Peptides (v.23, #3)

Fragment of human beta-fibrinogen induces a behavioral effect on mouse forced swimming by Yutaka Masuda; Shun Ohunuma; Junya Sugawara; Yoshihiko Kawarada; Toshihiro Sugiyama (409-411).
We detected a peptide having a behavioral activity on mouse forced swimming from sera of healthy volunteers without affective and psychotic diseases. The amino acid sequence was GVNDNEEGF, which was found in the sequence of human β-fibrinogen. The synthesized peptide also showed the behavioral activity dose-dependently, but human fibrinopeptide B (QGVDNEEGFFSAR) did not. The activity was decreased by dopamine 1 antagonist SCH-23390, but not by dopamine 2 antagonist sulpiride. These findings strongly suggest that metabolism of human β-fibrinogen induces the fragment affecting the mouse behavior via the dopamine1 neuronal activity.
Keywords: Human β-fibrinogen; Metabolism; Fragment; Amino acid sequence; Behavioral effect; Mouse forced swimming;

Structure and biological activities of synthetic peptides corresponding to bovine neutrophil β-defensin BNBD-12, GPLSC1GRNGGVC2IPIRC3 PVPMRQIGTC4 FGRPVKC5 C6RSW with disulfide connectivities C1-C5, C2-C4 and C3-C6 and its variants with one, two and three disulfide bridges have been investigated. Selective protection of cysteine thiols was necessary in the four and six cysteine containing peptides for the formation of disulfide connectivities as observed in BNBD-12. Circular dichroism (CD) spectra indicate that in aqueous medium, only a small fraction of molecules populate turn-like conformations. In the presence of micelles and lipid vesicles, the single, two and three disulfide containing peptides adopt β-hairpin or β-sheet structures. Antibacterial activity was observed for all the peptides, irrespective of the number of disulfide bridges or how they were connected. Our results suggest that a rigid β-sheet structure or the presence of three disulfide bridges does not appear to be stringent requirements for antibacterial activity in β-defensins.
Keywords: β-defensin; Synthetic peptides; Antibacterial activity; Peptide conformation;

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica by Todd Isaacson; AnaMaria Soto; Shawichi Iwamuro; Floyd C Knoop; J.Michael Conlon (419-425).
Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an α-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH2; temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 μM; japonicin-2, 12 μM; and temporin-1Ja > 100 μM. The MICs against S. aureus were japonicin-1, > 100 μM; japonicin-2, 20 μM; and temporin-1Ja, > 100 μM.
Keywords: Antimicrobial peptide; Amphibian skin; Rana; Japonicin; Temporin;

Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima by Ren Lai; Yong-Tang Zheng; Ji-Hong Shen; Guan-Jie Liu; Hen Liu; Wen-Hui Lee; Shao-Zhong Tang; Yun Zhang (427-435).
Two groups of antimicrobial peptides have been isolated from skin secretions of Bombina maxima. Peptides in the first group, named maximins 1, 2, 3, 4 and 5, are structurally related to bombinin-like peptides (BLPs). Unlike BLPs, sequence variations in maximins occurred all through the molecules. In addition to the potent antimicrobial activity, cytotoxicity against tumor cells and spermicidal action of maximins, maximin 3 possessed a significant anti-HIV activity. Maximins 1 and 3 were toxic to mice with LD50 values of 8.2 and 4.3 mg/kg, respectively. Peptides in the second group, termed maximins H1, H2, H3 and H4, are homologous with bombinin H peptides. cDNA sequences revealed that one maximin peptide plus one maximin H peptide derived from a common larger protein.
Keywords: Amphibian; Peptide; Antimicrobial; Maximin; Bombina maxima; cDNA; Sequence; HIV;

