Peptides (v.25, #8)

SPAG11/isoform HE2C, an atypical anionic β-defensin-like peptide by Hans Henning von Horsten; Bettina Schäfer; Christiane Kirchhoff (1223-1233).
A human caput epididymidal cDNA, HE2C, was cloned based on its homology to the known chimpanzee counterpart, suggesting that the encoded β-defensin-like peptide represented a conserved component of the innate epididymidal epithelial defense system in primates. An approximately 6 kDa HE2- related peptide was co-purified together with other HE2 isoforms from human seminal plasma by affinity chromatography. By its antibody reactivity as shown by Western blot analysis, this peptide was distinct from the more abundant HE2 isoforms and was concluded to correspond to HE2C. Similar to other HE2-encoded isoforms, the endogenous HE2C was proteolytically processed from a larger precursor by a furin-like prohormone convertase. This was confirmed by N-terminal sequencing. In order to study the structural and functional properties of HE2C it was recombinantly expressed in insect cells. Post-translational processing also occurred within these cells, yielding the mature processed HE2C peptide. Correct disulfide bonding of the recHE2C peptide was shown by p-aminophenylarsineoxide(PAPAO)-agarose binding assay. Purified recHE2C strongly bound to Escherichia coli DH5α and Bacillus subtilis; however, it did not exhibit microbicidal activity when tested in a radial diffusion assay against these bacteria. Different from the previously described β-defensins, the mature HE2C peptide has an anionic pI and an algebraic net charge of −1. Also, it lacks the amphipathic transitions, which, according to the Shai-Matzusaki-Huang model, are prerequisite for the membranolytic activity of antimicrobial peptides.
Keywords: Epididymis; β-Defensin-like peptide; Disulfide bonding; Radial diffusion assay;

A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean by Shao Yun Wang; Jin Hong Wu; T.B. Ng; Xiu Yun Ye; Ping Fan Rao (1235-1242).
A non-specific lipid transfer peptide (nsLTP) with antimicrobial activity was isolated from the mung bean (Phaseolus mungo) seeds. The procedure entailed aqueous extraction, ion exchange chromatography on CM-Sephadex and high performance liquid chromatography (HPLC) on POROS-HS-20. The peptide exhibited a molecular mass of 9.03 kDa in mass spectrometry. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum and Sclerotium rolfsii, and antibacterial action against Staphylococcus aureus but not against Salmonella typhimurium. The lipid binding of this peptide was very similar to that of a previously described lipid transfer protein extracted from wheat seeds and maize seeds, indicating that it possessed lipid transfer activity. The present findings add to the scarcity of the literature on leguminous nsLTPs.
Keywords: Mung bean; nsLTP; Antifungal; Antibacterial;

Long-sarafotoxins: characterization of a new family of endothelin-like peptides by Mirian A.F. Hayashi; Caroline Ligny-lemaire; Zvi Wollberg; Michaël Wery; Andrzej Galat; Tomohisa Ogawa; Bruno H. Muller; Hung Lamthanh; Yvon Doljansky; Avner Bdolah; Reto Stöcklin; Frédéric Ducancel (1243-1251).
Sarafotoxins (SRTXs) constitute a family of vasoactive peptides that were initially isolated from the venom of Atractaspis engaddensis, and that are structurally and functionally related to endothelins (ETs). Analysis of the venom of Atractaspis microlepidota microlepidota revealed several new SRTX molecules manifesting some new structural and functional characteristics. These novel SRTXs are longer by three amino acids than the previously described SRTXs, and are designated here “long-SRTXs”. Six isoforms, derived from new poly-cistronic precursors, have been identified so far in the venom of this snake. One of these isoforms, designated SRTX-m, was chemically synthesized and its biological properties were studied. Our results show that SRTX-m induces toxicity in mice, mostly due to vasoconstriction, and also that it has a lower toxicity and potency than the more potent SRTX described up to now: sarafotoxin-b (SRTX-b) from A. engaddensis.
Keywords: Long-sarafotoxins; Endothelins; Vasoactives peptides; cDNA; Mass spectrometry; Atractaspis microlepidota microlepidota;

