Peptides (v.24, #9)

Triggered by receptor binding of gp120, the human immunodeficiency virus type 1 (HIV-1) gp41 changes its conformation to a fusogenic six-helix bundle structure. In the present study, this core conformation modeled by the peptides derived from the gp41 N- and C-terminal heptad repeat regions was determined by fluorescence native polyacrylamide gel electrophoresis and size exclusion high-performance liquid chromatography (HPLC). Two previously described small molecule HIV-1 fusion inhibitors significantly blocked the six-helix bundle formation. It suggests that these biophysical techniques can be used in a novel way to study the conformational change of gp41 during virus entry into cells and to identify HIV-1 fusion inhibitors.
Keywords: HIV-1 gp41; HIV-1 fusion inhibitors; Native PAGE; Fluorescence; HPLC;

Comparative activities of cecropin A, melittin, and cecropin A–melittin peptide CA(1–7)M(2–9)NH2 against multidrug-resistant nosocomial isolates of Acinetobacter baumannii by Andrea Giacometti; Oscar Cirioni; Wojciech Kamysz; Giuseppina D’Amato; Carmela Silvestri; Maria Simona Del Prete; Jerzy Łukasiak; Giorgio Scalise (1315-1318).
The in vitro activity of three polycationic peptides, cecropin A, melittin, and cecropin A–melittin hybrid peptide CA(1–7)M(2–9)NH2, alone and in combination with various clinically used antimicrobial agents, was investigated against 32 nosocomial isolates of Acinetobacter baumannii. Antimicrobial activities were measured by MIC, MBC and bacterial killing assay. The peptides demonstrated different ranges of inhibitory values: overall, the organisms were more susceptible to CA(1–7)M(2–9)NH2 (MIC range, 0.25–16 mg/l) than to cecropin A (0.50–32 mg/l) and melittin (0.50–32 mg/l). Synergy was observed when CA(1–7)M(2–9)NH2 and melittin were combined with β-lactam antibiotics.
Keywords: Cecropin A; Melittin; Acinetobacter baumannii; Susceptibility; Antimicrobial peptides;

The cDNA encoding prothoracicotropic hormone (PTTH), the brain neuropeptide that stimulates the prothoracic glands to synthesize ecdysone, was cloned from the corn earworm Helicoverpa zea (Hez). The amino acid sequence deduced from the cDNA indicates a molecular structure that is distinct from the PTTH’s reported in other Lepidoptera, but all contain an identical proteolytic cleavage site and the seven cysteine residues that are essential for activity. Northern hybridization shows a single mRNA present in the brain–subesophageal ganglion complex. Using RT–PCR, we observed constant amounts of PTTH mRNA during larval development but large fluctuations at pupation and prior to adult eclosion.
Keywords: Prothoracicotropic hormone; cDNA structure; Developmental expression; Helicoverpa zea;

Insect diapause-specific peptide from the leaf beetle has consensus with a putative iridovirus peptide by Hiromasa Tanaka; Kenji Sato; Yoshimi Saito; Tetsuro Yamashita; Masanobu Agoh; Junji Okunishi; Eiichi Tachikawa; Koichi Suzuki (1327-1333).
Diapause and hibernation during periods of environmental adversity are essential features of the life cycle in many organisms, yet the molecular basis for these events differs among animals. We have identified an endogenous diapause/hibernation-specific peptide, from the leaf beetle Gastrophysa atrocyanea. This peptide provides antifungal activity, acts as a N-type voltage-gated Ca2+ channel blocker, and has a new consensus sequence with an unknown polypeptide encoded in the insect iridescent virus. These results indicate that the diapause-specific peptide may be utilized as a probe to analyze and compare functional and evolutional aspects of the life cycles of insects and iridoviruses.
Keywords: Diapause-specific peptide; Homology to viral peptide; Antifungal activity; Ca2+ channel blocker; Leaf beetle;

Histochemical localization of the PBAN receptor in the pheromone gland of Heliothis peltigera by Miriam Altstein; Orna Ben-Aziz; Kalpana Bhargava; Qijing Li; Manuela Martins-Green (1335-1347).
The presence of the pyrokinin (PK)/ Pheromone biosynthesis activating neuropeptide (PBAN) receptor in pheromone gland cells of Heliothis peltigera females was demonstrated, and its spatial distribution in the ovipositor was visualized with two photo-affinity biotinilated ligands: BpaPBAN1-33NH2 and BpaArg27-PBAN28-33NH2. Light microscopy histological studies revealed that the gland is contained within the inter-segmental membrane (ISM) between the 8th and 9th abdominal segments. The gland was found to be composed of a single layer of columnar epithelial cells positioned under the inter-segmental cuticle. Similar epithelial cells were also found in the dorsal and ventral regions of the 9th abdominal segment. All regions containing the glandular cells bound both ligands, indicating presence of the PK/PBAN receptor. The patterns obtained with both ligands were similar, hinting at the possibility that either both ligands bind to the same receptor, or, that if there are two distinct receptors, their spatial distribution throughout the gland is very similar.
Keywords: PBAN receptor; Photo-affinity ligands; Heliothis peltigera; Pheromone gland; Insect neuropeptide;

