Peptides (v.23, #1)

A tridecapeptide possesses both antimicrobial and protease-inhibitory activities by Qingshun Li; Christopher B Lawrence; H Maelor Davies; Nicholas P Everett (1-6).
A 13-residue synthetic peptide (Rev4) was designed based on indolicidin, an antimicrobial peptide from bovine neutrophils. The synthetic peptide retains high antimicrobial activity. When tested for its stability in tobacco leaf extracts, Rev4 was highly stable compared to another antimicrobial peptide, magainin. When mixed with Rev4, magainin was protected from degradation by the leaf extract. Our results show that Rev4 is a potent protease inhibitor which selectively inhibits three out of four different types of proteases. Four other synthetic peptides were tested and the results were suggestive of no correlation between their antimicrobial and protease inhibitory activities.
Keywords: antimicrobial peptide; Protease inhibitor; Indolicidin; Magainin; Peptide stability;

A single-chained antifungal protein with a molecular weight of 6.5 kDa and displaying a novel N-terminal sequence was isolated from dried juvenile cicadas which are used in traditional Chinese medicine, by using ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and then gel filtration on a Superdex peptide column. The peptide, designated cicadin, exerted potent antifungal activity with IC50 values at nonomolar concentrations against a variety of fungi including Botrytis cinerea, Mycosphaerella arachidicola, Fusarium oxysporum, Rhizoctonia solani and Coprinus comatus. Cicadin suppressed the activity of HIV-1 reverse transcriptase and stimulated the proliferation of murine splenocytes.
Keywords: Antifungal peptide; Dried juvenile cicadas;

Plasmodium vivax Duffy binding protein peptides specifically bind to reticulocytes by Marisol Ocampo; Ricardo Vera; Luis Eduardo Rodriguez; Hernando Curtidor; Mauricio Urquiza; Jorge Suarez; Javier Garcia; Alvaro Puentes; Ramsés Lopez; Mary Trujillo; Elizabeth Torres; Manuel Elkin Patarroyo (13-22).
Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition.Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50–130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.
Keywords: Plasmodium vivax; Duffy binding protein; HARBP (High Activity Reticulocyte Binding Peptide); HAEBP (High Activity Erythrocyte Binding Peptide);

Dendroaspis natriuretic peptide (DNP) is a recently isolated 38 amino acid peptide that shares structural and functional properties with the other members of the natriuretic peptide family. The present study demonstrates the presence of DNP-like immunoreactivity in sections of rat aorta, carotid artery and renal vasculature and tubules. DNP-like immunoreactivity was detected in culture aortic vascular smooth muscle cells and medium and is regulated by endothelin-1, angiotensin II and sodium nitroprusside but not by transforming growth factor-β. Our observations indicate that DNP elicits a marked inhibitory effect on DNA synthesis in culture rat aortic vascular smooth muscle cells.
Keywords: CNP; DNP; Vascular smooth muscle;

The structure of a well-known neurohormone involved in homeostasis regulation and stress response, the crustacean hyperglycemic hormone, was investigated in the deep-sea hydrothermal vent crab Bythograea thermydron. The neuropeptide was isolated from neurohemal organs (sinus glands) and its biological activity checked using an homologous bioassay. Partial amino acid sequence was established by a combination of Edman chemistry and mass spectrometry. Then, the sequence of the cDNA encoding the hormone precursor was determined. The preprohormone is composed of a 29 amino acid signal peptide, followed by a 41 amino acid associated peptide flanking the 72 amino acid hyperglycemic hormone. Comparison of these data with other known crab hyperglycemic hormone and prohormone sequences was performed using phylogenetic analysis methods.
Keywords: Crustacean hyperglycemic hormone; Hydrothermal vents; MALDI-TOF mass spectrometry; X-organ-sinus gland complex; Endoproteinase Lys-C (EC 3.4.21.50);

