Peptides (v.24, #1)

Binding of cationic cell-permeable peptides to plastic and glass by Diane E. Chico; Randall L. Given; Brian T. Miller (3-9).
Cell-penetrating peptides derived from hydrophilic regions of the homeoprotein Antennapedia (Antp) or the transcription-regulating factor Tat have been used to transport several peptide and oligonucleotide cargoes into the interior of cells. Such vector peptides penetrate cells, in part, because they contain multiple lysine and arginine residues. Using radiolabeled peptide cargoes covalently linked to Antp- or Tat-related vectors, or to d-Arg heptamers, we found that a significant amount of the label remained tightly bound to plastic and glass surfaces. Binding of the labeled conjugates was due entirely to the cationic vector moieties. Under certain conditions, such non-specific binding could be mistaken for cellular penetration.
Keywords: Antennapedia; Tat; Poly-d-arginine; Vector peptides; Glass adsorption; Plastic adsorption; Radioiodination;

Two peptides, with a molecular mass of about 11 kDa and an N-terminal sequence abundant in arginine and glutamine residues, were isolated from wax gourd seeds. The isolation protocol included affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono-S and gel filtration on Superdex 75. The peptides, designated α- and β-benincasins, inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 20 and 320 pM, respectively. α-Benincasin exhibited weak antifungal activity toward Coprinus comatus and Physalospora piricola but not toward Mycosphaerella arachidicola.
Keywords: Arginine/glutamate-rich peptides; Seeds; Wax gourd; Purification;

Identification of a vasopressin-like immunoreactive substance in hydra by F Morishita; Y Nitagai; Y Furukawa; O Matsushima; T Takahashi; M Hatta; T Fujisawa; S Tunamoto; O Koizumi (17-26).
Vasopressin (VP)-like immunoreactivity has long been known in the hydra nervous system, but has not yet been structurally identified. In this study, using HPLC fractionation and an immunological assay, we have purified two peptides, FPQSFLPRGamide and SFLPRGamide, from Hydra magnipapillata. Both the peptides shared the same C-terminal structure, -PRGamide, with Arg-VP. The nonapeptide proved to be Hym-355, a peptide that stimulates neuronal differentiation in hydra. Detailed evaluation by competitive enzyme-linked immunosorbent assay (ELISA) and double immunostaining using anti-VP and anti-Hym-355 antibodies enabled us to conclude that the two peptides account for a major part of the VP-like immunoreactivity in hydra nerve cells.
Keywords: Hydra; Vasopressin; Oxytocin; Immunohistochemistry; ELISA; Cnidaria; Neuropeptide; Hormone; Neurotransmitter;

Isolation, identification and localization of a second beetle antidiuretic peptide by Richard A. Eigenheer; Ursula M. Wiehart; Susan W. Nicolson; Liliane Schoofs; Kathleen M. Schegg; J.Joe Hull; David A. Schooley (27-34).
We isolated from head extracts of Tenebrio molitor a peptide that inhibits fluid secretion by the Malpighian tubules of this insect. This second antidiuretic factor, ADFb, like the previously published ADFa, works through cyclic GMP as a second messenger. It has primary structure Tyr–Asp–Asp–Gly–Ser–Tyr–Lys–Pro–His–Ile–Tyr–Gly–Phe–OH with an EC50 of approximately 240 pM in a fluid secretion assay. This peptide is now the second sequenced endogenous insect ADF which inhibits Malpighian tubule fluid secretion. Immunohistochemical techniques show that the peptide is localized in the brain; it appears to be produced mainly in two pairs of bilaterally symmetrical cells in the protocerebrum.
Keywords: Water balance; Cyclic GMP; Fluid secretion; Antidiuretic factor; Tenebrio molitor; Malpighian tubules;