A cyclo peptide activates signaling events and promotes growth and the production of the bone matrix by S. Pallu; R. Bareille; M. Dard; H. Kessler; A. Jonczyk; M. Vernizeau; J. Amédée-Vilamitjana (1349-1357).
The interaction of bone cells and their underlying extracellular matrix impacts biological processes such as maintenance of tissue integrity. The biological recognition of the extracellular matrix by attached cells is mediated by the activity of integrins that recognize adhesive-specific domains. The most widely recognized adhesive motif is the RGD sequence, common to many of the adhesive matrix molecules. Here, we show that cyclo DFKRG which was previously selected to increase cell adhesion of human bone marrow stromal cells (HBMSC), increases both cell differentiation and mineralization through activation of tyrosine kinases, focal adhesion kinase (p125FAK) and Mitogen Activated Protein (MAP) kinases.
Keywords: Adhesion; RGD-peptide; Integrins; FAK; MAPK; Phosphorylation; Mineralization;

Analysis of conserved residues of the human puromycin-sensitive aminopeptidase by Michael W. Thompson; Louis B. Hersh (1359-1365).
The puromycin-sensitive aminopeptidase (ApPS) is a zinc metallopeptidase involved in the degradation of neuropeptides. Putative catalytic residues of the enzyme, Cys146, Glu338, and Lys396 were mutated, and the resultant mutant enzymes characterized. ApPS C146S exhibited normal catalytic activity, ApPS E338A exhibited decreased substrate binding, and ApPS K396I exhibited decreases in both substrate binding and catalysis. ApPS K396I and ApPS Y394F were analyzed with respect to transition state inhibitor binding. No effect was seen with the K396I mutation, but ApPS Y394F exhibited a 3.3-fold lower affinity for RB-3014, a transition state inhibitor, indicating that Tyr394 is involved in transition state stabilization.
Keywords: Aminopeptidase; Substrate binding; Catalysis; Mechanism; Mutagenesis; Catalytic residues; Mutagenesis; Bestatin; Puromycin;

Aminopeptidases in visceral organs during alterations in body fluid volume and osmolality by Elaine Gasparello-Clemente; Luis Casis; Adolfo Varona; Javier Gil; Jon Irazusta; Paulo Flávio Silveira (1367-1372).
Enzymatic cleavage of some peptides in the local environment could be included among the mechanisms related to the regulation of hydrosaline balance. In order to examine this hypothesis, we measured representative aminopeptidase activities in visceral organs of rats after applying certain hydrosaline challenges. Decreased levels (about 30%) of particulate puromycin-insensitive-neutral aminopeptidase in the renal medulla and of soluble acid aminopeptidase in the lung were observed under hyperosmolality and hypovolemia. Decreased levels (more than 45%) of particulate type-I-pyroglutamyl aminopeptidase in the heart were observed under altered volemia. These results indicate that aminopeptidases at these anatomical locations might be involved in the regulation of body fluid volume and osmolality.
Keywords: Peptidases; Osmoregulation; Volemia; Water-electrolyte homeostasis; Hydrosaline challenges;

Human galanin expresses amphipathic properties that modulate its vasoreactivity in vivo by Sumeet Dagar; Hayat Önyüksel; Syed Akhter; Aparna Krishnadas; Israel Rubinstein (1373-1380).
The purpose of this study was to determine whether human galanin, a pleiotropic 30-amino acid neuropeptide, expresses amphipathic properties in vitro and, if so, whether these properties modulate its vasoactive effects in the intact peripheral microcirculation. We found that human galanin aggregates in an aqueous solution and forms micelles with a critical micellar concentration (CMC) of 0.4 μM. In addition, the peptide interacted with model membrane as indicated by long and significant increase of the surface pressure of the biomimetic monolayer membrane in vitro. Interactions of human galanin with sterically stabilized phospholipid micelles (SMM) were not associated with a significant change in peptide conformation. Using intravital microscopy, we found that suffusion of human galanin alone elicited significant concentration-dependent vasoconstriction in the intact hamster cheek pouch. This response was amplified when human galanin in SSM was suffused onto the cheek pouch. The effects of human galanin alone and in SSM were mediated by galanin receptors because galantide, a galanin receptor antagonist, abrogated galanin-induced vasoconstriction. Collectively, these data show that human galanin expresses amphipathic properties in the presence of phospholipids which in turn amplifies its vasoactive effects in the intact peripheral microcirculation.
Keywords: Neuropeptide; CMC; Sterically stabilized phospholipid micelles; Microcirculation; Vasodilation; Hamster cheek pouch; Receptor antagonist;