A novel bombesin-related peptide was isolated from skin secretions of Chinese red belly toad Bombina maxima. Its primary structure was established as pGlu-Lys-Lys-Pro-Pro-Arg-Pro-Pro-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH2. The amino-terminal (N-terminal) 8-residue segment comprising four prolines and three basic residues is extensively different from bombesins from other Bombina species. The peptide was thus named proline rich bombesin (PR-bombesin). PR-bombesin was found to elicit concentration-dependent contractile effects in the rat stomach strip, with both increased potency and intrinsic activity as compared with those of [Leu13]bombesin. Analysis of different bombesin cDNA structures revealed that an 8 to 14- nucleotide fragment replacement in the peptide coding region (TGGGGAAT in the cDNAs of multiple bombesin forms from Bombina orientalis and CACCCCGGCCACCC in the cDNA of PR-bombesin) resulted in an unusual Pro-Pro-Arg-Pro-Pro motif in the N-terminal part of PR-bombesin.
Keywords: Bombesin; PR-bombesin; Amphibian; Bombina maxima; cDNA; Sequence;

Cladistic analysis of anuran POMC sequences by Jasem Alrubaian; Phillip Danielson; David Walker; Robert M Dores (443-452).
Procedures for performing cladistic analyses can provide powerful tools for understanding the evolution of neuropeptide and polypeptide hormone coding genes. These analyses can be done on either amino acid data sets or nucleotide data sets and can utilize several different algorithms that are dependent on distinct sets of operating assumptions and constraints. In some cases, the results of these analyses can be used to gauge phylogenetic relationships between taxa. Selecting the proper cladistic analysis strategy is dependent on the taxonomic level of analysis and the rate of evolution within the orthologous genes being evaluated. For example, previous studies have shown that the amino acid sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and β-endorphin, can be used to resolve phylogenetic relationships at the class and order level. This study tested the hypothesis that POMC sequences could be used to resolve phylogenetic relationships at the family taxonomic level. Cladistic analyses were performed on amphibian POMC sequences characterized from the marine toad, Bufo marinus (family Bufonidae; this study), the spadefoot toad, Spea multiplicatus (family Pelobatidae), the African clawed frog, Xenopus laevis (family Pipidae) and the laughing frog, Rana ridibunda (family Ranidae). In these analyses the sequence of Australian lungfish POMC was used as the outgroup. The analyses were done at the amino acid level using the maximum parsimony algorithm and at the nucleotide level using the maximum likelihood algorithm. For the anuran POMC genes, analysis at the nucleotide level using the maximum likelihood algorithm generated a cladogram with higher bootstrap values than the maximum parsimony analysis of the POMC amino acid data set. For anuran POMC sequences, analysis of nucleotide sequences using the maximum likelihood algorithm would appear to be the preferred strategy for resolving phylogenetic relationships at the family taxonomic level.
Keywords: Bufo marinus POMC; Maximum Parsimony Analysis; Maximum Likelihood Analysis;

Novel analogs of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), have been synthesized and characterized in vitro and in vivo. The N-terminal amino acid D-phenylalanine in aSvg-30 was replaced by a D-tyrosine residue for specific radioactive labeling with 123I. Additionally, Met17 of aSvg-30 was substituted by norleucine and the N-terminus of the peptide was acetylated to increase in vivo metabolic stability. The aSvg-30 analogs were tested for their ability to displace [125I-Tyr0]Svg in binding experiments and to inhibit Svg-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for the human CRF1 (hCRF1), hCRF and hCRF receptor. Ac-[D-Tyr11, His12, Nle17Svg(11–40), named K31440, showed high specific binding to hCRF (K i = 1.48 ± 0.34 nM) and hCRF (K i = 2.05 ± 0.61 nM) but not the hCRF1 receptor (K i = 288 ± 13 nM) and decreased Svg-stimulated cAMP activity in hCRF2-expressing cells in a similar fashion as aSvg-30. In biodistribution studies specific uptake of 123I-K31440 was detected after 1 h in small intestine of BALB/c nude mice. These data demonstrate that 123I-K31440 may serve as a useful tool to detect native CRF2 receptors and elucidate their role in gastrointestinal disorders and diseases such as irritable bowel syndrome or cancer.
Keywords: CRF (corticotropin-releasing factor); Ucn (urocortin); Svg (sauvagine); aSvg-30 (antisauvagine-30); HEK (human embryonic kidney); cAMP (adenosine 3‘; 5′- cyclic monophosphate); RPHPLC (reverse-phase HPLC); receptor imaging; SPECT (single photon emission computed tomography); 123I, iodine-123;

