Peptides (v.21, #3)

A synthetic peptide of human apoprotein E with antibacterial activity by Masachika Azuma; Taiki Kojimab; Itsuo Yokoyama; Hisao Tajiri; Kazuhiro Yoshikawa; Shinsuke Saga; Carlos A Del Carpio (327-330).
In recent years, several endogenous mammalian antibacterial peptides have been described. An amphipathic cationicα-helix is a common feature in many cases; therefore, other peptides with this characteristic might also possess antibiotic activity. In fact, a 30-mer peptide of apoprotein E 133–162 (LRVRLASHLRKLRKRLLRDADDLQKRLAVY) was found to have antibiotic activity comparable to those of a classic antibiotic (Gentamicin) and a neutrophil-derived antibiotic peptide (CAP18). Calculation of cationicity, hydrophobicity, and hydrophobic moment and the helical wheel diagram of apoprotein E 133–162 revealed close similarities to CAP18.
Keywords: Antibiotic peptides; Apoprotein E; Amphipathic; Cationic; α-Helix;

A hyperglycemic peptide hormone from the Caribbean shrimp Penaeus (litopenaeus) schmitti by A Huberman; M.B Aguilar; I Navarro–Quiroga; Laida Ramos; Isis Fernández; F.M White; D.F Hunt (331-338).
From a crude extract of the sinus glands of the shrimp Penaeus (litopenaeus) schmitti a peptide with hyperglycemic activity in a homologous bioassay was isolated and characterized by a combination of automatic Edman degradation, enzymatic digestions, TLC of dansyl-amino acids, and mass spectrometry. Its Mr is 8359.4 Da by MS, which coincides with the deduced sequence. Its N-terminus is free and its C-terminus is amidated. It has 6 Cys residues in conserved positions compared with other known CHHs. This is the first sinus gland hormone from an Atlantic Ocean shrimp characterized to date. It has a remarkable 90% sequence similarity to the Indo-Pacific shrimp P. (marsupenaeus) japonicus Pej-VII hyperglycemic hormone.
Keywords: Sinus gland; Shrimp; Hyperglycemic hormone; CHH; Penaeus; Western Atlantic; Evolution;

Organization of proenkephalin in amphibians: cloning of a proenkephalin cDNA from the brain of the anuran amphibian, Spea multiplicatus by Stephanie Lecaude; Jasem Alrubaian; Cristina Sollars; Catherine Propper; Phillip Danielson; Robert M Dores (339-344).
Cloning of a proenkephalin cDNA from the pelobatid anuran amphibian, Spea multiplicatus, provides additional evidence that Leu-enkephalin, although present in the brain of anuran amphibians, is not encoded by the proenkephalin gene. The S. multiplicatus proenkephalin cDNA is 1375 nucleotides in length, and the open reading frame contains the sequences of seven opioid sequences. There are five copies of the Met-enkephalin sequence, as well as an octapeptide opioid sequence (YGGFMRNY) and a heptapeptide opioid sequence (YGGFMRF). In the proenkephalin sequence of S. multiplicatus the penultimate opioid is a Met-enkephalin sequence rather than the Leu-enkephalin present in mammalian sequences. The same order of opioid sequences also is observed for the proenkephalin sequence of the pipid anuran amphibian, Xenopus laevis. Hence, from a phylogenetic standpoint the organization of tetrapod proenkephalin has been remarkably conserved. What remains to be resolved is whether the Leu-enkephalin sequence found in mammalian proenkephalin is an ancestral trait or a derived trait for the tetrapods. Unlike the proenkephalin precursor of X. laevis, all of the opioid sequences in the S. multiplicatus proenkephalin cDNA are flanked by paired-basic amino acid proteolytic cleavage sites. In this regard the proenkephalin sequence for S. multiplicatus is more similar to mammalian proenkephalins than the proenkephalin sequence of X. laevis. However, a comparison of the proenkephalin sequences in human, X. laevis, and S. multiplicatus revealed several conserved features in the evolution of the tetrapod proenkephalin gene. By contrast, a comparison of tetrapod proenkephalin sequences with the partial sequence of a sturgeon proenkephalin cDNA indicates that the position occupied by the penultimate opioid sequence in vertebrate proenkephalins may be a highly variable locus in this gene.
Keywords: Proenkephalin; Met-enkephalin; Leu-enkephalin; Amphibian;

