analgesia in morphine tolerant rats by Shaifali Bhalla; George Matwyshyn; Anil Gulati (553-561).
Several neurotransmitter mechanisms have been proposed to play a role in the development of morphine tolerance. The present study provides evidence for the first time that endothelin (ET) antagonists can restore morphine analgesia in morphine tolerant rats. Tolerance to morphine was induced by subcutaneous implantation of six morphine pellets during a 7-day period. The degree of tolerance to morphine was measured by determining analgesic response (tail-flick latency) and hyperthermic response to morphine sulfate (8 mg/kg, subcutaneously (s.c.)) in placebo and morphine pellet implanted rats. The maximal tail-flick latency in morphine pellet-vehicle treated rats (7.54 s) was significantly lower (P<0.05) when compared to placebo pellet-vehicle treated rats (10 s), indicating that tolerance developed to the analgesic effect of morphine. In separate sets of experiments, ET antagonists, BQ123 (10 μg, intracerebroventricularly (i.c.v.)) and BMS182874 (50 μg, i.c.v.) were administered in placebo and morphine tolerant rats. BQ123 was injected twice daily for 7 days and once on day 8. BMS182874 was administered only on day 8. Morphine (8 mg/kg, s.c.) was administered 30 min after BQ123 or BMS182874 administration. It was found that both BQ123 and BMS182874 potentiated morphine analgesia in placebo and morphine tolerant rats. BQ123 potentiated tail-flick latency by 30.0% in placebo tolerant rats and 94.5% in morphine tolerant rats compared to respective controls. BMS182874 potentiated tail-flick latency by 30.2% in placebo tolerant rats and 66.7% in morphine tolerant rats. Morphine-induced hyperthermic effect was also potentiated by BQ123 and BMS182874. The duration of analgesic action was also prolonged by BQ123 and BMS182874. The effect of BMS182874 was less as compared to BQ123. BQ123 and BMS182874 are selective ETA receptor antagonists. Therefore, it is concluded that ETA receptor antagonists restore morphine analgesia in morphine tolerant rats.
Keywords: Morphine; Analgesia; BQ123 (cyclo(-d-Trp-d-Asp-Pro-d-Val-Leu)); BMS182874 (5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyd)-1-naphthalenesulfonamide); Central nervous system; Tolerance; Dependence; Rats; Opiates; Endothelin;

Roles of different peptide fragments derived from proadrenomedullin in the regulation of vascular tone in isolated rat aorta by Juxiang Li; Yongsheng Ren; Xianhong Dong; Guangzhen Zhong; Shengying Wu; Chaoshu Tang (563-568).
The effects of proadrenomedullin N-terminal 20 peptide (PAMP) and adrenotensin (ADT) on adrenomedullin (ADM)-induced vasodilation were investigated in aortic rings from rat. ADM (10−9 to 10−7  M) relaxed the aorta preconstricted with phenylephrine in a concentration-dependent manner. Denudation of endothelium or pretreatment with nitric oxide synthase (NOS) inhibitor, l-NAME, attenuated the vasodilatory action of ADM. ADM-induced vasorelaxation in the aortic rings with endothelium was converted to contraction by PAMP, but not by ADT. The ADM-induced vasodilation was not affected by PAMP in aorta rings without endothelium or in intact aortic rings pretreated with l-NAME. ADM-stimulated nitrite production and NOS activity of the aortas, which was inhibited by PAMP, ADT or PAMP plus ADT. ADM, PAMP, and ADT increased the cyclic adenosine monophosphate (cAMP) contents in vascular tissue. The combination of ADM with PAMP or ADT caused a smaller increase in cAMP level as compared with that of PAMP or ADT alone. These results show that ADM-induced endothelium-dependent vasodilation could be converted to vasoconstriction in the presence of PAMP, probably through a NO-dependent pathway. There was no indication that cAMP was involved in the converting effect of PAMP on ADM vasodilator action.
Keywords: Proadrenomedullin N-terminal 20 peptide (PAMP); Adrenomedullin (ADM); Adrenotensin (ADT); Aortic ring; Nitric oxide; Cyclic AMP;