Characterization of a growth hormone-releasing hormone binding site in the rat renal medulla by Luce Boulanger; Nathalie Girard; Julie Strecko; Pierrette Gaudreau (43-50).
Receptor binding analysis was performed in the renal medulla from 2-month-old rats, an extrapituitary tissue containing the highest level of GHRH receptor mRNA. At 4°C, in the presence of a cocktail of protease inhibitors, binding of [125I-Tyr10]hGHRH NH2 to medullary homogenates was specific, time-dependent, reversible and saturable (Kd: 28 nM; Bmax: 30 fmol/mg prot.). In these experimental conditions, no change of binding parameters could be detected in the course of aging. The structure-affinity profile was different in the two tissues and chemical cross-linking revealed the presence of 65-, 55- and 38-kDa 125I-GHRH-labeled complexes in the renal medulla compared to 65-, 47- and 28-kDa radioactive complexes in the anterior pituitary. It is suggested that GHRH binding sites, and possibly the receptor, may be different in the two tissues.
Keywords: Growth hormone-releasing hormone; 125I-GHRH; Binding site; Structure-affinity; Kidney; Aging;

Effect of angiotensin-(1–7) on jejunal absorption of water in rats by E.L Borges; B.M Cabral; A.A Braga; M.J Neves; R.A.S Santos; E Rogana (51-56).
The effect of angiotensin-(1–7) on jejunal water absorption in rats was investigated. The jejunal sac of anesthetized rats was filled with two ml of tyrode solution containing 3.7 MBq of tritiated water. A femoral vein was cannulated for administration of peptides and drugs. Infusion of Ang-(1–7) at the dose of 0.7 ng/kg.min produced a significant increase in jejunal water absorption compared to control (32% increase). The Ang-(1–7) antagonist A-779 abolished the effect of Ang-(1–7) on water absorption. A reduction of the Ang-(1–7) effect was also produced by treatment with the AT1 receptor antagonist, losartan or the AT2 receptor antagonist, PD123.177. The increase in jejunal water absorption produced by Ang-(1–7) was blocked by the nitric oxide synthase inhibitor, L-NAME and by indomethacin. These data suggest that the effect of Ang-(1–7) on the jejunal loop is mediated by activation of a multiple angiotensin receptors and/or by an atypical angiotensin receptor. Furthermore, the effect of Ang-(1–7) on jejunal water absorption is mediated by nitric oxide and by a cyclooxygenase-dependent mechanism.
Keywords: Angiotensin-(1–7); A-779; L-NAME (NG -nitro-L-arginine methyl ester); Losartan; Tritiated water;

1A-779 attenuates angiotensin-(1–7) depressor response in salt-induced hypertensive rats by Mohamed.A Bayorh; Danita Eatman; Marcus Walton; Robin R Socci; Myrtle Thierry-Palmer; Nerimiah Emmett (57-64).
Chronic infusion of angiotensin-(1–7) [Ang-(1–7)] lowers blood pressure in salt-induced and spontaneously hypertensive (SHR) rats. In the present study, we have examined the acute effect of Ang-(1–7) in salt-induced hypertension using Dahl salt-sensitive rats placed on low (0.3%) or high (8.0% NaCl) salt diets for 2 weeks. Rats fed a high salt diet showed a greater rise in BP than those fed a low salt diet. Ang-(1–7) (24 μg/kg) reduced mean arterial pressure (MAP), enhanced the release of prostacyclin and nitric oxide, and suppressed thromboxane A2 levels. A-779 (48 μg/kg, i.v), a selective Ang-(1–7) antagonist, partially blocked these effects of Ang-(1–7). The Ang-(1–7)-induced depressor response observed in these animals was related to an increase in vasodilatory prostanoids, a decrease in the constrictor prostanoid thromboxane A2, and an increase in nitric oxide levels in both plasma and isolated aortic rings.
Keywords: Angiotensin-(1–7); Dahl rats; A779; Blood pressure; Nitric Oxide; Prostaglandins;

Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs by C.R Nakaie; E.G Silva; E.M Cilli; R Marchetto; S Schreier; T.B Paiva; A.C.M Paiva (65-70).
Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50°C, l h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC1-AngII and TOAC0-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes.
Keywords: Angiotensin analogs; Bradykinin analogs; Peptide synthesis; Spin labeled peptides; TOAC spin label; Electron paramagnetic resonance;