Comparison of central administration of corticotropin-releasing hormone and urocortin on food intake, conditioned taste aversion, and c-Fos expression☆ by Stephen C Benoit; Todd E Thiele; Stephen C Heinrichs; Paul A Rushing; Kathleen A Blake; Randy J Steeley (345-351).
Corticotropin-releasing hormone (CRH) is a potent regulator of the hypothalamic-pituitary-adrenal axis, and reduces food intake when administered into the third cerebral ventricle (i3vt). However, CRH also promotes conditioned taste aversion (CTA) learning which indicates that its anorectic effects are accompanied by aversive consequences that would reduce food intake independently of energy regulation. Urocortin (Ucn) is a closely related mammalian peptide that binds to both identified CRH receptor subtypes and also reduces food intake when administered i3vt. The present experiments compared the aversive consequences of i3vt administration of CRH and Ucn at doses that produced comparable decrements in food intake. Experiment 1 found that 1.0 μg Ucn and 2.0 μg CRH produced similar reductions in food intake. Experiment 2 demonstrated that, at these doses, CRH but not Ucn promoted robust and reliable CTA learning. A third experiment showed comparable increased c-Fos-like immunoreactivity after Ucn and CRH in forebrain and hindbrain structures associated with food intake. It is concluded that Ucn, at doses that reduce food intake to levels like that observed after administration of CRH, do not produce similarly aversive consequences.
Keywords: CRH receptor; CTA; Saccharine; Aversion; Body weight; Feeding;

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on proliferation of lymphoblastoid cells☆ by Tatyana N Lepikhova; Elena V Navolotskaya; Tatyana A Zargarova; Rosa I Nurieva; Valery M Lipkin; Vladimir P Zav’yalov (353-357).
Influence of the ACTH-like peptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH corresponding to the sequence 11–20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain on growth of MT-4 human T-lymphoblastoid cell line was investigated. It was found that the ACTH-like peptide at concentration range 10−11 –10−7 M inhibits the proliferation of MT-4 cells. Labeled ACTH ‘address segment’ [125I]ACTH (13–24) was used to establish that MT-4 cells express specific receptors for ACTH (K d = 97 pM). The ACTH-like peptide and human ACTH (but not IgG1 heavy chain) were shown to compete with [125I]ACTH (13–24) for binding to these receptors (K i1 = 0.38 nM and K i2 = 0.34 nM).
Keywords: ACTH (Adrenocorticotropin); MT-4 cells; Receptor; Peptide; Growth inhibition;

Allergen inhalation challenge induces decrease of serum neutral endopeptidase (NEP) in asthmatics by N Tudoric; M Zhang; M Kljajic–Turkalj; J Niehus; B Cvoriscec; K Jurgovsky; G Kunkel (359-364).
There is accumulating evidence that tachykinins are implicated in inflammation, including asthma. Therefore, we hypothesized that the neutral endopeptidase (NEP), under challenge conditions, could be affected. Serum from 21 asthmatics and six healthy volunteers was sampled before, 30, and 120 min after allergen challenge. NEP-IR was determined using an ELISA and was found in all subjects. Compared to prechallenge, no difference was seen between asthmatics and controls; however, under challenge conditions, NEP-IR in asthmatics was significantly lower (30 min, P = 0.058; 120 min, P = 0.0017, respectively). This finding supports indirectly the hypothesis that tachykinins are released during allergen exposure, and suggests a regulatory role of NEP.
Keywords: Allergen; Bronchoprovocation; Neutral endopeptidase;