Glucagon-like peptide-1 (7–36) amide prevents the accumulation of pyruvate and lactate in the ischemic and non-ischemic porcine myocardium by Mohammad Kavianipour; Mario R Ehlers; Klas Malmberg; Gunnar Ronquist; Lars Ryden; Gerhard Wikström; Mark Gutniak (569-578).
Glucagon-like peptide-1 (7–36) amide (GLP-1) has been studied as a treatment option in diabetic patients. We investigated the effect of recombinant GLP-1 infusion on hemodynamic parameters, myocardial metabolism, and infarct size during normoxic conditions as well as during ischemia and reperfusion using an open-chest porcine heart model. In the presence of rGLP-1, interstitial levels of pyruvate and lactate decreased during ischemia and reperfusion both in ischemic and non-ischemic tissue. Moreover, rGLP-1 infusion resulted in increased plasma insulin levels and decreased blood glucose levels. Neither hemodynamic variables nor the consequent infarct size were influenced by rGLP-1 infusion. We conclude that rGLP-1 altered myocardial glucose utilization during ischemia and reperfusion. It did not exert any untoward hemodynamic effects.
Keywords: Microdialysis; Pig; Myocardium; Glucagon-like peptide-1 (7–36) amide; Ischemia;

The tripeptide thyrotropin releasing hormone (TRH) has multiple interesting and complex physiological effects. One of these is the cerebrovasodilating effect, which has been described under several different conditions. The final mechanism for this effect is unknown. In the present study, we found an initial atropine-resistant cerebral vasodilation (24%) elicited by the NOS inhibitor L-NAME in the rat. D-NAME and 7-NI did not produce this effect. TRH (300 μg kg−1, i.v.) induced an increase in cerebral blood flow by 62%. L-NAME reduced this effect significantly. The cerebrovasodilating mechanism of TRH, at least in part, is endothelial NO dependent as the neuronal 7-NI NOS inhibitor does not affect the TRH response.
Keywords: Nitric oxide; L-NAME; 7-NI; TRH; Cerebral blood flow; Peptides;

Identification of essential amino acids in Humanin, a neuroprotective factor against Alzheimer’s disease-relevant insults by Yohichi Yamagishi; Yuichi Hashimoto; Takako Niikura; Ikuo Nishimoto (585-595).
Humanin (HN) is a secretory peptide that inhibits neurotoxicity by various Alzheimer’s disease-relevant insults. We have so far identified that the substitution of Leu9 for Arg nullifies the extracellular secretion of HN. Here we comprehensively investigate the amino acid requirement of HN essential for its secretion and for its neuroprotective function. Intracellulary expressed HN-EGFP (EGFP N-terminally fused with HN) was extracellularly secreted, whereas neither EGFP nor (L9R)HN-EGFP was secreted at all. While Ala substitution of neither residue affected HN secretion, Arg substitution revealed that the two structures—Leu9–Leu11 and Pro19–Va120—were essential for the secretion of full-length HN. In the Leu9–Leu11 domain, the Leu10 residue turned out to play a central role in this function, because the Asp substitution of Leu10, but not Leu9 or Leu11, nullified the secretion of HN. Utilizing Ala-scanned HN constructs, we also investigated a comprehensive structure–function relationship for the neuroprotective function of full-length HN, which revealed (i) that Pro3, Ser7, Cys8, Leu9, Leu12, Thr13, Ser14, and Pro19 were essential for this function and (ii) that Ser7 and Leu9 were essential for self-dimerization of HN. These findings indicate that HN has activity similar to a signal peptide, for which the Leu9–Leu11 region, particularly Leu10, functions as a core domain, and suggest that self-dimerization of HN is a process essential for its neuroprotective function.
Keywords: Alzheimer’s disease; Humanin; Secretion; Dimerization; Neuronal death; Amyloid precursor protein; Signal peptide; Structure–function relationship; Core domain; Secretion-defective mutant; Rescue factor;

Neural basis of orexigenic effects of ghrelin acting within lateral hypothalamus by Pawel K Olszewski; Dehong Li; Martha K Grace; Charles J Billington; Catherine M Kotz; Allen S Levine (597-602).
Ghrelin stimulates feeding when administered centrally and peripherally. The lateral hypothalamus (LH) is thought to mediate ghrelin-induced hyperphagia. Thus, we examined central mechanisms underlying feeding generated by LH ghrelin. We determined that 0.3 nmol of LH-injected ghrelin was the lowest dose increasing food consumption and it induced Fos immunoreactivity (IR; a marker of neuronal activation) in feeding-related brain areas, including the hypothalamic paraventricular, arcuate, and dorsomedial nuclei, amygdala, and nucleus of the solitary tract. Also, LH ghrelin induced Fos IR in LH orexin neurons. We conclude that the LH, as part of larger central circuitry, integrates orexigenic properties of ghrelin.
Keywords: Ghrelin; Food intake; Paraventricular nucleus of the hypothalamus; Lateral hypothalamus; Arcuate nucleus; Nucleus of the solitary tract; Dorsomedial nucleus of the hypothalamus; Central nucleus of the amygdala; Orexin; Hypocretin; Fos; Immunohistochemistry;