Neuropeptide Y-induced angiogenesis in aging by Joanna Kitlinska; Edward W Lee; Sharareh Movafagh; Jennifer Pons; Zofia Zukowska (71-77).
Age-related changes in NPY-driven angiogenesis were investigated using Matrigel and aortic sprouting assays in young (2 months.) and aged (18 months.) mice. In both assays, NPY-induced vessel growth decreased significantly with age. In parallel, aged mice showed reduced expression (RT-PCR) of Y2 receptors and the NPY converting enzyme, dipeptidyl peptidase IV (DPPIV), in spleens. Aging of human microvascular endothelial cells in vitro led to a loss of their mitogenic responses to NPY accompanied by a lack of NPY receptor mRNAs. Thus, NPY-dependent angiogenesis is impaired with age, which is associated with a decreased expression of endothelial NPY receptors (Y2) and DPPIV.
Keywords: Neuropeptide Y; Angiogenesis; Aging;

Parathyroid hormone effects on signaling pathways in endothelial cells vary with peptide concentration by Doug Throckmorton; Doris Kurscheid-Reich; Oscar R Rosales; Jose Rodriguez-Commes; Raquel Lopez; Bauer Sumpio; Qing Zhong; Ke-Hong Ding; Richard McCarthy; Paula Q Barrett; Carlos M Isales (79-85).
We have previously reported that parathyroid hormone (PTH) has specific effects on a human umbilical vein endothelial cell line. Further studies were performed to characterize the signaling cascades initiated by PTH. We report that PTH induced the appearance of voltage sensitive calcium channels. Furthermore, PTH increased ceramide but not diacylglycerol content. Since elevations in [Ca2+]i and phospholipid turnover are signals for the activation of protein kinase C (PKC), the cells were screened for PKC isoforms. PTH induced a redistribution of the PKCϵ to the particulate fractions of cell homogenates. In summary, PTH induced PKC translocation through a calcium-phospholipid pathway in an endothelial cell line.
Keywords: PTH; Protein kinase C; Calcium channels; Ceramide; Diacylglycerol;

The role of Ca2+/cAMP-dependent signal transduction and transcription factor CREB in mediating NPY- Y1 receptor function was investigated in SK-N-MC cells. The Y1 receptor agonist, [Leu31,Pro34]-NPY, inhibited forskolin-stimulated cAMP production which was insensitive to thapsigargin or the CaM kinase II inhibitor, KN-93. Although activation of the Y1 receptor leads to an increase in CREB phosphorylation, [Leu31,Pro34]-NPY inhibited CREB phosphorylation in KN-93-treated cells. SK-N-MC cells were also transfected with PathDetectR cis-CRE and trans-CREB/trans-cFos reporter genes to monitor the role of Ca2+/cAMP signals, triggered by Y1 receptor, on reporter gene activity. Treatment of the cis-CRE-luciferase expression vector-transfected cells with [Leu31,Pro34]-NPY increased reporter gene activity by ∼2 fold through a KN-93 sensitive pathway. In contrast, the peptide inhibited forskolin-stimulated luciferase activity. Consistently, [Leu31,Pro34]-NPY induced trans-CREB mediated luciferase activity through a CaM kinase dependent pathway, and inhibited forskolin-stimulated luciferase gene expression. However, no effect of the peptide was observed on trans-cFos- mediated luciferase activity. These findings suggest that the NPY Y1 receptor induces the expression of CRE containing target genes through the CaM kinase-CREB pathway, and inhibits CRE containing genes when cellular cAMP levels are elevated.
Keywords: Y1 receptor; cAMP; CaM kinase II; Phosphorylation; Transcription factor; Reporter gene;

Fragments of the B-loop of the epidermal growth factor family of peptides are reported to have mitogenic and angiogenic properties but appear to fail to compete with radioiodinated EGF in receptor binding. In this study, 11 analogs of a fragment of the B-loop of EGF-related peptides from several species were synthesized to study binding to A431 human epidermoid carcinoma using both 125I-EGF and [3′4′-3H-Tyr22,29, Abu20,31]EGF(20–31)-NH2. Specific binding sites were found for the human fragment and 8 analogs at a density five times higher than that of the EGF receptors. Analogs did not compete with 125I-EGF for binding to the EGF receptor. The novel binding site may mediate the biological effects of the fragments. The primary rather than secondary structure of the fragments appears to determine affinity.
Keywords: B-loop fragment; Epidermal growth factor; Specific binding; Transforming growth factor alpha;