Peptidase activities in human semen by David Fernández; Asier Valdivia; Jon Irazusta; Carmen Ochoa; Luis Casis (461-468).
Enkephalins are one of the opioids present in human semen and to date their function in this tissue remains unknown. The present work studies enkephalin-degrading enzyme activities, puromycin-sensitive alanyl aminopeptidase (AAP-S), puromycin-insensitive alanyl aminopeptidase N (Ap N) and neprilysin (NEP) in human seminal fractions. AAP-S activity was not detected in any fractions, whereas Ap N appeared in soluble and particulate sperm fractions in seminal fluid and in prostasome fraction. With regard to NEP activity, this was exclusively located in prostasome membranes. The high activity values observed in the prostasome fraction suggested that these peptidases and their substrates could be involved in seminal physiology.
Keywords: Aminopeptidase; Neprilysin; Enkephalin; Semen; Spermatozoa; Prostasome;

High airway-to-blood transport of an opioid tetrapeptide in the isolated rat lung after aerosol delivery by Ann Tronde; Eva Krondahl; Hans von Euler-Chelpin; Per Brunmark; Ursula Hultkvist Bengtsson; Gunilla Ekström; Hans Lennernäs (469-478).
The airway-to-blood absorption of the μ-selective opioid tetrapeptide agonist Tyr-D-Arg-Phe-Phe-NH2 (MW 631) was investigated in the isolated, perfused, and ventilated rat lung model. The lung metabolism of the peptide was compared after airway and vascular delivery. The concentrations of the parent tetrapeptide and five of its metabolites in the perfusate and in bronchoalveolar lavage fluid were analyzed by LC-MS.The metabolism of the peptide was higher after delivery to the airways compared to vascular delivery. However, the tetrapeptide was highly transported from the air-to-blood side to an extent of 47.8 ± 10.7% in 2 h. In conclusion, the results prompt investigations of the pulmonary route as a successful alternative to parenteral delivery for this tetrapeptide.
Keywords: Opioid peptide; Peptide metabolism; First-pass metabolism; Drug delivery; Absorption; Permeability; Inhalation; Pulmonary; Lung; Isolated perfused lung; Rat; Caco-2;

Regional differences in bioavailability of an opioid tetrapeptide in vivo in rats after administration to the respiratory tract by Eva Krondahl; Ann Tronde; Stefan Eirefelt; Heidi Forsmo-Bruce; Gunilla Ekström; Ursula Hultkvist Bengtsson; Hans Lennernäs (479-488).
TArPP (Tyr-d-Arg-Phe-Phe-NH2), 1–10 μmol/kg, was administered to anesthetized rats by nasal microinfusion, intratracheal microinfusion, intratracheal nebulization, aerosol inhalation, and i.v. bolus and infusion. Plasma concentrations of TArPP and its deamidated metabolite were determined by LC-MS-MS.Regional differences in bioavailability (F), first-pass metabolism, and absorption rate were found for TArPP after delivery to the respiratory tract. Absorption was rapid after both pulmonary and nasal administration (tmax ∼ 10–20 min). After nasal microinfusion, F was 52 ± 9%. For all the pulmonary groups, F was higher (72–114%). First-pass metabolism of TArPP was lower in the lung than in the nasal cavity. It is evident that the pulmonary route is attractive for successful systemic delivery of small, hydrophilic and enzymatic susceptible peptides.
Keywords: Drug delivery; Respiratory tract; Nasal; Pulmonary; Inhalation; Regional absorption; First-pass metabolism; Bioavailability; Pharmacokinetics; Opioid peptide; Deamidation; Rat;