Convertases are proteases responsible for the bioactivation of many proteins and peptides having a potential role in ontogenesis. As a model to study regulation of convertases in embryo, we use the P19 embryonal carcinoma cell line, which can differentiate into various cell types. The expression of convertase PC2 and its specific binding peptide 7B2 are co-induced during neuronal differentiation of P19 cells. We investigated the possibility that expression of both proteins may be coregulated by T3 and dexamethasone, activators of nuclear receptors, isobutylmethylxanthine, and dibutyryl cAMP, activators of protein kinase A, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. Western blotting results show that expression of PC2 and 7B2 can be upregulated by modulators of the protein kinases, and upregulation needs not be strictly stoichiometric.
Keywords: Regulation; Expression; Convertase PC2; Helper peptide 7B2; Neuronal differentiation; P19 Embryonal carcinoma;

Isatin (Indole-2, 3-dione) inhibits natriuretic peptide-induced hyperthermia in rats☆ by Imre Pataki; Ágnes Adamik; Gyula Telegdy (373-377).
The effects of an endogenous indole, isatin (indole-2, 3-dione), on the hyperthermia induced by atrial natriuretic peptide (ANP-28), brain natriuretic peptide (BNP-32), and C-type natriuretic peptide (CNP-22) were investigated in rats. Intracerebroventricular administration of each peptide in a dose of 1 μg caused elevations in colon temperature 30 and 60 min after injection. An intraperitoneal (i.p.) injection of isatin (50 mg/kg) abolished the natriuretic peptide-induced hyperthermia. These data reinforce the possible involvement of natriuretic peptides in thermoregulatory processes in the central nervous system, and suggest that isatin might counteract their hyperthermic effect in vivo.
Keywords: Isatin; Natriuretic peptide; Hyperthermia; Rat;

CCK/dopamine interactions in Fawn-Hooded and Wistar–Kyoto rat brain by Daniel J Lodge; Jennifer L Short; Linda D Mercer; Philip M Beart; Andrew J Lawrence (379-386).
The aim of this study was to compare the actions of CCK neuropeptides within the nucleus accumbens (N.Acc) of alcohol preferring (Fawn-Hooded, FH) and alcohol nonpreferring (Wistar–Kyoto, WKY) rats. CCK-8S (30–300 nM) facilitated the K+ stimulated release of [3H]dopamine (DA) from N.Acc prisms in both rat strains, whereas CCK-4 (30 nM–1 μM) caused a significant decrease of evoked [3H]DA in the FH rat only. A scattered distribution of CCK-A and -B receptor immunopositive varicose fibers were visualized throughout the N.Acc of both rat strains along with a topographic distribution of CCK receptor positive cells throughout the ventral mesencephalon.
Keywords: CCK receptors; Dopamine; Nucleus accumbens; Mesolimbic system; Immunohistochemistry; Alcohol-preferring rat;

Recent data generated from adult male and female rats indicates that leptin is capable of stimulating luteinizing hormone (LH) secretion via a hypothalamic action. Consequently, we hypothesized that this peptide may similarly play a role in controlling LH secretion during late juvenile and peripubertal development; hence, contributing to hypothalamic-pituitary function during sexual maturation. Therefore, this study was conducted to determine if leptin is capable of stimulating LH release during this critical time of development and, if so, to determine whether this action is due to an effect at the hypothalamic level. Results showed that leptin, when administered directly into the brain third ventricle (3V), can stimulate (P < 0.01) LH release in late juvenile animals at doses of 0.01–1.0 μg. A higher dose of 10 μg was ineffective in stimulating LH release. Immunoneutralization of luteinizing hormone-releasing hormone (LHRH) via 3V administration of LHRH antiserum to late juvenile animals indicated a hypothalamic site of action, since the leptin-induced LH release was blocked in the animals that received anti-LHRH, but not in the control animals that received normal rabbit serum. Leptin did not significantly stimulate LH release from animals in first proestrus, estrus, or diestrus. We also report that the serum levels of leptin increase (P < 0.05) during the late juvenile period of development, then decrease (P < 0.05) once the animal enters the peripubertal period. Collectively, our results show that leptin is capable of acting centrally to stimulate LH release, but only during late juvenile development; thus, we suggest the peptide likely plays a facilitatory role on late juvenile LH secretion, but does not drive the LHRH/LH releasing system to first ovulation and hence, sexual maturity.
Keywords: Leptin; Luteinizing hormone; Pubertal development;