We previously reported that a synthetic peptide induced humoral autoimmunity to thyrotropin receptor (TSH-R) and the expansion of antigen-specific type 2 helper T (Th2) cell population in mice . The peptide corresponds to the human TSH-R C-terminal region. In the present study, we undertook a similar approach in patients with Graves’ disease (GD). Peripheral lymphocytes from 5 healthy controls and 11 GD patients were prepared and analyzed for cytokines from helper T cells by an antigen specific enzyme-linked immuno spot (ELISPOT) assay. In GD patients, the total number of IL-4 producing cells increased significantly and the number of interferon-γ(IFN-γ) producing cells decreased. Further, co-incubation with several of the 20 kinds of TSH-R extracellular peptides increased the number of IL-4 producing cells in patients with GD. Such stimulatory peptides appear frequently in a TSH-R sequence. These peptides did not affect the numbers of IFN-γ producing cells significantly. These results indicated that GD patients have an expanded Th2 population responding to TSH-R and the dominance of the humoral immune system in such patients.
Keywords: Autoimmunity; Thyrotropin receptor; Helper T cell; Cytokines; ELISPOT; Graves’ disease;

Expression and distribution of calcitonin receptor-like receptor in human hairy skin by S Hagner; R.V Haberberger; D Overkamp; R Hoffmann; K.H Voigt; G.P McGregor (109-116).
Calcitonin gene-related peptide and adrenomedullin exert potent effects in skin but their cellular targets are unknown. This study aimed to identify the cellular location of calcitonin receptor-like receptor (CRLR) which is pharmacolocically identical to CGRP receptor-1, a putative molecular target of CGRP and adrenomedullin. RT-PCR analysis of human hairy skin revealed the presence of CRLR mRNA and immunohistochemical analysis, employing a previously characterized polyclonal antibody raised to CRLR, provided novel evidence of the cellular distribution of CRLR. Extensive and specific CRLR-immunostaining was detected in arteriolar smooth muscle and venular endothelium and is consistent with CGRP’s putative role in neurogenic inflammation. Novel targets for CGRP and/or adrenomedullin were identified, including capillary endothelium, hair follicles and sweat glands.
Keywords: Calcitonin receptor-like receptor; Calcitonin gene-related peptide; Adrenomedullin; Human; Skin; Immunohistochemistry; Vasculature; Capillaries;

Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats by Roberto Ciccocioppo; Carlo Polidori; Lorena Antonelli; Severo Salvadori; Remo Guerrini; Maurizio Massi (117-125).
Chronic intracerebroventricular (ICV) treatment with nociceptin/orphanin FQ (NC), the endogenous ligand for the opioid receptor-like 1 (ORL1) receptor, reduces ethanol intake in alcohol-preferring rats and abolishes the rewarding properties of ethanol in the place conditioning paradigm. To pharmacologically characterize the receptor involved, the present study evaluated the effect on ethanol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats of ICV injections for 8 days of NC or of the NC analogs NC(1–17)NH2, NC(1–13)NH2, NC(1–12)NH2 and [Nphe1]NC(1–13)NH2. In vitro studies indicate that NC, NC(1–17)NH2, NC(1–13)NH2 and NC(1–12)NH2 are agonists, while [Nphe1]NC(1–13)NH2 is a selective antagonist at the ORL1 receptor. Freely feeding and drinking rats were offered 10% ethanol 30 min/day at the beginning of the dark phase of the light cycle. NC significantly attenuated ethanol intake at 500 or 1000 ng/rat (210 or 420 pmol/rat). NC(1–17)NH2, markedly reduced ethanol intake, but its effect was statistically significant at 1000 (420 pmol/rat), not at 500 ng/rat (210 pmol/rat). After the end of treatment ethanol drinking promptly came back to baseline level. Ethanol consumption was also reduced by NC(1–13)NH2; however, its effect was less potent and pronounced. NC(1–12)NH2 did not modify ethanol intake at doses up to 4000 ng/rat (2339 pmol/rat). Water and food consumption were not modified. Treatment with [Nphe1]NC(1–13)NH2, 66 or 99 μg/rat, did not modify ethanol intake; however, [Nphe1]NC(1–13)NH2, 66 μg/rat, given just before 1000 ng/rat of NC(1–17)NH2, abolished the effect of the agonist. The present results show that the 13 aminoacid N-terminal sequence of NC is essential for the effect on ethanol intake and indicate that [Nphe1]NC(1–13)NH2 acts as an antagonist to block the effect of NC. These findings provide further evidence that selective agonists at the ORL-1 receptor attenuate ethanol intake in alcohol-preferring rats and suggest that the NC/ORL1 system may represent an interesting target for treatment of alcohol abuse.
Keywords: Nociceptin; Orphanin FQ; ORL1 receptors; Ethanol intake;