Bioactive properties of certain amphipathic peptides are amplified when self-associated with sterically stabilized micelles (SSM) composed of polyethylene glycol (PEG)-conjugated phospholipids. The purpose of this study was to determine the effects of amphipathic peptide molecular mass and PEG chain length on vasoreactivity evoked by vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, and pituitary adenylate cyclase-activating peptide1–38 (PACAP1–38), a 38-amino acid neuropeptide, associated with PEGylated phospholipid micelles in vivo. Both peptides were incubated for 2 h with SSM composed of PEG with molecular mass of 2000 or 5000 grafted onto distearoyl-phosphatidylethanolamine (DSPE-PEG2000 or DSPE-PEG5000) before use. We found that regardless of peptide molecular mass, PEG chain length had no significant effects on peptide–SSM interactions. Using intravital microscopy, VIP associated with DSPE-PEG5000 SSM or DSPE-PEG2000 SSM incubated at 25 °C evoked similar vasodilation in the intact hamster cheek pouch microcirculation. Likewise, PACAP1–38-induced vasodilation was PEG chain length-independent. However, SSM-associated PACAP1–38 evoked significantly smaller vasodilation than that evoked by SSM-associated VIP (P < 0.05) at 25 °C. When the incubation temperature was increased to 37 °C, SSM-associated PACAP1–38-induced vasodilation was now similar to that of SSM-associated VIP. This response was associated with a corresponding increase in α-helix content of both peptides in the presence of phospholipids. Collectively, these data indicate that for a larger amphipathic peptide, such as PACAP1–38, greater kinetic energy or longer incubation period is required to optimize peptide–SSM interactions and amplify peptide bioactivity in vivo.
Keywords: Neuropeptide; DSPE-PEG; Sterically stabilized micelles; Microcirculation; Arteriole; Vasomotor tone; Hamster;

In vitro circadian ANP secretion by gene transferring cells encapsulated in polycaprolactone tubes: gene chronotherapy by Zhengrong Wang; Liguo Chen; Chaomin Wan; Yi Qu; G. Cornélissen; F. Halberg (1259-1267).
A new insofar as chronobiologic therapeutic approach by atrial natriuretic peptide (ANP) for hypertension and/or congestive heart failure (CHF) is based on the release of ANP from ANP cDNA transfected Chinese Hamster Ovary (CHO) cells encapsulated in polycaprolactone (PCL) tubes. ANP secretion was maintained for at least 6 months. The encapsulated cells remained viable during culturing. Control cells without transferred ANP cDNA were negative. ANP secretion is circadian periodic, peaking around 04:18, shifted to around 07:56 by melatonin treatment. The encapsulation technique, based on principles of chronotherapy, may provide a more efficient gene therapy, applicable for eventual human implantation of gene transferred cells.
Keywords: ANP; Circadian rhythm; CHO cells; Chronobiology; Congestive heart failure; Encapsulation; Hypertension; Melatonin; Polycaprolactone;

Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects by Giuseppina Mazzocchi; Giuliano Neri; Marcin Rucinski; Piera Rebuffat; Raffaella Spinazzi; Ludwik K. Malendowicz; Gastone G. Nussdorfer (1269-1277).
Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), two subtypes of which have been identified and named GHS-R1a and GHS-R1b. Evidence has been provided that ghrelin and its receptors are expressed in the adrenal gland, and we have investigated the possible role of the ghrelin system in the functional regulation of the human adrenal cortex. Reverse transcription–polymerase chain reaction detected the expression of both subtypes of GHS-Rs exclusively in the zona glomerulosa (ZG). Ghrelin did not significantly affect either basal or agonist-stimulated aldosterone secretion from cultured ZG cells. In contrast, ghrelin raised proliferative activity and decreased apoptotic deletion rate of ZG cells, the maximal effective concentration being 10−8  M. The growth effects of 10−8  M ghrelin on cultured ZG cells were not affected by either the protein kinase (PK)A and PKC antagonists H-89 and calphostin-C or the mitogen-activated PK (MAPK) p38 antagonist SB-293580, but were abolished by both the tyrosine kinase (TK) and MAPK p42/p44 antagonists tyrphostin-23 (10−5  M) and PD-98059 (10−4  M), respectively. Ghrelin (10−8  M) enhanced TK and MAPK p42/p44 activities of ZG cells. Preincubation with 10−5  M tyrphostin-23 blocked the ghrelin-induced stimulation of both TK and MAPK p42/p44, while preincubation with 10−4  M PD-98059 only annulled MAPK p42/p44 stimulation. Collectively, our findings allow us to conclude that ghrelin, acting via GHS-Rs exclusively located in the ZG, enhances the growth of human adrenal cortex, through a mechanism involving the activation of the TK-dependent MAPK p42/p44 cascade.
Keywords: Ghrelin; GHS-R; Adrenal cortex; Cell proliferation; Apoptosis; Humans;