Isolation and characterization of novel tachykinins from the posterior salivary gland of the common octopus Octopus vulgaris by Atsuhiro Kanda; Eiko Iwakoshi-Ukena; Kyoko Takuwa-Kuroda; Hiroyuki Minakata (35-43).
Two novel tachykinins (OctTK-I: Lys–Pro–Pro–Ser–Ser–Ser–Glu–Phe–Ile–Gly–Leu–Met–NH2 and OctTK-II: Lys–Pro–Pro–Ser–Ser–Ser–Glu–Phe–Val–Gly–Leu–Met–NH2) were isolated from the posterior salivary gland of the octopus (Octopus vulgaris) using a contraction assay of the carp rectum. These peptides had in common the pentapeptide sequence –Phe–X-Gly–Leu–Met–NH2 at the C-terminal and induced immediate contractions on the carp rectum and the guinea-pig ileum. cDNAs encoding their precursor proteins were cloned. The OctTK gene was expressed in the posterior salivary gland and the expression was localized in mucus-secreting cells of the gland. The results suggested that OctTKs might be secreted as a venomous substance acting on vertebrates such as fishes, which are the prey or natural enemies of the octopus.
Keywords: Octopus tachykinins; cDNA cloning; RT-PCR and Southern blot analysis; In situ hybridization;

Deamidase inactivates a d-amino acid-containing Aplysia neuropeptide by F Morishita; O Matsushima; Y Furukawa; H Minakata (45-51).
Membrane-catalyzed degradation of the cardioexcitatory peptide, Asn-D-Trp-Phe-NH2 (N(d)WF-NH2), which was previously isolated from Aplysia, was investigated in relation to its inactivation mechanism. The principal degradation was deamidation of the C-terminal amide, producing biologically inert Asn-D-Trp-Phe-OH (N(d)WF-OH). Among membrane fractions prepared from different organs, the fraction from the ganglia showed the highest specific activity of the deamidation reaction. The deamidase activity was inhibited by Ebelactone B and the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), while the degradation of the synthetic stereoisomer, Asn-Trp-Phe-NH2 (N(l)WF-NH2), was sensitive to the divalent cation-chelating agent, o-phenanthroline, and aminopeptidase inhibitors, amastatin and bestatin. The presence of D-Trp residue in the second position of N(d)WF-NH2 endows this peptide not only with stereospecific bioactivity but also peptidase stability. The deamidation reaction seems to be the major inactivation mechanism for this peptide.
Keywords: d-Amino acid; Deamidase; Inactivation; Neuropeptide;

In this study, four somatostatin-like receptor (Sst) cDNAs were identified from goldfish pituitary, using RT-PCR screening and rapid amplification of cDNA ends (RACE) strategies. These include two type-five like Sst (Sst5B and Sst5C) and two type-three like Sst receptors (Sst3A and Sst3B), designated based on their amino acid sequence similarities to the known mammalian and fish Sst5 and Sst3. Both Sst5C and Sst3A mRNAs are widely expressed in all brain regions and pituitary; however, Sst3B expression is restricted to forebrain and Sst5B expression is mainly detected in pituitary and spinal cord.
Keywords: Somatostatin receptor; cDNA; Tissue distribution; Brain; Pituitary; Goldfish;

Evidence for the existence of a new receptor for CGRP, which is not CRLR by Madhu Chauhan; Chandra S. Thota; Sudhir Kondapaka; Sunil Wimalawansa; Chandra Yallampalli (65-71).
Receptors for calcitonin gene-related peptide (CGRP), a neuropeptide known to be the most potent vasodilator, are abundantly expressed in cerebellum. A monoclonal antibody to cerebellar CGRP receptors specifically detects a 66 kDa protein from rat cerebellum and other rat and human tissues, but not from SK-N-MC cells which express calcitonin receptor-like receptor (CRLR), a recently described component of CGRP receptors. In contrast, mRNA expression for CRLR was abundant in SK-N-MC cells, but it was undetectable in rat cerebellum. Furthermore, the antibody could not detect any immunoreactive protein in HEK 293 cells transiently transfected with CRLR and receptor activity-modifying protein 1 (RAMP1) indicating the possible existence of another CGRP receptor, which does not involve CRLR. Due to the absence of biochemical or structural data on the existence of a CGRP2 receptor and the new data provided in this paper, we suggest to identify the two CGRP receptors as CGRP-A and CGRP-B.
Keywords: CGRP; CGRP receptors; CRLR; RAMPs;