Albutensin A and complement C3a decrease food intake in mice by Kousaku Ohinata; Akio Inui; Akihiro Asakawa; Keiji Wada; Etsuko Wada; Masaaki Yoshikawa (127-133).
Albutensin A (Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg) derived from serum albumin dose-dependently decreased food intake after intracerebroventricular (10–50 nmol/mouse) or peripheral (0.3–1.0 μmol/mouse) administration in fasted conscious ddY mice. Albutensin A delayed gastric emptying and elevated blood glucose levels. Although albutensin A showed low affinity for bombesin receptor, it decreased food intake in bombesin receptor knockout mice, indicating that its inhibitory effect on feeding was not mediated through bombesin receptor. Then, we investigated whether the albutensin A-induced decrease in food intake was mediated by complement C3a and C5a receptors, because albutensin A had affinities for these receptors. Des-Arg-albutensin A, lacking affinity for C3a and C5a receptors, did not inhibit food intake. We found for the first time that centrally administered C3a (10–100 pmol/mouse) by itself decreased food intake in fasted mice. In contrast, C5a increased food intake after central injection. Based on these results, we conclude that the inhibitory effect of albutensin A on food intake is mediated through the C3a receptor.
Keywords: Albutensin A; Food intake; Blood glucose; Gastric emptying; Complement C3a; Bombesin;

Glucose-insensitivity induced by Ca2+ toxicity in islet β-cells and its prevention by PACAP by Kazuhiro Yanagida; Kazuro Yaekura; Terukatsu Arima; Toshihiko Yada (135-142).
The present study examined whether a sustained increase in cytosolic Ca2+ concentration ([Ca2+]i) causes glucose-insensitivity in β-cells and whether it could be modulated by pituitary adenylate cyclase-activating polypeptide (PACAP), a pancreatic insulinotropin. Rat single β-cells were cultured for 2 days with sustained increases in [Ca2+]i, followed by determination of the [Ca2+]i response to glucose (8.3 mM) as monitored with fura-2. High K+ (25 mM) produced sustained increases in [Ca2+]i in β-cells, which were inhibited by nifedipine, a Ca2+ channel blocker. After culture with high K+, the incidence and amplitude of [Ca2+]i responses to glucose were markedly reduced. This glucose-insensitivity was prevented by the presence of nifedipine or PACAP-38 (10−13 M and 10−9 M) in high K+ culture. PACAP-38 attenuated high K+-induced [Ca2+]i increases. In conclusion, sustained increases in [Ca2+]i induce glucose-insensitivity (Ca2+ toxicity in β-cells) and it is prevented by PACAP possibly in part due to its Ca2+-reducing capacity.
Keywords: Rat pancreas; Islet β-cells; High K+; Cytosolic Ca2+ increase; Glucose-insensitivity; Ca2+ toxicity; PACAP; Insulinotropin; Diabetes; Cytoprotection;