Octreotide: a new approach to the management of acute abdominal hypertension by Ayhan Kaçmaz; Ali Polat; Yılmaz User; Metin Tilki; Sırrı Özkan; Göksel Şener (1381-1386).
Acutely increased intra-abdominal pressure (IAP) may lead to abdominal compartment syndrome (ACS), which ischaemia/reperfusion (I/R) injury plays an important role. The main goal of the management of ACS is to lower the intra-abdominal pressure despite reperfusion injury. Octreotide (OCT), a synthetic somatostatin analogue, lowers the splanchnic perfusion. The aim of this study was to investigate whether OCT improves the reperfusion injury after decompression of acute abdominal hypertension.Under anesthesia, a catheter was inserted intraperitoneally and using an aneroid manometer connected to the catheter, IAP was kept at 20 mmHg (ischemia group; I) for 1 h. In the I/R group, pressure applied for an hour was decompressed and 1 h reperfusion period was allowed. In another group of I/R, OCT was administered (50 μg/kg i.p.) immediately before the decompression of IAP. The results demonstrate that kidney and lung tissues of malondialdehyde (MDA; an end product of lipid peroxidation) levels and myeloperoxidase (MPO; index of tissue neutrophil infiltration) activity were elevated, while glutathione (GSH; a key to antioxidant) levels were reduced in I/R group (P<0.001). Moreover, OCT treatment applied in the I/R group reduced the elevations in blood urea nitrogen (BUN) and serum creatinine levels. Our results implicate that IAP causes oxidative organ damage and OCT, by reducing splanchnic perfusion and controlling the reperfusion of abdominal organs, could improve the reperfusion-induced oxidative damage. Therefore, its therapeutic role as a “reperfusion injury-limiting” agent must be further elucidated in IAP-induced abdominal organ injury.
Keywords: Abdominal hypertension; Octreotide; Myeloperoxidase; Lipid peroxidation; Glutathione;

Sequence, distribution and quantification of the motilin precursor in the cat by Luo Xu; Inge Depoortere; Leen Thielemans; Zhong Huang; Ming Tang; Theo L Peeters (1387-1395).
Keywords: Competitive PCR; mRNA; Homology; Brain; Thyroid;

We recently reported that neuropeptide Y (NPY) protein levels and cAMP responsive element binding (CREB) protein phosphorylation are lower in amygdaloid structures during ethanol withdrawal after chronic exposure. Furthermore, we reported that normalization of CREB phosphorylation by infusing protein kinase A (PKA) activator into the central amygdala prevents anxiety-like effects in rats during ethanol withdrawal. Here we investigated whether normalization of CREB phosphorylation by infusing PKA activator (Sp-cAMP) into the central amygdala also normalizes the expression of NPY during ethanol withdrawal. Sprague–Dawley male rats were cannulated targeting the central amygdala and then treated either with Lieber-DeCarli ethanol diet or control diet for 15 days. Subsequently ethanol-fed rats were withdrawn for 0 and 24 h. The control-diet fed and ethanol-withdrawn rats were infused twice with PKA activator or inhibitor (Rp-cAMP). The protein and mRNA levels of NPY were determined in amygdaloid structures using gold-immunolabeling and the in situ RT-PCR procedure. It was found that chronic ethanol treatment has no effect on mRNA and protein levels of NPY in the central, medial, or basolateral amygdala. On the other hand, ethanol withdrawal produced significant reductions in mRNA and protein levels of NPY in the central and medial but not in the basolateral amygdala. The reductions in mRNA and protein levels of NPY were normalized in the central amygdala by infusion with PKA activator in ethanol-withdrawn rats. On the other hand, PKA-inhibitor infusion does not have any effect on mRNA and protein levels of NPY in the central amygdala of ethanol-withdrawn rats, but significantly decreased the expression of NPY in the central amygdala of control-diet fed rats. These results suggest that the decreased cellular expression of NPY in the central amygdala may play an important role in the CREB-mediated regulation of anxiety-like behaviors during ethanol withdrawal.
Keywords: Neuropeptide Y; Ethanol withdrawal; Ethanol dependence; CREB; Rat brain;