The melanocortin agonist melanotan II increases insulin sensitivity in OLETF rats by Ryouichi Banno; Hiroshi Arima; Ikuko Sato; Masayuki Hayashi; Motomitsu Goto; Yoshihisa Sugimura; Takashi Murase; Yutaka Oiso (1279-1286).
Effects of peripheral administration of melanotan II (MTII), a melanocortin agonist, on insulin sensitivity and glucose tolerance were examined in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Subcutaneous administration of MTII with osmotic mini-pumps decreased food intake and body weight in OLETF rats. MTII group showed more sensitivity to insulin compared with that allowed to eat ad libitum or pair-fed group in insulin tolerance tests on day 9. MTII group also showed significantly lower glucose values than ad libitum group in glucose tolerance tests on days 11 and 23. Thus, MTII increased insulin sensitivity and improved glucose tolerance in OLETF rats.
Keywords: Melanocortin agonist; Hyperphagia; Obesity; Insulin resistance; OLETF rats;

Galanin inhibits gastric acid secretion through a somatostatin-independent mechanism in mice by Laura Piqueras; Yvette Taché; Vicente Martinez (1287-1295).
The role of somatostatin in galanin-induced inhibition of gastric acid secretion in urethane-anesthetized mice was investigated by using immunoneutralization of endogenous somatostatin and somatostatin receptor type 2 (SSTR2) knockout mice. Intravenous galanin (10 and 20 μg/kg/h) inhibited pentagastrin-stimulated gastric acid secretion by 47 and 33%, respectively. Somatostatin antibody injected i.v. increased acid secretion by 3.5-fold over basal levels but did not modify the antisecretory effects of galanin. Urethane-anesthetized SSTR2 knockout mice had a basal secretion 14-fold higher than wild-type animals, that was inhibited by galanin (10 and 20 μg/kg/h) by 49 and 31% respectively. In mice galanin inhibits gastric acid secretion through a somatostatin-independent mechanism.
Keywords: Galanin; Somatostatin; Somatostatin receptors; SSTR2; Acid secretion; Immunoneutralization; Pentagastrin;

Circadian phase difference of leptin in android versus gynoid obesity by Federico Perfetto; Roberto Tarquini; Germaine Cornélissen; Giorgio Mello; Alessio Tempestini; Paola Gaudiano; Francesco Mancuso; Franz Halberg (1297-1306).
A circadian rhythm in serum leptin, measured every 4 h for 24 h, characterizes normal-weight women (N = 14), and women with gynoid (N = 17) or android (N = 26) obesity, peaking around midnight (P < 0.05), but differing by about 3 h between android and gynoid women (P < 0.01). Obesity is associated with a higher MESOR (rhythm-adjusted mean; P < 0.001) and a smaller relative circadian amplitude (P < 0.05). Gynoid obesity is associated with a larger circadian amplitude of cortisol (P < 0.05), whereas android obesity is associated with a larger circadian amplitude and a higher MESOR of insulin (P < 0.05). Understanding putative mechanisms underlying different body fat distribution may lead to improved chronotherapeutic measures.
Keywords: Body fat distribution (android versus gynoid); Circadian rhythm; Cortisol; Insulin; Leptin;