Effects of α-MSH on kainic acid induced changes in core temperature in rats 2 2 In memory of the late Professor Sven Ahlenius. by M. Oprica; Å. Forslin Aronsson; C. Post; C. Eriksson; S. Ahlenius; L.M. Popescu; M. Schultzberg (143-149).
The effects of intraperitoneal (i.p.) administration of kainic acid (KA) and α-melanocyte-stimulating hormone (α-MSH) alone or in combination, on core temperature of freely moving rats were examined. KA or saline was administered once (10 mg/kg) and α-MSH or saline was given repeatedly i.e. 10 min before and 10, 30 and 60 min after the administration of saline or KA. Two doses of α-MSH were used: 0.5 and 2.5 mg/kg. KA alone produced a biphasic effect on core temperature, i.e. an initial short-lasting hypothermia followed by hyperthermia that lasted about 6 h. The higher dose of α-MSH had a potentiating effect on KA-induced hypothermia, while the lower dose of α-MSH increased the hyperthermia produced by KA. α-MSH administered alone produced a late (3 h), dose-dependent increase in core temperature. It is conceivable that repeated administration of α-MSH in the doses used in our study may cause a cumulative effect in raising body temperature for a limited period of time.The previously described interactions between KA and α-MSH, respectively, with dopaminergic and serotoninergic systems may account for the effects on core temperature in rats observed in our study.
Keywords: excitotoxic amino acid; hyperthermia; hypothermia; inflammation; melanocortin receptors;

Melanin concentrating hormone increase hippocampal synaptic transmission in the rat by Mariana Varas; Mariela Pérez; Oscar Ramı́rez; Susana Rubiales de Barioglio (151-155).
A retrograde facilitation has been demonstrated in the one trial step-down inhibitory avoidance of melanin-concentrating hormone (MCH), when it was infused into rat hippocampal formation. Considering the high density of specific binding sites for the MCH peptide on the hippocampus and the participation of this structure on learning and memory processes we have studied the effects of MCH on the hippocampal synaptic transmission. For this purpose, slices of rat hippocampus were perfused with different concentration of MCH. The main result of the present study was a long-lasting potentiation on the hippocampal evoked response on dentate gyrus induced by MCH (4–11 μM) at 30, 60 and 120 min with a maximum effect at 120 min. Previous perfusion of DL - 2- amino - 5 phosphonovaleric acid (APV, 20 μM) was unable to impair the increased hippocampal evoked response induced by MCH 4 μM. On the other hand, the channel blocker Dizocilpine (MK-801, 10 μM) completely impaired the increased hippocampal synaptic plasticity induced by MCH perfusion. We postulate the increased hippocampal synaptic efficacy induced by MCH as one of the mechanisms underlying the retrograde facilitation on the inhibitory avoidance paradigm, observed after MCH hippocampal microinjection. We cannot rule out other MCH neurochemical mechanism and other areas of the brain involved in the MCH effects.

Investigation of substance P transport across the blood-brain barrier by Anita L Freed; Kenneth L Audus; Susan M Lunte (157-165).
The transport of substance P (SP) was investigated using the bovine brain microvessel endothelial cell culture model of the blood-brain barrier (BBB). The samples were derivatized precolumn with naphthalene dialdehyde, then analyzed by cyclodextrin-modified micellar electrokinetic chromatography with laser-induced fluorescence detection. SP crossed the BBB in both the apical-to-basolateral and basolateral-to-apical directions through an active transport mechanism. The transport of SP from the apical side was demonstrated to be via transcytosis. The N-terminal (SP1–4) and C-terminal (SP3–11) fragments were also found to permeate the BBB from the apical side.
Keywords: Substance P; Transport; Blood-brain barrier; Brain microvessel endothelial cells; Capillary electrophoresis; Laser induced fluorescence detection;