Increased susceptibility to LTP generation and changes in NMDA-NR1 and -NR2B subunits mRNA expression in rat hippocampus after MCH administration by Mariana Marcela Varas; Mariela F Pérez; Oscar A Ramı́rez; Susana R de Barioglio (1403-1411).
The present study attempts to determine which mechanisms underlie the retrograde facilitation of memory induced by microinjection hippocampal melanin-concentrating hormone (MCH) on the inhibitory avoidance paradigm. Previous reports using this test on the hippocampus suggest that NMDA receptor-mediated mechanisms are involved in memory processing and are also necessary for the induction of long-term potentiation (LTP) of the hippocampal dentate gyrus. In addition, alterations in expression of synaptic NMDA subunits in the hippocampus have been associated with memory formation of an inhibitory avoidance task. We have studied the effects of the neuropeptide upon the electrophysiological parameters using hippocampal slices from rats injected with the peptide and tested in step-down tests as well as possible changes in the mRNA expression of NMDA receptor subunits. We postulate that the increased facility to induce LTP, and the overexpression of this N-methyl-d-aspartate mRNA receptor subunits induced by MCH, could be behind the retrograde facilitation observed after MCH hippocampal microinjection.
Keywords: Melanin-concentrating hormone; Inhibitory avoidance task; Long-term potentiation; Expression of NMDA receptor subunits;

The neuroprotective peptide NAP inhibits the aggregation of the beta-amyloid peptide by Osnat Ashur-Fabian; Yael Segal-Ruder; Ehud Skutelsky; Douglas E. Brenneman; Ruth A. Steingart; Eliezer Giladi; Illana Gozes (1413-1423).
Alzheimer’s disease (AD) is characterized by brain plaques containing the beta-amyloid peptide (Aβ). One approach for treating AD is by blocking Aβ aggregation. Activity-dependant neuroprotective protein contains a peptide, NAP that protects neurons in culture against Aβ toxicity. Here, NAP was shown to inhibit Aβ aggregation using: (1) fluorimetry; (2) electron microscopy; (3) high-throughput screening of Aβ deposition onto a synthetic template (synthaloid); and (4) Congo Red staining of neurons. Further assays showed biotin–NAP binding to Aβ. These results suggest that part of the neuroprotective mechanism exerted by NAP is through modulation of toxic protein folding in the extracellular milieu.
Keywords: Aβ aggregation; Activity-dependent neuroprotective protein; Alzheimer’s disease; Brain-cortical cultures; Vasoactive intestinal peptide; NAP;

Synthesis and biological evaluation of oxytocin analogues containing l-α-t-butylglycine [Gly(Bu t )] in positions 8 or 9 by Maria Fragiadaki; Vassiliki Magafa; Jiřina Slaninová; Paul Cordopatis (1425-1431).
We report the solid phase synthesis and some pharmacological properties of seventeen new oxytocin (OT) analogues. Basic modification at positions 8 and/or 9 (introduction of l-α-t-butylglycine [Gly(Bu t )]) was combined with d-Cys6, d-Tyr(Et)2, Mpa1 or Pen1 modifications and their various combinations. We also present properties of two previously reported re-synthesized analogues ([Gly(Bu t )8]OT and [Mpa1, Gly(Bu t )8]OT). The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OTR.
Keywords: Oxytocin analogues; l-α-t-Butylglycine; d-Cysteine; β-Mercaptopropionic acid; Penicillamin; Biological activity;

Synthesis and binding characteristics of a novel enkephalin analogue, [3H]Tyr-d-Ala-Gly-Phe-d-Nle-Arg-Phe by Fanni Tóth; Judit Farkas; Géza Tóth; Mária Wollemann; Anna Borsodi; Sándor Benyhe (1433-1440).
The endogenous opioid heptapeptide (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF) has been shown to interact with multiple opioid as well as non-opioid sites in mammalian brain membranes. To increase the stability and bioavailability of MERF, new synthetic derivatives with d-amino acid substitutions were prepared and studied. One of the new compounds in this series, Tyr-d-Ala-Gly-Phe-d-Nle-Arg-Phe (DADN), had only moderate affinity in competing with [ 3 H ]MERF, whereas it displayed the highest potency in producing antinociception following intrathecal administration. DADN was radiolabeled with 41 Ci/mmol specific activity. Specific binding of [ 3 H ]DADN was saturable, stereoselective and of high affinity. Chemical stability, increased μ-receptor selectivity, and hydrophobicity of the peptide all contribute to the effectiveness observed in biochemical and pharmacological studies.
Keywords: Opioid receptors; Met-enkephalin-Arg6-Phe7; Radiolabeling; Ligand binding; Rat brain;