Ultrastructure of orexin-1 receptor immunoreactivities in the spinal cord dorsal horn by Jian-Lian Guan; Qing-Ping Wang; Tomoko Hori; Fumiko Takenoya; Haruaki Kageyama; Seiji Shioda (1307-1311).
The ultrastructural properties of orexin 1-receptor-like immunoreactive (OX1R-LI) neurons in the dorsal horn of the rat spinal cord were examined using light and electron microscopy techniques. At the light microscopy level, the most heavily immunostained OX1R-LI neurons were found in the ventral horn of the spinal cord, while some immunostained profiles, including nerve fibers and small neurons, were also found in the dorsal horn. At the electron microscopy level, OX1R-LI perikarya were identified containing numerous dense-cored vesicles which were more heavily immunostained than any other organelles. Similar vesicles were also found within the axon terminals of the OX1R-LI neurons. The perikarya and dendrites of some of the OX1R-LI neurons could be seen receiving synapses from immunonegative axon terminals. These synapses were found mostly asymmetric in shape. Occasionally, some OX1R-LI axon terminals were found making synapses on dendrites that were OX1R-LI in some cases and immunonegative in others. The synapses made by OX1R-LI axon terminals were found both asymmetric and symmetric in appearance. The results provide solid morphological evidence that OX1R is transported in the dense-cored vesicles from the perikarya to axon terminals and that OX1R-LI neurons in the dorsal horn of the spinal cord have complex synaptic relationships both with other OX1R-LI neurons as well as other neuron types.
Keywords: Orexin receptor; Rat; Synapse; Axon terminal; Dense-cored vesicles;

Vascular and brain neuropeptide Y in banded and spontaneously hypertensive rats by M.J. Morris; C.F. Tortelli; D.P. Hart; L.M.D. Delbridge (1313-1319).
Debate exists regarding the relative importance of neuropeptide Y (NPY) in the pathogenesis of genetic and non-genetic hypertension. NPY concentrations were compared in conduit, mesenteric and renal vasculatures and in hypothalamic and medullary regions of age-matched normotensive control, aortic banded and spontaneously hypertensive rats (SHRs). Lower NPY concentrations were measured in the pre-optic area of banded rats compared to controls and SHR. Renal vein NPY levels were reduced in banded animals, whereas renal artery levels were decreased in SHR. In mesenteric arteries, NPY concentration was selectively increased in SHR. These findings suggest that local hemodynamic alterations influence endogenous levels of this potent vasoconstrictor.
Keywords: Neuropeptide Y; Hypertension; Central nervous system; Blood vessels; Aortic coarctation; SHR;

Adrenomedullin as a sensitive marker for coronary and peripheral arterial complications in patients with atherosclerotic risks by Yoshihiko Suzuki; Takeshi Horio; Hiroshi Nonogi; Tokio Hayashi; Kazuo Kitamura; Tanenao Eto; Kenji Kangawa; Yuhei Kawano (1321-1326).
Plasma adrenomedullin (AM) levels are elevated in various pathological states including cardiovascular and inflammatory diseases. The present study investigated whether an increased AM level is a marker of vascular complications in patients with atherosclerotic risks. In 114 patients with cardiovascular risks and/or diseases including ischemic heart disease (IHD) and peripheral arterial disease (PAD), plasma AM concentration and other inflammatory markers such as high sensitive C-reactive protein (CRP) and interleukin (IL)-6 were examined. The plasma AM level was not altered by the absence or presence of each of four major risk factors, i.e., hypertension, diabetes mellitus, hyperlipidemia, and smoking and its level was not significantly correlated with blood pressure, plasma glucose, or serum lipid levels. The patients with IHD had a significantly higher concentration of plasma AM than those without IHD. The AM level in subjects with PAD was also increased significantly compared with those without PAD. The plasma AM was strongly correlated with inflammatory parameters such as CRP and IL-6. Among AM, CRP, and IL-6, however, only AM was an independent predictor for both IHD and PAD by multiple logistic regression analysis. Our findings suggest the possibility that plasma AM is a novel sensitive marker for the presence of vascular lesions in patients with atherosclerotic risks.
Keywords: Hormone; Peptide; Atherosclerosis; Vascular disease; Inflammation;