[D-Trp34] neuropeptide Y is a potent and selective neuropeptide Y Y5 receptor agonist with dramatic effects on food intake☆ by Eric M Parker; Ambikaipakan Balasubramaniam; Mario Guzzi; Deborra E Mullins; Brian G Salisbury; Sulaiman Sheriff; Melanie B Witten; Joyce J Hwa (393-399).
The neuropeptide Y (NPY) Y5 receptor has been proposed to mediate several physiological effects of NPY, including the potent orexigenic activity of the peptide. However, the lack of selective NPY Y5 receptor ligands limits the characterization of the physiological roles of this receptor. Screening of several analogs of NPY revealed that [D-Trp34]NPY is a potent and selective NPY Y5 receptor agonist. Unlike the prototype selective NPY Y5 receptor agonist [D-Trp32]NPY, [D-Trp34]NPY markedly increases food intake in rats, an effect that is blocked by the selective NPY Y5 receptor antagonist CGP 71683A. These data demonstrate that [D-Trp34]NPY is a useful tool for studies aimed at determining the physiological roles of the NPY Y5 receptor.
Keywords: Neuropeptide Y; Neuropeptide Y receptor; Y5 receptor; [D-Trp34]NPY; [D-Trp32]NPY; Food intake;

Two different photoinsulins, the radioactive NϵB29-(4-azidosalicyloyl) insulin and the novel biotinylated NϵB29-(4-azidotetrafluorobenzoyl-biocytinyl) insulin, were synthesized in order to study the binding stoichiometry of insulin and the insulin receptor in a direct approach. Both derivatives were cross-linked simultaneously to the (αβ)2 receptor. Insulin-receptor conjugates were formed that carry a radioactive label as well as a biotin label. The doubly labeled complexes were isolated by streptavidin-affinity chromatography. Analysis of both markers, the radioactive 125I marker and the biotin marker, proved the existence of a covalent complex of one receptor molecule and two ligands. Thus, orthogonal photocross-linking is introduced as a method for the isolation and analysis of bivalent receptor complexes.
Keywords: Insulin; Insulin receptor; Photoaffinity labeling; Biotin; Bivalence;

The effects of angiotensin II (ANG II) microinjected unilaterally (left or right) and bilaterally (left and right) at a dose of 0.5 μg (0.5 nmol) into the CA1 hippocampal area of male Sprague Dowley rats on learning and memory (shuttle box) were studied. Bilateral microinjections of ANG II improved learning, i.e. increased the number of avoidances during the two training days as compared to the respective controls microinjected with saline. ANG II facilitated learning and memory, especially when microinjected into the left CA1 hippocampal area as compared to the respective controls microinjected with saline. Left-side microinjection of ANG II increased the number of avoidances on the first and second training day as compared to the right-side microinjection of ANG II. These findings suggest asymmetric effects of ANG II on cognitive processes in hippocampus.
Keywords: Angiotensin II; Asymmetry; Hippocampus; Learning and memory; Rat;