Sensory nerves and neuropeptides in uterine cervical ripening by J.J Collins; S Usip; K.E McCarson; R.E Papka (167-183).
At the time of parturition (fetal delivery) the uterine cervix must “ripen,” becoming soft, pliable, and dilated to accommodate the fetus’ delivery. The fundamental processes underlying cervical ripening remain poorly understood. Knowledge that abundant autonomic and sensory nerves supply the uterine cervix, that transection of afferent nerves supplying the cervix blocks parturition, and that some of the changes in the cervix resemble those seen in inflammatory reactions suggests nerves may have a role in the cervical ripening changes. The present study utilized immunohistochemistry, plasma extravasation, and solution hybridization-nuclease protection assay to elucidate the complement of primary afferent nerves and some receptors in the rat cervix during pregnancy, and to determine if they may have roles in the ripening process at term. This study revealed an abundance of nerves associated with the cervical vasculature and myometrial smooth muscle containing immunoreactivity for substance P, calcitonin gene-related peptide, secretoneurin, and nitric oxide synthase throughout pregnancy. Many of these are small unmyelinated capsaicin-sensitive C-fibers. Substance P- (NK1-) and calcitonin gene-related peptide receptors were apparent on uterine cervix vasculature from pregnant, parturient, and postpartum rats. NK1 receptor mRNA was maximal at 20 days of pregnancy. Plasma extravasation of i.v. administered Evans Blue or Monastral Blue was most pronounced at parturition (shortly after NK1 mRNA is maximal); this was similar to plasma extravasation evoked by i.v. administration of substance P or capsaicin-treatment. This study revealed new data about the nervous system of the rat uterine cervix and that these nerves and their transmitters could very well be part of a neurogenic inflammatory process involved in cervical ripening.
Keywords: Neurogenic inflammation; Substance P; Calcitonin gene-related peptide; Secretoneurin; Nitric oxide; NK1 receptors; Calcitonin gene-related peptide receptors; Plasma extravasation;

Neuropeptide enzyme hydrolysis in allergic human saliva by Federica Albo; Riccardo Antonangeli; Antonella Cavazza; Mario Marini; L.Giorgio Roda; Paolo Rossi (185-192).
The activity of neuropeptide-degrading enzymes, and possible variations in this activity under allergic conditions, was examined in human saliva obtained from allergic volunteers and from an age- and sex-matching group of healthy controls, using leucine enkephalin as model substrate. The results obtained indicate that, under experimental conditions, the substrate was partially hydrolyzed by all three classes of enzymes known to degrade it in human saliva: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In the presence of saliva obtained from allergic donors, a large increase in the activity of aminopeptidases, and a more limited increase in the activity of dipeptidylaminopeptidases, induced an increase of substrate hydrolysis with respect to that measured in the controls. The activity of all substrate-active enzymes, the allergy-associated variations in this activity, and the amount of substrate hydrolyzed, were found to be different in male and female saliva. Specifically, in the controls the gender-related differences in substrate hydrolysis were mainly caused by the higher activity of aminopeptidases observed in male as compared to female saliva. In contrast, in allergic saliva, a greater increase in the activity of aminopeptidases in female saliva reduced the gender-related differences in the pattern of hydrolysis, which was also different from that observed in the controls.
Keywords: Human saliva; Neuropeptides; Enzyme hydrolysis; Allergy; Gender-related differences;

Submandibular gland tripeptide FEG (Phe-Glu-Gly) and analogues: keys to structure determination by Essam Metwally; Jose M Pires; Graham J Moore; Dean A Befus; Joseph S Davison; Ronald Mathison (193-199).
This study examined the structure activity relationship of NH3-Phe-Glu-Gly-COO (FEG), a potent inhibitor of intestinal anaphylaxis. The inhibition by FEG analogues of antigen-provoked contractions of isolated ileal segments obtained from ovalbumin-sensitized rats was determined and molecular modeling performed. A combination of aromaticity of the first residue, minimal extension of the carboxyl group on residue 2, and underivatized N and C termini were essential for biological activity. FEG, WEG, WDG and the d-enantiomeric forms of FEG (feG) and YEG (yeG) retained biological activity. By considering dipole moments, the structural and conformational features critical to biological activity were established as the glutamyl-carboxyl group/Phe side chain and carboxyl/amino termini interactions. Analysis of Ramachandran plots for position 1 sidechains indicate that mobility of the aromatic sidechain must be restricted to retain biological activity. The anti-anaphylactic effects of FEG, characterized by specific structural and conformational restrictions, indicate a selective interaction with a receptor for this peptide in the intestine.
Keywords: Intestinal anaphylaxis; Structure activity relationships; Salivary glands; Dipole moments; Ramachandran plot; Molecular dynamics;