Transport of angiotensin peptides across the Caco-2 monolayer by Hui-Lee Chua; Seetharama Jois; Meng-Kwoon Sim; Mei-Lin Go (1327-1338).
The bidirectional transport of the angiotensin peptides—des-Asp-angiotensin I (DAAI), angiotensins III and IV—were studied using human intestinal Caco-2 monolayers. The peptides had low permeability rates but were relatively stable to enzymatic hydrolysis. DAAI was transported by diffusion while angiotensins III and IV were transported by an energy requiring, carrier-mediated process. The physicochemical properties and solution conformations of the peptides were investigated in an attempt to establish structure–transport correlations. Among the three peptides, DAAI was the most hydrophobic, had the highest hydrogen bonding potential and was the only peptide to have a random solution conformation, as determined from circular dichroism, two-dimensional 1 H NMR and molecular modelling. On the other hand, the more hydrophilic angiotensin IV had less hydrogen bonding potential and a solution conformation characterized by a β turn. These factors may influence the transport characteristics of DAAI and angiotensin IV.
Keywords: Angiotensin peptides; des-Asp-angiotensin I; Caco-2 monolayer; Mechanisms of transport; Structure–permeability correlations; Solution conformation;

Differential levels of “urotensin-II-like” activity determined by radio-receptor and radioimmuno-assays by Nambi Aiyar; Brian Guida; Zhaohui Ao; Jyoti Disa; Diane Naselsky; David J. Behm; Jui-Lan Su; Frederick C. Kull; Stephen A. Douglas (1339-1347).
Plasma and urinary levels of “urotensin(U)-II-like” substances determined in healthy human volunteers were 12.4 ± 0.6 ng/ml and 2.2 ± 0.3 ng/ml by RIA, an order of magnitude lower than that seen by RRA, 167.5 ± 9.5 ng/ml and 65.2 ± 4.3 ng/ml. HPLC demonstrated the existence of at least three prominent activity peaks in plasma and urine, the more hydrophobic of which did not co-elute with U-II, degradation products or URP. RRA and RIA recognized these peaks with contrasting efficacy. As such, published levels of “U-II-like” activity should be interpreted with caution until a better understanding is obtained regarding what species specific RIA and RRA assay reagents interact with.
Keywords: Urotensin-II; UT receptor; Vasoactive peptide; Radioimmunoassay; Radioreceptor assay; Hypertension;

Study in vitro and in vivo of nociceptin/orphanin FQ(1–13)NH2 analogues substituting N-Me-Gly for Gly2 or Gly3 by Li-xiang Chen; Quan Fang; Qiang Chen; Jia Guo; Zhuan-zi Wang; Yong Chen; Rui Wang (1349-1354).
In the present study, two analogues containing N-Me-Gly (Sarcosine, Sar) were synthesized to further investigate the structural–activity relationships of orphanin FQ/nociceptin (OFQ/NC, NC). The replacement of Gly2 or Gly3 with Sar increased the flexibility and decreased the hydrophobicity of the N-terminal tetrapeptide. The activity of the analogues was investigated in a series of assays in vivo and in vitro. [Sar2]NC(1–13)NH2 was found to (1) produce dose-dependent inhibition of the electrically induced contraction in MVD assay (pEC50 = 6.14); (2) produce significant hyperalgesia effects in a dose-dependent manner when intracerebroventricularly (i.c.v.) injected in mice. The inhibitive effects of [Sar2]NC(1–13)NH2 in MVD assay could be significantly antagonized by [Nphe1]NC(1–13)NH2, and partially antagonized by naloxone; the hyperalgesic effect of [Sar2]NC(1–13)NH2 could be significantly antagonized by naloxone, and partially antagonized by [Nphe1]NC(1–13)NH2. On the contrary, [Sar3]NC(1–13)NH2 showed no effects in these assays. All the findings suggest that the flexibility of the peptide bond between Phe1 and Gly2 and between Gly2 and Gly3 play an important role in NC–OP4 receptor interaction, and the hydrophobicity of the N-terminal tetrapeptide showed no significant effect on this interaction. The present work also helps to provide a novel method to elucidate structural and conformational requirements of the opioid peptide–receptor interaction.
Keywords: Orphanin FQ/nociceptin; Sarcosine; Mouse vas deferens (MVD); Tail-flick test; Naloxone; [Nphe1]NC(1–13)NH2;