Evidence for a paracrine role of adrenomedullin in the physiological resetting of aldosterone secretion by rat adrenal zona glomerulosa by Giovanna Albertin; Ludwick K Malendowicz; Cinzia Tortorella; Giuseppina Mazzocchi; Gastone G Nussdorfer (413-417).
Adrenomedullin (ADM) has been recently found to directly inhibit agonist-stimulated aldosterone secretion by dispersed zona glomerulosa (ZG) cells and to stimulate basal catecholamine release by adrenomedullary fragments. In light of the fact that catecholamines enhance aldosterone secretion acting in a paracrine manner, we have investigated whether these two effects of ADM may interact when the integrity of the adrenal gland is preserved. ADM increased basal aldosterone output by adrenal slices containing a core of adrenal medulla, and the effect was blocked by the β-adrenoceptor antagonist l-alprenolol. In contrast, ADM evoked a moderate inhibition of K+-stimulated aldosterone production, and the blockade was complete in the presence of l-alprenolol. The in vivo bolus injection of ADM did not affect plasma aldosterone concentration (PAC) in rats under basal conditions. Conversely, when rat ZG secretory function was enhanced (by sodium restriction or infusion with angiotensin-II [ANG-II]) or depressed (by sodium loading or infusion with the angiotensin-converting enzyme inhibitor captopril), ADM evoked a sizeable decrease or increase in PAC, respectively. The prolonged infusion with the ADM receptor antagonist ADM(22–52) caused a further enhancement of PAC in sodium-restricted or ANG-II-treated rats, and a further moderate decrease of it in sodium-loaded or captopril-administered animals. RIA showed that ADM plasma concentration did not exceed a concentration of 10−11 M in any group of animals. Under basal conditions, ADM adrenal content was 1.2–2.0 pmol/g, which may give rise to local concentrations higher than 10−8 M (i.e. well above the minimal effective ones in vitro). ADM adrenal concentration was markedly increased (from two-fold to three-fold) by both ZG stimulatory and suppressive treatments. Collectively, our findings suggest that in vivo 1) ADM, in addition to directly inhibit aldosterone secretion, may enhance it indirectly by eliciting catecholamine release, the two actions annulling each other under basal conditions; 2) under conditions leading to enhanced aldosterone secretion, the direct inhibitory effect of ADM prevails over the indirect stimulatory one, and the reverse occurs when aldosterone secretion is decreased; and 3) the modulatory action of ADM on the aldosterone secretion has a physiological relevance, endogenous ADM being locally synthesized in adrenals.
Keywords: Adrenomedullin; Adrenal gland; Aldosterone secretion; Rat;

Surface-active properties of vasoactive intestinal peptide☆ by Hayat Önyüksel; Bavish Bodalia; Varun Sethi; Sumeet Dagar; Israel Rubinsteina (419-423).
The purpose of this study was to determine whether human vasoactive intestinal peptide (VIP) aggregates in aqueous solution and, if so, whether the peptide interacts with a biomimetic phospholipid monolayer and increases surface pressure. Using a custom-made Teflon trough containing HEPES buffer (pH 7.4) at room temperature and a surface tensiometer, we found that the critical micellar concentration (CMC) of VIP is 0.4 μM. Surface pressure of a dipalmitoylphosphatidylcholine (DPPC) monolayer spread over the HEPES buffer declined significantly over 120 min because of phospholipid decomposition. However, injection of VIP at concentrations above CMC into the subphase of the monolayer elicited a significant concentration-dependent increase in surface pressure that persisted for 120 min (P < 0.05). Unlike VIP, injection of [8Arg]-vasopressin at an equimolar concentration only prevented the time-dependent decline in DPPC monolayer surface pressure. Taken together, these data indicate that human VIP aggregates in aqueous solution and expresses surface-active properties at physiological concentrations in vitro. We suggest that these attributes could have a role in modulating the bioactive effects of the peptide in vivo.
Keywords: Surface tension; Monolayer; Critical micellar concentration; Surfactant; Phospholipids; Neuropeptides; Vasopressin;