Leptin fails to reduce ethanol intake in Marchigian Sardinian alcohol-preferring rats by Carlo Polidori; Fabio Luciani; Amalia Fedeli; Nori Geary; Maurizio Massi (1441-1444).
Leptin, a hormone secreted by the adipocytes and involved in feeding and energy balance control, has been proposed to modulate alcohol craving in mice and humans. This study evaluated whether leptin modulates alcohol intake in Marchigian Sardinian alcohol-preferring (msP) rats. Rats were offered 10% ethanol either 2 h per day at the beginning of dark period of the 12:12 h light/dark cycle, or 24 h per day. Leptin was injected into the lateral ventricle (LV), the third ventricle (3V), or intraperitoneally (IP) once a day, 1 h before the onset of the dark period. Neither acute nor chronic (9 days) leptin injections (1 or 8 μg per rat) into the LV or 3V modified ethanol intake in male msP rats, offered ethanol 2 h per day. Chronic LV injection of leptin (8 or 32 μg per rat in male rats and 8 or 16 μg per rat in female rats for 7 days), or chronic IP injections of leptin (1 mg/kg in male rats for 5 days) failed to modify the intake of ethanol, offered 24 h per day. Finally, chronic LV leptin injections (8 or 32 μg per rat for 12 days) did not modify ethanol intake in male msP rats, adapted to ad libitum access to ethanol and then tested after a 6-day period of ethanol deprivation. In contrast, in most of these conditions leptin significantly reduced food intake. These data do not support a role for leptin in alcohol intake, preference, or craving in msP rats.
Keywords: Leptin; Ethanol intake; Food intake; Alcohol-preferring rats;

In this investigation, substance P (SP) and neurokinin A (NKA) concentrations have been determined in the ovary of control prepubertal mice, and prepubertal mice injected with pregnant mare serum (PMS) gonadotropin , an equine gonadotropin with predominant FSH action, or with PMS followed by human chorionic gonadotropin (hCG), which produces heavily luteinized ovaries after the stimulation with PMS. Control animals were injected with saline. The ovaries of animals treated with gonadotropins were heavier than the control ovaries, the combination of PMS plus hCG produced significantly heavier ovaries than PMS alone. The concentrations of SP and NKA in the ovaries of the animals treated with PMS or PMS/hCG were significantly lower than in control ovaries. No significant differences in ovarian tachykinin concentrations were observed between PMS and PMS/hCG-treated animals. The total ovarian content of SP was lower in PMS-injected animals as compared with the controls. The total ovarian content of NKA was not significantly different in the three groups of animals studied. These results show that ovaries stimulated with gonadotropins have lower concentrations of tachykinins than normal ovaries at the same age. It is therefore evident that gonadotropins can affect tachykinin stores in the ovaries of mice.
Keywords: Tachykinins; NKA; Substance P; PMS; hCG; Ovary;

The PAR-1-activating peptide facilitates pepsinogen secretion in rats by Naoyuki Kawao; Kaori Hiramatsu; Naoki Inoi; Ryotaro Kuroda; Hiroyuki Nishikawa; Fumiko Sekiguchi; Atsufumi Kawabata (1449-1451).
Protease-activated receptor-2 (PAR-2) is abundantly expressed in gastric mucosal chief cells, facilitating pepsinogen secretion. In the present study, we investigated whether PAR-1, a thrombin receptor, could modulate pepsinogen secretion in rats. The PAR-1-activating peptide TFLLR-NH2 as well as the PAR-2-activating peptide SLIGRL-NH2, administered i.v. repeatedly at 1-h intervals, significantly increased gastric pepsinogen secretion over 2–4 h (after two to four doses). In contrast, the control peptide FTLLR-NH2, given in the same manner, had no such effect. Thus, PAR-1, like PAR-2, might function to facilitate pepsinogen secretion, suggesting a novel role of the thrombin-PAR-1-pathway in the stomach.
Keywords: Protease-activated receptor-1 (PAR-1); Pepsinogen secretion; Thrombin;

Erratum to “Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes” by Javier E. Garcı&#x0301;a; Alvaro Puentes; Ramsés López; Ricardo Vera; Jorge Suárez; Luis Rodrı&#x0301;guez; Hernando Curtidor; Marisol Ocampo; Diana Tovar; Martha Forero; Adriana Bermudez; Jimena Cortés; Mauricio Urquiza; Manuel E. Patarroyo (1453).