Dansyl-PQRamide, a possible neuropeptide FF receptor antagonist, induces conditioned place preference by E.Y.-K. Huang; J.-Y. Li; C.-H. Wong; P.P.-C. Tan; J.-C. Chen (489-496).
Neuropeptide FF (NPFF) is an endogenous anti-opioid peptide. NPFF could potentiate the naloxone-precipitated morphine withdrawal syndromes in morphine-dependent rats, indicating the possible involvement of the endogenous NPFF system in opioid analgesia and dependence. The present study was performed to examine the effects of dansyl-PQRamide (dns-PQRa), a putative NPFF antagonist, on conditioned place preference (CPP), in addition, its interaction with the opioid system. Two CPP experiments were conducted. First, rats were treated with dns-PQRa (4–13 mg/kg, i.p.) and paired with the non-preferred compartment while the vehicle was paired with the preferred compartment. Second, similar to experiment 1 except naloxone (1 mg/kg, i.p.) was given 10 min prior to each dns-PQRa administration. The post-drug place preference was examined after 4 alternative pairings. Another group of animals after repetitive dns-PQRa treatments were analyzed for levels of neurotransmitters in discrete brain areas. Dns-PQRa (4–13 mg/kg, i.p.) induced a significant dose-dependent CPP. The dns-PQRa-induced CPP was completely blocked by pretreatment with 1 mg/kg i.p. naloxone, while naloxone alone did not induce any place aversion. The chronic dns-PQRa-treated (13 mg/kg, i.p., b.i.d.) rats caused a significant increase in 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in the olfactory tubercle compared to the vehicle-treated controls. There was also an increase in the turnover of serotonin in the olfactory tubercle, nucleus accumbens and medial prefrontal cortex. These results suggest that blockade of the NPFF system produces rewarding, possibly via an inhibition of the anti-opioid action of NPFF. These results also reveal a close relationship between NPFF, drug rewarding and the dopaminergic and serotoninergic neurons in the mesolimbic system.
Keywords: Neuropeptide FF; Naloxone; Conditioned place preference; Olfactory tubercle; Dopamine; Serotonin;

Nociceptin, OP4 receptor ligand in different models of experimental epilepsy by Andrzej Rubaj; Witold Zgodzinski; Katarzyna Gustaw; Maria Sieklucka-Dziuba (497-505).
The anticonvulsive activity of nociceptin, endogenous OP4 receptors agonist was investigated in pentylenetetrazole (PTZ), N-methyl D-aspartic acid (NMDA), bicucculine (BCC) and electrically evoked seizure models of experimental epilepsy. Nociceptin, at the dose of 10 nmol, suppressed the clonic seizures induced by PTZ, NMDA and BCC. [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1–13)-NH2 which has been proposed to be selective antagonist OP4 receptors, did not prevent the action of nociceptin. The effect of [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1–13)-NH2 on seizures induced by PTZ, NMDA and BCC was very similar to that of nociceptin. These data support the hypothesis that it possesses agonistic properties. Naloxone did not reverse the anticonvulsive action of nociceptin as well as [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1–13)-NH2 which excludes the participation of opioid receptor in this action. On the other hand in the electroconvulsive model of generalized seizures, nociceptin as well as [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1–13)-NH2 influenced neither the electroconvulsive threshold nor the maximal electroshock test. The data suggest that nociceptin and [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1–13)-NH2 can exert anticonvulsive action. These properties depend on OP4 but not opioid receptors activation.
Keywords: Nociceptin; [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1–13)-NH2; Pentylenetetrazole; N-methyl D-aspartic acid; Bicucculine; Electrically evoked seizures;

The neuropeptide calcitonin gene-related peptide (CGRP) may play a role in neurogenic inflammation, tissue remodeling of the uterine cervix, promoting vasodilation, parturition, and processing of sensory information in the spinal cord. CGRP-immunoreactive nerves of the cervix and spinal cord have been studied but cellular identification of the CGRP receptor has received little attention. CGRP-receptor component protein (CGRP-RCP) is a small protein associated with the CGRP receptor; thus, immunostaining for the CGRP-RCP can be used to identify sites of the CGRP receptor. We determined sites of CGRP-RCP immunoreactivity relative to the presence of CGRP-ir nerve fibers in the female rat uterine cervix, spinal cord, and dorsal root ganglia. CGRP-RCP immunoreactivity was expressed in the dorsal horn of the spinal cord, venules of the uterine cervix, and perikarya of sensory neurons in dorsal root ganglia. CGRP-immunoreactive fibers were adjacent to CGRP-RCP-immunoreactive vessels in the cervix and among CGRP-RCP-immunoreactive structures in the dorsal horn of the spinal cord. This suggests CGRP-RCP is associated with structures innervated by CGRP nerves and these interactions may be changed in tissues in response to an appropriate stimulus.
Keywords: Calcitonin gene-related peptide; Calcitonin gene-related peptide receptor; Neurogenic inflammation;