Structure–activity relationships of the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 at the mouse melanocortin receptors by Jerry Ryan Holder; Zhimin Xiang; Rayna M Bauzo; Carrie Haskell-Luevano (73-82).
The melanocortin pathway is involved in the regulation of several physiological functions including skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. This melanocortin pathway consists of five known G-protein coupled receptors, endogenous agonists derived from the proopiomelanocortin (POMC) gene transcript, the endogenous antagonists Agouti and the Agouti-related protein (AGRP) and signals through the intracellular cAMP signal transduction pathway. The melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) located in the brain are implicated as participating in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as α-melanocyte stimulation hormone (α-MSH). All the endogenous (POMC-derived) melanocortin agonists contain the putative message sequence “His-Phe-Arg-Trp.” Herein, we report 12 tetrapeptides, based upon the template Ac-His6-DPhe7-Arg8-Trp9-NH2 (α-MSH numbering) that have been modified at the Arg8 position by neutral, basic, or acidic amino acid side chains. These peptides have been pharmacologically characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. The most notable results of this study include the observation that removal of the guanidinyl side chain moiety results in decreased melanocortin receptor potency, but that this Arg8 side chain is not critical for melanocortin receptor agonist activity. Additionally, incorporation of the homoArg8 residue results in 56-fold MC4R versus MC3R selectivity, and the Orn8 residue results in 123-fold MC4R versus MC5R and 63-fold MC5R versus MC3R selectivity.
Keywords: Melanocortin; Melanotropin; POMC; α-MSH; MTII; SHU9119; Solid-phase peptide synthesis; G-protein coupled receptors;

Bombesin receptor subtype-3 modulates plasma insulin concentration by Kouji Matsumoto; Kazuyuki Yamada; Etsuko Wada; Takanori Hasegawa; Yoshihiro Usui; Keiji Wada (83-90).
Mice lacking a functional bombesin receptor subtype-3 (BRS-3) develop mild obesity. However, the origin of obesity in BRS-3 knockout (KO) mice remains unclear. We used a strain-crossing strategy to investigate the physiological role of the BRS-3 pathway. We crossed female heterozygous BRS-3 KO mice (X−/X) and male KK-Ay mice (Ay/+) to obtain BRS-3 KO/KK-Ay hybrid animals. In X−/Y:Ay/+ mice, plasma insulin concentrations were significantly higher, and on the oral glucose tolerance test, the additional secretion of insulin was impaired compared to other genotypes. Our results indicate that the BRS-3 pathway contributes to the regulation of plasma insulin concentrations.
Keywords: Bombesin receptor subtype-3; Insulin; Obesity; Diabetes; Knockout mouse; KK-Ay; Orphan G-protein coupled receptor;

Endogenous amylin contributes to the anorectic effects of cholecystokinin and bombesin by A Mollet; S Meier; V Grabler; S Gilg; E Scharrer; T.A Lutz (91-98).
Previous studies indicated that amylin contributes to the anorectic effects of cholecystokinin (CCK) and bombesin (BBS), possibly by enhancing the release of pancreatic amylin or by modulating their anorectic actions within the central nervous system (CNS).To elucidate the interaction between amylin and CCK or BBS, respectively, we investigated the influence of an IP injection of CCK or BBS on feeding in amylin-deficient mice (IAPP−/−). The anorectic effects of CCK and BBS were nearly abolished in IAPP−/− mice compared to wildtype (WT) mice (e.g. 20 μg/kg CCK, 1-h food intake: WT/NaCl 0.53±0.03 g; WT/CCK 0.16±0.03 g (P<0.001); IAPP−/−/NaCl 0.49±0.05 g; IAPP−/−/CCK 0.39±0.04 g). Acute amylin replacement restored the anorectic effect of CCK in IAPP−/− mice.To find out whether CCK or BBS enhance the feeding-induced release of pancreatic amylin, we injected rats with CCK-8 (0.5–50 μg/kg) or BBS (5 μg/kg) and measured plasma amylin levels after injections. Neither CCK nor BBS increased the plasma amylin level in rats. We suggest that the mediation of the anorectic effects of CCK and BBS by amylin is not dependent on a CCK- or BBS-induced release of pancreatic amylin, but may rather be due to a modulation of their effects by amylin within the CNS.
Keywords: Amylin; CCK; BBS; Food intake; Interaction; Plasma amylin level; Amylin-deficient mice;

Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice by Edward W Lee; Derrick S Grant; Sharareh Movafagh; Zofia Zukowska (99-106).
Which of Y1–Y5 receptors (Rs) mediate NPY’s angiogenic activity was studied using Y2R-null mice and R-specific antagonists. In Y2R-null mice, NPY-induced aortic sprouting and in vivo Matrigel capillary formation were decreased by 50%; Y1R-antagonist blocked the remaining response. NPY-induced sprouting was equally inhibited by Y2R- (and Y5R- but less by Y1R-) antagonists in wild type mice. Spontaneous and NPY-induced revascularization of ischemic gastrocnemius muscles were similarly reduced in Y2R-null mice. Thus, NPY-induced angiogenesis, spontaneous and ischemic, is primarily mediated by Y2Rs. However, Y5Rs and, to a lesser degree Y1Rs, also may play a role in NPY-mediated angiogenesis.
Keywords: Neuropeptide Y; Angiogenesis; Y2KO mice; Y2 receptor;

Synthesis and angiogenetic activity in the chick chorioallantoic membrane model of thymosin beta-15 by Vassiliki Koutrafouri; Leondios Leondiadis; Nikolas Ferderigos; Konstantinos Avgoustakis; Evangelia Livaniou; Gregory P. Evangelatos; Dionyssis S. Ithakissios (107-115).
Thymosin beta-15 (Tβ15), a 44 amino acid peptide (MW=5173) localized in human prostate and breast cancer tissues was successfully synthesized in multigram quantities using Fmoc solid-phase peptide synthesis. The synthesized product was shown to have the right structure by ESI and MALDI mass spectral techniques and amino acid analysis. Relatively high yield was achieved, which might be due to enhanced acid stability of the p-cyanotrityl resin used. The effect of the synthesized Tβ15 on the angiogenesis process was investigated using the chick chorioallantoic membrane (CAM) in vivo model. At concentrations above 1 μg/10 μl per disc, Tβ15 exhibited a positive effect on angiogenesis, comparable to the effect of the intense angiogenetic factor phorbol 12-myristate 13-acetate at a standard concentration of 0.1 μg/10 μl per disc. The results of this study contribute to the further elucidation of the biological regulatory role of thymosin peptides and provide helpful information in the investigation of their possible therapeutic potential.
Keywords: Thymosin beta-15; Fmoc solid phase peptide synthesis; p-Cyanotrityl resin; Angiogenesis; CAM;

Effects of adrenomedullin on the contraction of gastric arteries during reserpine-induced gastric ulcer by Salvatore Salomone; Antonina Caruso; Vincenza Maria Cutuli; Nunzio Guido Mangano; Agata Prato; Matilde Amico-Roxas; Alfredo Bianchi; Giuseppe Clementi (117-122).
Adrenomedullin (100 ng/kg, s.c.) prevents reserpine-induced damage of gastric mucosa. In the model of in vitro gastric arteries from reserpine-treated rats, adrenomedullin pre-treatment resulted in a decrease of the vasoconstriction in response to 5-hydroxytryptamine. In contrast, adrenomedullin pre-treatment of rat with intact gastric mucosa did not affect the vasoconstriction to 5-hydroxytryptamine. In the presence of the NOS inhibitor N G-nitro-l-arginine, the responsiveness to 5-hydroxytryptamine in gastric arteries from rats treated with reserpine+adrenomedullin was enhanced to the same level of rats treated with reserpine alone. The anti-ulcer effect of adrenomedullin could therefore be related, at least in part, to an increase of blood flow at the gastric mucosa, by a mechanism involving nitric oxide.
Keywords: Adrenomedullin; Gastric ulcer; Gastric artery; Nitric oxide; 5-Hydroxytryptamine;