Digestive motor effects and vascular actions of CGRP in dog are expressed by different receptor subtypes by Marie-Claude L’Heureux; Serge St-Pierre; Louise Trudel; Victor Plourde; Raymond Lepage; Pierre Poitras (425-430).
Calcitonin gene-related peptide (CGRP) is a 37 AA peptide localized in blood vessels and nerves of the GI tract. Activation of CGRP receptors (subtypes 1 or 2) usually induces vasodilation and/or muscle relaxation, but its effects in dog and on gastroduodenal motility are still unclear. This study looked for the effect of CGRP and the antagonist CGRP8–37, specific for CGRP type 1 receptor, 1) on GI motility (interdigestive and postprandial), and 2) on hemodynamy, in conscious dogs. During the interdigestive period, the infusion of CGRP1–37 (200 pmol/kg/h) or CGRP8–37 (2000 pmol/kg/h) did not modify the duration of the migrating motor complex nor the release nor the motor action of plasma motilin. The gastric emptying of a solid meal (15 g meat/kg) was reduced by the administration of CGRP1–37 (AUC: 2196 ± 288.6 versus 3618 ± 288.4 with saline or T 1 2 : 78 ± 7.3 versus 50 ± 4.3 min; P < 0.01) and this effect was reversed by the antagonist CGRP8–37. CGRP1–37 significantly (P < 0.01) diminished arterial pressures (118 ± 1.6/64 ± 1.4 vs. 125 ± 1.4/75 ± 1.2 mmHg with saline) and accelerated the basal cardiac rhythm (110 ± 1.4 versus 83 ± 1.6 beats/min). However, CGRP8–37 failed to block the cardiovascular effects of CGRP1–37. In dog, CGRP could influence digestive motility by slowing the gastric emptying of a meal through an action on CGRP-1 receptors. Hemodynamic effects of CGRP were not blocked by CGRP8–37 and seem therefore mediated by CGRP-2 receptor subtype.
Keywords: Regulatory peptides; Gastroduodenal motility; Gastric emptying; Motilin; Migrating motor complex;

The post-prandial release of glucagon-like peptide-1 (GLP-1) from the distal gut appears to involve a neural reflex that arises from the proximal gut. The neuropeptide calcitonin gene-related peptide (CGRP)’s potent stimulatory effect on GLP-1 release was characterized, using the isolated vascularly perfused rat ileum. CGRP, but not its homolog amylin, induced a dose-dependent and sustained release of GLP-1. This effect was greatly reduced in the presence of CGRP(8–37), was abolished by galanin, potentiated by luminal glucose and unaffected by atropine. GIP enhanced, but did not potentiate, this effect. The results reveal how CGRP is involved in the complex regulation of GLP-1 release.
Keywords: CGRP; GLP-1; Perfused ileum;

Effects of oral casokefamide on plasma levels, tolerance, and intestinal transit in man by Ewert Schulte–Frohlinde; Wolfgang Reindl; Daniela Bierling; Christian Lersch; Victor Brantl; Hansjörg Teschemacher; Volker Schusdziarra (439-442).
Food-derived opioid peptides such as β-casomorphins are of interest for treatment of chronic diarrhea. The β-casomorphin analog casokefamide was administered orally at doses of 5.5, 8.0, and 16.0 mg to 10 healthy male volunteers, respectively. Dose-dependent increases of plasma levels with a maximum of 350 fmol/l were determined. No side-effects due to casokefamide has been observed. In comparison to placebo, casokefamide showed a trend toward prolongation of oro-caecal transit time. Orally applied casokefamide is well tolerated and may represent a useful tool for treatment of diarrhea in the future.
Keywords: β-Casomorphin; Casokefamide; Motility; Human study; Plasma levels; Opioid peptides;

Rodent submandibular gland peptide hormones and other biologically active peptides by Catherine Rougeot; Isabelle Rosinski-Chupin; Ronald Mathison; François Rougeon (443-455).
The cervical sympathetic trunk-submandibular gland neuroendocrine axis plays an integral role in physiological adaptations and contributes to the maintenance of systemic homeostasis, particularly under the ‘stress conditions’ seen with tissue damage, inflammation, and aggressive behavior. The variety of polypeptides, whose release from acinar and ductal cells is under sympathetic nervous system control, offers coordinated and progressive levels of endocrine communication. Proteolytic enzymes (e.g. the kallikreins and furin maturases) are involved in the conversion of inactive precursors (e.g. Pro-EGF and SMR1) into biologically active molecules (e.g. EGF, SMR1-pentapeptide), which act on local or distant targets and thereby modulate the homeostatic process.
Keywords: Endocrine peptides; Submandibular gland; EGF; SMR1-Peptides;