Urocortin (Ucn), a newly identified member of the corticotropin-releasing factor (CRF) family, is not only expressed in the brain, but also abundantly present in the peripheral tissues, especially in the gastrointestinal tract (GI) as determined by radioimmuoassay. In order to determine the precise localization of urocorin in the GI, we mapped the distribution of urocortin-like immunoreactivity (ir) in the GI of the rat using an immunofluorescence histochemical technique. Ucn, both in the brain and the peripheral tissues, is involved in the regulatory control of host-defense mechanism during stress. In order to study the possible involvement of the sympathetic system in the expression of GI urocortin in response to stress, we examined the effect of chemical sympathectomy on urocortin-ir and its colocalization with tyrosine hydroxylase (TH). UCn was expressed in all parietal cells of the stomach, myenteric and submucosal plexuses as well as in cells in Lieberkühn crypts of the small and large intestine. Most of the acid secreting parietal cells contained both Ucn and TH. Chemical sympathectomy did not affect Ucn immunoreactivity of parietal cells.
Keywords: Stomach; Dopamine; Gastric ulcer; Stress ileus; Double immunofluorescence labeling;

Interaction of xenin with the neurotensin receptor of guinea pig enteral smooth muscles by Gerhard E. Feurle; Jörg W. Metzger; Alexandra Grudinski; Gerd Hamscher (523-529).
Xenin, a 25 aminoacid peptide, interacts with the neurotensin receptor subtype 1 of intestinal muscles of the guinea pig. Replacement of the C-terminal Lys -Arg peptide bond in xenin 6 by a reduced pseudo-peptide bond augmented binding affinity to isolated jejunal and colonic muscle membranes by factors of 7.7 and 21.0 respectively; the potency to contract the jejunum and to relax the colon was increased by factors of 3.2 and 1.3. The C-terminus Trp-Ile-Leu (WIL) of xenin, in contrast to the C-terminus Tyr-Ile-Leu (YIL) of neurotensin, bound competitively to the muscle membranes. WIL blocked the contractile action of xenin in the jejunum and was synergistic with the relaxing action in the colon. The Lys -Arg motif and Trp in the C-terminus of xenin are essential structures in the action of xenin on the enteral smooth muscle receptors.
Keywords: Xenin; Neurotensin; Neurotensin receptor; Smooth muscle; Guinea pig; Intestine; Reduced Ψ; pseudo-peptide bond;

Ghrelin-producing cells exist as two types of cells, closed- and opened-type cells, in the rat gastrointestinal tract by Ichiro Sakata; Kazuaki Nakamura; Mami Yamazaki; Maki Matsubara; Yuijiro Hayashi; Kenji Kangawa; Takafumi Sakai (531-536).
Ghrelin was recently isolated from the rat stomach as an endogenous ligand for the growth-hormone secretagogue receptor (GHS-R) and is known to exist in the gastrointestinal tract and hypothalamus. In this study, we investigated in detail the distribution and morphologic characteristics of ghrelin-containing cells (ghrelin cells) in the gastrointestinal tract by immunohistochemistry and in situ hybridization. Ghrelin cells were found to be localized in the mucous membrane of the stomach, duodenum, ileum, cecum and colon but not in myenteric plexus, and they can be classified into open- and closed-type cells. The greatest number of ghrelin cells was found in the stomach, and it was found that the number of the opened-type cells gradually increased in the direction from stomach to the lower gastrointestinal tract. These results suggest that the two types of ghrelin cells may be distinctly regulated and play different physiological roles in various regions of the gastrointestinal tract.
Keywords: Ghrelin; Rat; Gastrointestinal tract; Immunohistochemistry; In situ hybridization;

Comparative study of enterostatin sequence in five rat strains and enterostatin binding proteins in rat and chicken serum by Yingjen Jeffrey Wu; David Hughes; Ling Lin; Doug H Braymer; David A York (537-544).
Enterostatin, a pentapeptide derived from the precursor protein procolipase has been shown to inhibit dietary fat intake and to reduce body fat after chronic administration in rats. We repeat that the enterostatin amino acid sequence from the genomic DNA of 5 different rat strains is APGPR. 125I-APGPR bound to three proteins (300, 205 and 60 kDa) in rat serum and one 60 kDa protein in chicken serum. These serum binding proteins were also eluted by APGPR affinity chromatography. Western blot analysis of serum protein identified enterostatin-like immunoreactivity associated with the same molecular weight bands. Our results demonstrate the enterostatin sequence in rat is APGPR and suggest the presence of enterostatin binding proteins in rat and chicken serum.
Keywords: Enterostatin; Gene sequence; Binding protein; Antibody; Procolipase; Fat;