Octreotide improves burn-induced intestinal injury in the rat by Göksel Şener; A.Özer Şehirli; Handan Şatiroǧlu; Ayhan Kaçmaz; Gül Ayanoǧlu-Dülger; Berrak Ç. Yeǧen (123-127).
The local thermal trauma activates a number of systemic mediator cascades, e.g. a complement activation, cytokine production, resulting in a generalized sequestration and a priming of local and systemic neutrophils and macrophages. We aimed to determine the possible protective effect of octreotide (OCT), a synthetic somatostatin analogue, against burn-induced intestinal tissue damage possibly by inhibiting neutrophil infiltration.Under brief ether anaesthesia, shaved dorsum of the rats was exposed to 90 °C bath for 10 s to induce burn injury. Rats were decapitated either 3, 24 or 72 h after burn injury. Octreotide (10 μg/kg) or saline was administered subcutaneously (s.c.) immediately after the burn injury. In the 24- and 72-h burn groups, OCT injections were repeated three times daily. In the sham group the same protocol was applied except that the dorsum was dipped in a 25 °C water bath for 10 s Malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) activity were determined in the intestinal tissue. The results demonstrate that burn injury results in significant neutrophil accumulation, as evidenced by increases in MPO activity. The increase in MDA and the concomitant decrease in GSH levels demonstrate the role of oxidative mechanisms in burn injury. OCT may have some beneficial therapeutic effects by reducing neutrophil-dependent injury and related lipid peroxidation following burn trauma.
Keywords: Burn; Octreotide; Myeloperoxidase; Lipid peroxidation; Glutathione;

The inter-relationship between central vasopressin and the pineal gland in the modulation of active avoidance behavior was investigated. In sham-operated (SO) rats, intracerebroventricular (i.c.v) application of 10 ng arginine vasopressin (AVP) after both the last acquisition and the first extinction trials prolonged the extinction of the active avoidance response; application of 50 ng of the V1 antagonist, d(CH2)5Tyr(Me)AVP (AAVP) was without effect in both experiments. In contrast to the SO in pinealectomized (PX) rats neither AVP nor AAVP influenced the extinction of the avoidance response. Intraseptal infusion of 200 pg AVP or 5 ng AAVP either after the last acquisition or the first extinction trial was without effect in both SO and PX rats. Comparison of the acquisition trials revealed no differences between SO and PX rats.
Keywords: Active avoidance; Learning; Vasopressin; Septum; Pineal gland; Behavior; Rat;