Inverse agonist activity of agouti and agouti-related protein by Biao-Xin Chai; Richard R. Neubig; Glenn L. Millhauser; Darren A. Thompson; Pilgrim J. Jackson; Gregory S. Barsh; Chris J. Dickinson; Ji-Yao Li; Yu-Mei Lai; Ira Gantz (603-609).
Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83–132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87–120) and ASIP [90–132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125 I -AgRP (86–132) versus radiolabeled agonist 125 I -NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.
Keywords: Melanocortin; Obesity; Hypothalamus; Pigmentation; JKC-363; JKC-366;

Glucose-dependent insulinotropic peptide stimulates proliferation and TGF-β release from MG-63 cells by Qing Zhong; Ke-Hong Ding; Anthony L. Mulloy; Roni J. Bollag; Carlos M. Isales (611-616).
Glucose-dependent insulinotropic peptide (GIP) is known to modulate alkaline phosphatase activity and collagen type I message in osteoblastic-like cells. GIP effects on cell proliferation are not known. We report that GIP dose dependently stimulated 3 H -thymidine incorporation in the osteoblastic-like cell line MG-63. Furthermore, GIP increased message and secretion of transforming growth factor beta (TGF-β), an agent known to regulate osteoblastic proliferation and differentiation. However, when GIP was added to MG-63 cells concurrently with a TGF-β neutralizing antibody, there was no effect on 3 H -thymidine incorporation in these cells. These data demonstrate that GIP stimulates osteoblastic-like cell proliferation but that this effect is not mediated by TGF-β.
Keywords: GIP; TGF-β; Incretin; Osteoblast;

Conformation-dependent effects of VIP on nociception in rats by David C. Yeomans; Hayat Önyüksel; Sumeet Dagar; Hiroyuki Ikezaki; Ying Lu; Israel Rubinstein (617-622).
The purpose of this study was to determine whether intrathecal injection of aqueous (random coil) vasoactive intestinal peptide (VIP) and VIP self-associated with sterically stabilized phospholipid micelles (α-helix VIP) at the lower lumbar vertebral level modulates foot withdrawal latency to low and high rate noxious radiant skin heating in anesthetized rats. We found that intrathecal random coil VIP evoked a significant bimodal, concentration-dependent response, early potent antinociception followed by hyperalgesia, during exposure to low and high rates of skin heating (P<0.05). Intrathecal α-helix VIP elicited a qualitatively similar response to that of random coil VIP except that the rate of decay of antinociception was faster and slower at low and high rates of skin heating, respectively. In addition, a low concentration of α-helix VIP evoked a potent late antinociception not observed with random coil VIP. Taken together, these data indicate that VIP modulates somatosensory processing in the lumbosacral spinal cord of rats in a complex fashion, and that this response is dependent, in part, on the conformation of VIP in the vicinity of target cells in the peripheral nervous system.
Keywords: Pain; C-fibers; A-δ fibers; Neuropeptide; Phospholipids; Skin heating; Foot withdrawal latency;

Antiadhesive peptides as the inhibitors of Mycobacterium kansasii phagocytosis by Ignacy Z Siemion; Zbigniew Wieczorek (623-628).
Initial entry of Mycobacteria into the cells depends upon the formation of a molecular complex between Antigen 85 (Ag85), located on the bacterial cell wall, and serum protein-fibronectin (FN) [Nat. Struct. Biol. 7 (2000) 141; Nat. Struct. Biol. 7 (2000) 94]. Therefore, a way of preventing a Mycobacteria invasion could be to inhibit the interaction between fibronectin and leucocyte cellular receptors of the integrin type. We found that some antiadhesive peptides (such as RGDVY and GRGD), derived of fibronectin and human leucocyte antigen DQ (HLA-DQ) sequences, are in fact very potent inhibitors of Mycobacterium kansasii phagocytosis. This observation may open new prospects in the search for tuberculosis therapy.
Keywords: Mycobacterial diseases; Fibonectin adhesion inhibition; RGDVY peptide;

Support Vector Machine for predicting α-turn types by Yu-Dong Cai; Kai-Yan Feng; Yi-Xue Li; Kuo-Chen Chou (629-630).
Tight turns play an important role in globular proteins from both the structural and functional points of view. Of tight turns, β-turns and γ-turns have been extensively studied, but α-turns were little investigated. Recently, a systematic search for α-turns classified α-turns into nine different types according to their backbone trajectory features. In this paper, Support Vector Machines (SVMs), a new machine learning method, is proposed for predicting the α-turn types in proteins. The high rates of correct prediction imply that that the formation of different α-turn types is evidently correlated with the sequence of a pentapeptide, and hence can be approximately predicted based on the sequence information of the pentapeptide alone, although the incorporation of its interaction with the other part of a protein, the so-called “long distance interaction”, will further improve the prediction quality.
Keywords: Long distance interaction; Support Vector Machine; Tight turns;