Galanin (GAL) is a biologically active peptide that is involved in a variety of physiological functions. The purpose of this study was to evaluate whether porcine and rat galanin radioligands could be used as probes to discriminate GAL receptors (GALR) subtypes using a cell line, RINm5F, that express multiple GALR subtypes. Data from parallel equilibrium binding experiments using the same RINm5F membrane homogenates reveal that [125I]pGAL labels 20% more GALRs with a 2-fold lower affinity than those values identified when using [125I]rGAL. Competition studies using various GAL peptides showed different rank order of potencies depending on the radioligand used. Preincubation of RINm5F membranes with GppNHp, a non-hydrolizable GTP analog, prior to radioligand labeling suggests that a portion of GALRs is precoupled to G proteins. In addition, receptors labeled by [125I]rGAL appear more sensitive to GppNHp-induced uncoupling of G proteins than those labeled by [125I]pGAL. In conclusion, our data suggest that pGAL and rGAL radioligands define different pharmacological profiles of GALRs, and hence, these ligands can be used as pharmacological tools to discriminate GALR subtypes. Additionally, our data suggests that GALRs exist in a precoupled state with their respective G-proteins prior to interaction with the agonist.
Keywords: Galanin; Precoupling; Adenylate cyclase; G-protein; GppNHp; Pertussis toxin;

Effect of a regulatory mutation on the rat atrial natriuretic peptide gene transcription by Speranza Rubattu; Rosangela Giliberti; Paola De Paolis; Rosita Stanzione; Paola Spinsanti; Vanessa Venturelli; Massimo Volpe (555-560).
To investigate the functional relevance of a regulatory mutation affecting the enhancer element PEA2 of the rat ANP gene we transfected rat cardiomyocytes and aortic endothelial cells with either the mutant or the wild-type ANP promoter construct (-683 +54) and performed CAT assays both at baseline and in response to Phenylephrine and Angiotensin II. In the myocardial cells we also determined the DNA/nuclear protein interaction through electrophoretic mobility shift assay. These studies showed a significantly lower degree of ANP transcription in the presence of the mutant PEA2 site, thus demonstrating its functional significance and the biological relevance of ANP gene structural alterations.
Keywords: Atrial natriuretic peptide; ANP promoter; PEA2; Gene mutation;

Mechanisms transducing the aldosterone secretagogue signal of endothelins in the human adrenal cortex by Paola G Andreis; Giuliano Neri; Cinzia Tortorella; Francesco Aragona; Gian Paolo Rossi; Gastone G Nussdorfer (561-566).
Evidence has been provided that the 21-amino acid hypertensive peptide endothelin (ET)-1 exerts a potent secretagogue effect on human adrenocortical zona glomerulosa (ZG), acting through two receptor subtypes, called ETA and ETB, the signaling mechanism(s) of which has (have) not yet been investigated. Collagenase dispersed human ZG cells were obtained from normal adrenals of patients undergoing nephrectomy/adrenalectomy for renal cancer. The selective ETA- and ETB-receptor activation was obtained by exposing dispersed cells to ET-1 plus the ETB-receptor antagonist BQ-788 and to the ETB-receptor agonist BQ-3020, respectively. The phospholipase (PL) C inhibitor U-73122 abolished ETA receptor-mediated secretory response, but only partially prevented the ETB receptor-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin, the calmodulin inhibitor W-7 and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes. When added together, calphostin-C and wortmannin or W-7 abolished ETA-mediated secretory response, but only decreased ETB-mediated one. The ETB receptor-, but not the ETA receptor-mediated aldosterone response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. ETA-receptor activation raised inositol triphosphate (IP3) production from dispersed ZG cells, while ETB-receptor stimulation enhanced both IP3 and prostaglandin-E2 production. Collectively, our findings indicate that ETs stimulate aldosterone secretion from human ZG cells, acting through ETA receptors exclusively coupled to PLC/PKC-dependent pathway and ETB receptors coupled to both PLC/PKC- and COX-dependent cascades.
Keywords: Endothelins; Endothelin receptors; Human adrenal cortex; Aldosterone secretion;