Secretin self-assembles and interacts spontaneously with phospholipids in vitro by Salil Gandhi; Israel Rubinstein; Takaya Tsueshita; Hayat Onyuksel (201-204).
Secretin, a 27-amino acid neuropeptide, is a member of the secretin/glucagon/vasoactive intestinal polypeptide (VIP) superfamily of amphipathic peptides. The peptide modulates gastrointestinal and neuronal function and is currently being evaluated for the treatment of autism. However, as most peptides, it has a short circulation half-life. Previously, we have shown that VIP self-assembles in aqueous environment and interacts with a biomimetic phospholipid membrane. These in vitro characteristics increase VIP half-life and bioactivity in vivo. The purpose of this study was to investigate whether secretin exhibits similar properties in vitro by forming micelles in aqueous solution and interacting with phospholipids. Results of this study demonstrated that secretin self-assembles to form micelles in HEPES buffer at 25°C above ≈0.4 μM. Additionally, secretin interacts with a biomimetic phospholipid membrane as indicated from a significant increase in membrane surface pressure (from 25.5 ± 1.3 to 32.5 ± 3.0, P < 0.05). Importantly, the peptide undergoes conformational transition from predominantly random coil in saline to α-helix in the presence of phospholipid, distearoyl-phosphatidylcholine-poly(ethylene) glycol (mol mass 2000) micelles. We suggest that these distinct biophysical attributes could modulate secretin bioactivity in vivo.
Keywords: Surface tension; Monolayer; Critical micellar concentration; Micelle; Circular dichroism; Neuropeptide; Vasopressin; DSPE-PEG;

Support vector machines for predicting the specificity of GalNAc-transferase by Yu-Dong Cai; Xiao-Jun Liu; Xue-Biao Xu; Kuo-Chen Chou (205-208).
Support Vector Machines (SVMs) which is one kind of learning machines, was applied to predict the specificity of GalNAc-transferase. The examination for the self-consistency and the jackknife test of the SVMs method were tested for the training dataset (305 oligopeptides), the correct rate of self-consistency and jackknife test reaches 100% and 84.9%, respectively. Furthermore, the prediction of the independent testing dataset (30 oligopeptides) was tested, the rate reaches 76.67%.
Keywords: Support Vector Machines; GalNAc-transferase; Self-consistency; Jackknife test;

FMRFamide-related peptides (FaRPs) are expressed throughout the animal kingdom and regulate a multitude of physiological activities. FaRPs have an RFamide C-terminal consensus structure that is important for interaction with the receptor. The ease of genetic manipulation and availability of genomic sequences makes Drosophila melanogaster an important experimental organism. Multiple classes of FaRPs encoded by different genes have been identified within this species. Here, we review FMRFamide-containing peptides encoded by the D. melanogaster FMRFamide gene in order to review the data on the expression, regulation, and activity of these peptides as well as acknowledge further endeavors required to elucidate FaRP signaling.
Keywords: dFMRFa; FaRPs; FMRFamide; heartbeat; myogenic; myotropins; neuropeptide;

Extracellular matrix molecules, long-term potentiation, memory consolidation and the brain angiotensin system by John W Wright; Enikö A Kramár; Starla E Meighan; Joseph W Harding (221-246).
Considerable evidence now suggests an interrelationship among long-term potentiation (LTP), extracellular matrix (ECM) reconfiguration, synaptogenesis, and memory consolidation within the mammalian central nervous system. Extracellular matrix molecules provide the scaffolding necessary to permit synaptic remodeling and contribute to the regulation of ionic and nutritional homeostasis of surrounding cells. These molecules also facilitate cellular proliferation, movement, differentiation, and apoptosis. The present review initially focuses on characterizing the ECM and the roles of cell adhesion molecules (CAMs), matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), in the maintenance and degradation of the ECM. The induction and maintenance of LTP is described. Debate continues over whether LTP results in some form of synaptic strengthening and in turn promotes memory consolidation. Next, the contribution of CAMs and TIMPs to the facilitation of LTP and memory consolidation is discussed. Finally, possible roles for angiotensins, MMPs, and tissue plasminogen activators in the facilitation of LTP and memory consolidation are described. These enzymatic pathways appear to be very important to an understanding of dysfunctional memory diseases such as Alzheimer’s disease, multiple sclerosis, brain tumors, and infections.
Keywords: Extracellular matrix; Cell adhesion molecules; Matrix metalloproteinases; Long-term potentiation; Hippocampus; Angiotensins;