Modulation of anxiety-related behaviors by μ- and κ-opioid receptor agonists depends on the social status of mice by Natalia N. Kudryavtseva; Mirjam A.F.M. Gerrits; Damira F. Avgustinovich; Michael V. Tenditnik; Jan M. Van Ree (1355-1363).
This study was aimed to determine the effects of μ- and κ-opioid receptor activation in relation to the social status of mice, being a winner with repeated experience of victories or a loser with repeated experience of social defeats. The behaviors of the animals were assessed in a social encounter test measuring the communicative behavior towards a familiar and an unfamiliar partner behind a perforated transparent partition (partition test) and in an elevated plus-maze test estimating the anxiety level of mice. Placebo and graded doses of the μ-opioid receptor agonist DAMGO (0.5 and 2 mg/kg s.c.) and the κ-opioid receptor agonist U-50,488H (0.6, 1.25, and 2.5 mg/kg s.c.) were administered to the control mice, winners and losers in two experiments. In the partition test, the winners spent somewhat more time and the losers less time than the controls in the vicinity of their partner probably related to a lower and higher level of anxiety respectively. In the plus-maze test the losers appeared to have a somewhat higher anxiety level than the controls and winners. In both tests DAMGO produced anxiogenic-like effects in the winners and the controls, but not in the losers. Winners hardly responded to treatment with U-50,488H, while the losers responded dose dependently with an anxiolytic-like effect in both tests. It is concluded that anxiety-like responses in mice are differentially affected by stimulation of μ- and κ-opioid receptors and that the effects depend on the social status of the animals.
Keywords: Opioids; Anxiety; Anxiolytic; Submission; Aggression; Mice; κ-Opioid receptors; U-50,488H; μ-Opioid receptor; DAMGO; Social status;

A ribonuclease with a temperature optimum of about 70 °C and a pH optimum of 6.5 was isolated from fruiting bodies of the mushroom Pleurotus eryngii. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and S-Sepharose. It possessed a molecular mass of 16 kDa, and exhibited higher ribonucleolytic activity toward poly A and poly G and lower ribonucleolytic activity toward poly C and poly U. Its N-terminal sequence was distinctly different from those of other mushroom ribonucleases, and resembled that of Pleurotus tuber-regium only by 40%. Furthermore, its thermostability characteristics, polyhomoribonucleotide specificity and molecular mass were dissimilar to those of other mushroom ribonucleases.
Keywords: Ribonuclease; Mushroom; Pleurotus eryngii; Fruiting body;

Identification of an Adrenomedullin precursor fragment in plasma of sepsis patients by Joachim Struck; Chen Tao; Nils G. Morgenthaler; Andreas Bergmann (1369-1372).
Adrenomedullin and PAMP are potent vasodilatory peptides derived from a common larger precursor peptide. Elevation of circulating levels of both peptides has been described for diseases involving dysfunction of the cardiovascular system. However, the reliable quantification has been hampered by their short half-life times and – as known for Adrenomedullin – the existence of a binding protein. Here we report the identification of another peptide derived from the Adrenomedullin precursor, termed proADM 45–92, which is present in large concentrations in plasma of septic shock patients. This peptide is produced in stoichiometric amounts to Adrenomedullin and PAMP, but – contrary to them – is apparently non-functional and stable. Thus, proADM 45–92 represents a suitable diagnostic target which could be used to assess the concentrations of Adrenomedullin gene products released into the bloodstream.
Keywords: Adrenomedullin; PAMP; Sepsis; Plasma; Mass spectrometry; Immunoassay;