In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone. We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare. In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems. Neither recombinant precursors showed activity. Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide. These results indicate that the cDNA encodes the hormone.
Keywords: Androgenic gland hormone; Arrangement of disulfide bridges; Armadillidium vulgare; Baculovirus expression system; Crustacea; Heterodimeric glycopeptide; Isopoda; Post-translational modification; Sex differentiation;

A synthetic peptide with estrogen-like activity derived from a phage-display peptide library by Natarajan Venkatesh; Yehudith Zaltsman; Dalia Somjen; Batya Gayer; Ettickan Boopathi; Roni Kasher; Tikva Kulik; Ephraim Katchalski-Katzir; Fortune Kohen (573-580).
We describe a novel approach to develop peptides with estrogen like activity using a monoclonal antibody specific to estradiol (mAb E2-15) for the affinity selection of phage displayed peptides from a combinatorial peptide library. Based on the sequences of the selected phage, we synthesized a 15-mer linear peptide LPALDPTKRWFFETK which was derivatized to a 23 mer cyclic peptide CAELPALDPTKRWFFETKPPPPC. Both peptides displayed estrogen-like activity according to the following criteria:(i) in inhibiting the binding of [3H]estradiol to mAb E2-15 and to estrogen receptor (ER)α; (ii) in inducing transcriptional activity in MCF7 human breast cancer cells transfected with an estrogen receptor element luciferase construct and (iii) in causing an increase in creatine kinase specific activity in rat tissues in vivo. This approach can be employed to design peptide mimetic for other hormones as well.
Keywords: Steroid-mimetic peptides; Monoclonal anti-estradiol antibody; Phage-display peptide library; Estrogen receptor α; Estrogen receptor β;

Post-trial administration of vasopressin in humans does not enhance memory formation (vasopressin and memory consolidation) by Steffen Gais; Marcel Sommer; Stefan Fischer; Boris Perras; Jan Born (581-583).
Many animal studies show an enhancing effect of vasopressin (VP) on memory, but not all human studies could confirm this finding. This study examined the influence of post-learning administration of VP (40 IU, intranasally) on the consolidation of declarative memories in healthy humans during different intervals of sleep and waking. We could not find any effect of VP on memory consolidation, but EEG activity indicated a significant arousing influence of VP. Results suggest that if VP affects memory function it might do so primarily at the stage of encoding of the materials to be learned but it leaves unaffected processes of consolidation.
Keywords: Vasopressin; Memory consolidation; Sleep;

A new member of the AKH/RPCH family was isolated from the corpora cardiaca of the firebug Pyrrhocoris apterus. It is the second adipokinetic peptide identified in this species. The peptide was characterized and its structure was deduced from the multiple MSN electrospray mass spectra as that of an octapeptide with the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2. The peptide differs from the original P. apterus AKH (Pya-AKH) by one amino acid in position 3. Topical application and/or injection of the peptide induced lipid mobilization, but was inactive in mobilization of carbohydrates.
Keywords: AKH/RPCH family; Bug; Hormone; Electrospray mass spectrometry; Insect neuropeptide; Lipid mobilization; Pya-AKH;

Alzheimer’s disease through the eye of a mouse by John E Morley; Sue A Farr; Vijaya B Kumar; William A Banks (589-599).
There is now ample evidence that β-amyloid proteins decrease memory. The SAMP8 mouse (P8) develops an early decline in the ability to learn and to retain new information. The studies reviewed here suggest that this is due to overproduction of β-amyloid. Both antibodies to β-amyloid and specific antisense to the amyloid precursor protein reverse these deficits in the P8 mouse. This antisense can cross the blood brain barrier. It is hypothesized that the overproduction of β-amyloid leads to a decline in Δ9 desaturase activity with an alteration in membrane fatty acids. This results in altered membrane mobility leading to a decline in neurotransmitter activity and a decreased release of acetylcholine. This decreased cholinergic activity results in a decreased ability of the P8 mouse to learn and retain new information.
Keywords: SAMP8; Alzheimer’s; Beta amyloid; Memory; Antisense; Δ9 desaturase;