Substance P(1–7) affects the expression of dopamine D2 receptor mRNA in male rat brain during morphine withdrawal by Qin Zhou; Per-Anders Frändberg; Anna M.S. Kindlundh; Pierre Le Grevès; Fred Nyberg (147-153).
Previous studies have confirmed an important role of the undecapeptide substance P (SP) in opioid reward and dependence. It is further shown that the SP N-terminal metabolite SP1–7 may attenuate the intensity of opioid withdrawal in mice. In this study we have investigated the effect of the heptapeptide fragment on the expression of the brain dopamine D2 receptor mRNA and on the withdrawal reaction, as well, in morphine-dependent rats. Male Sprague–Dawley rats were randomly distributed into two groups. Guide cannula was implanted and aimed at the lateral ventricle and animals were subsequently made opioid dependent by two daily injections of morphine (10 mg/kg) for 7 days. Half an hour before naloxone challenge (2 mg/kg) one group of rats received an injection of SP1–7 (28 nmol per rat) and the other, serving as control, was injected with saline through the cannula. Animals were decapitated 4 h following SP1–7 or saline injections. The results indicated that the level of the dopamine D2 receptor transcript was significantly reduced by SP1–7 in nucleus accumbens and frontal cortex but not altered in the striatum. In behavioral tests it was found that the heptapeptide attenuated several somatic withdrawal symptoms. The observed reduction in the receptor transcript in nucleus accumbens and frontal cortex is suggested to reflect an increased dopamine activity in these areas, which in turn may counteract the withdrawal reaction.
Keywords: Substance P; SP1–7; Morphine withdrawal; Dopamine; D2 receptor mRNA; Nucleus accumbens; Frontal cortex;

In the present study we investigated the role of hypothalamic histamine H1 receptors in the mediation of peripheral amylin’s anorectic effect. Rats with chronically implanted bilateral cannulas were infused into the ventromedial hypothalamus (VMH) with the specific histamine H1 receptor antagonists pyrilamine (PYR, 104 nmol/rat) or chlorpheniramine (CPA, 52 nmol/rat), respectively, combined with an intraperitoneal (IP) injection of amylin (5 μg/kg). Amylin’s inhibitory effect on food intake (i.e. 50% reduction in cumulative food intake 30 min after ingestion) was markedly reduced by CPA and PYR (e.g. amylin and CPA: 5% reduction versus control). We therefore suggest an important role of hypothalamic H1 receptors in the signal transduction of peripheral amylin’s anorectic action.
Keywords: Food intake; Amylin; Histamine H1 receptors; VMH; Chlorpheniramine; Pyrilamine;

Given a nascent protein sequence, how can one predict its signal peptide or “Zipcode” sequence? This is an important problem for scientists to use signal peptides as a vehicle to find new drugs or to reprogram cells for gene therapy (see, e.g. K.C. Chou, Current Protein and Peptide Science 2002;3:615–22). In this paper, support vector machines (SVMs), a new machine learning method, is applied to approach this problem. The overall rate of correct prediction for 1939 secretary proteins and 1440 nonsecretary proteins was over 91%. It has not escaped our attention that the new method may also serve as a useful tool for further investigating many unclear details regarding the molecular mechanism of the ZIP code protein-sorting system in cells.
Keywords: Support vector machine; Protein signal sequence; Jackknife test; Bench-mark window;

VIP as a trophic factor in the CNS and cancer cells by Terry W. Moody; Joanna M. Hill; Robert T. Jensen (163-177).
The effects of vasoactive intestinal peptide (VIP) on the proliferation of central nervous system (CNS) and cancer cells were investigated. VIP has important actions during CNS development. During neurogenesis, VIP stimulates the proliferation and differentiation of brain neurons. Addition of VIP to embryonic mouse spinal cord cultures increases neuronal survival and activity dependent neurotrophic factor (ADNF) secretion from astroglial cells. VIP is an integrative regulator of brain growth and development during neurogenesis and embryogenesis. Also, VIP causes increased proliferation of human breast and lung cancer cells in vitro. VIP binds with high affinity to cancer cells, elevates the cAMP and increases gene expression of c-fos, c-jun, c-myc and vascular endothelial cell growth factor. The effects of VIP on cancer cells are reversed by VIPhybrid, a synthetic VPAC1 receptor antagonist. VIPhyb inhibits the basal growth of lung cancer cells in vitro and tumors in vivo and potentiates the ability of chemotherapeutic drugs to kill cancer cells. Due to the high density of VPAC1 receptors in cancer cells, VIP has been radiolabeled with 123 I , 18 F and 99m Tc to image tumors. It remains to be determined if radiolabeled VIP analogs will be useful agents for early detection of cancer in patients.
Keywords: VIP; CNS; Cancer; Development; Proliferation;