Peptides (v.28, #6)

Announcement (III).

Ethnic differences in adrenocorticotropic hormone, cortisol and corticotropin-releasing hormone during pregnancy by Laura M. Glynn; Christine Dunkel Schetter; Aleksandra Chicz-DeMet; Calvin J. Hobel; Curt A. Sandman (1155-1161).
Significant ethnic disparities exist in reproductive outcomes. A potential contributing factor may be the functioning of the hypothalamic-pituitary-adrenal (HPA) axis and placenta during pregnancy. In the present study, levels of cortisol, ACTH and CRH were determined longitudinally from the plasma of 310 African American, Hispanic and non-Hispanic White women at 18–20, 24–26 and 30–32 weeks’ gestation. During pregnancy, African American women exhibited lower levels of cortisol than non-Hispanic women and higher levels of ACTH than Hispanic women. The trajectory of CRH increase also differed by ethnicity, with African Americans exhibiting the lowest levels both early and late in pregnancy. Higher levels of cortisol at 18–20 weeks were associated with higher levels of CRH at 30–32 weeks among the African American and Hispanic women, but not among non-Hispanic women. Ethnic differences persisted when adjusting statistically for sociodemographic and biomedical factors. The findings are consistent with the possibility that ethnic disparities in adverse birth outcomes may be due, in part, to differences in HPA axis and placental function.
Keywords: Pregnancy; Ethnicity; Race; Adrenocorticotropic hormone (ACTH); Cortisol; Corticotropin-releasing hormone (CRH); Preterm birth;

Oxytocin during pregnancy and early postpartum: Individual patterns and maternal–fetal attachment by Ari Levine; Orna Zagoory-Sharon; Ruth Feldman; Aron Weller (1162-1169).
Oxytocin (OT), a nanopeptide hormone, plays a role in the emergence of maternal behavior, yet few studies examined OT in humans across pregnancy and the postpartum. We followed healthy women at three points: first trimester of pregnancy, third trimester, and first postpartum month. Plasma OT levels showed high individual stability. A third of the sample showed consistent OT levels, whereas others showed increasing or decreasing trends or peak in late pregnancy. The increase in OT from early to late pregnancy correlated with higher maternal–fetal bonding. These data may help set standards for OT levels and underscore links with maternal–infant attachment.
Keywords: Oxytocin; Longitudinal; Pregnancy; Perinatal; Enzyme immuno-assay (EIA); Maternal–fetal attachment scale;

Neonatal oxytocin treatment modulates oxytocin receptor, atrial natriuretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression in rat heart by Hossein Pournajafi-Nazarloo; Adam Perry; Leila Partoo; Eros Papademeteriou; Fereidoun Azizi; C. Sue Carter; Bruce S. Cushing (1170-1177).
Oxytocin (OT) has been implicated in reproductive functions, induction of maternal behavior as well as endocrine and neuroendocrine regulation of the cardiovascular system. Here we demonstrate that neonatal manipulation of OT can modulate the mRNAs expression for OT receptor (OTR), atrial natriuretic peptide (ANP), endothelial nitric oxide synthase (eNOS) and estrogen receptor alpha (ERα) in the heart. On the first day of postnatal life, female and male rats were randomly assigned to receive one of the following treatments: (a) 50 μl i.p. injection of 7 μg OT; (b) 0.7 μg of OT antagonist (OTA); or (c) isotonic saline (SAL). Hearts were collected either on postnatal day 1 or day 21 (D1 or D21) and the mRNAs expression of OTR, ANP, inducible NOS (iNOS), eNOS, ERα and estrogen receptor beta (ERβ) were compared by age, treatment, and sex utilizing real time PCR. OT treatment significantly increased heart OTR, ANP and eNOS mRNAs expression on D1 in both males and females, ERα increased only in females. While there were significant changes in the relative expression of all types of mRNA between D1 and D21, there were no significant treatment effects observed in D21 animals. OTA treatment significantly decreased basal ANP and eNOS mRNAs expression on D1 in both sexes. The results indicate that during the early postnatal period OT can have an immediate effect on the expression OTR, ANP, eNOS, and ERα mRNAs and that these effects are mitigated by D21. Also with the exception of ERα mRNA, the effects are the same in both sexes.
Keywords: Oxytocin; Heart; Oxytocin receptor; NOS; Estrogen receptors;

Fetal functional capabilities in response to maternal hypertonicity associated with altered central and peripheral angiotensinogen mRNA in rats by Caiping Mao; Juanxiu Lv; Hong Zhu; Yun Zhou; Rongzheng Chen; Xin Feng; Yugui Cui; Chen Wang; Pengpeng Hui; Feichao Xu; Zhice Xu (1178-1184).
Although a number of studies have shown neural, hormonal, and behavioral capabilities in the control of body fluid regulation under conditions of dehydration in adults, limited information is available on the development of fetal functional abilities in response to osmotic challenge in rats. This study was performed to investigate the influence of maternal hypertonicity on fetal osmoregulatory capabilities at late gestational time in rats. Maternal and fetal plasma osmolality and blood sodium levels were determined and compared at continuous time points from 0.5 to 9 h following maternal injection of hypertonic NaCl. Subcutaneous administration of hypertonic saline evoked a rise in plasma osmolality and sodium concentrations in maternal rats and fetuses associated with an up-regulation in angiotensinogen gene mRNA in the fetal liver and down-regulation of the same gene in the fetal brain. The increased levels of fetal blood osmolality and sodium were less than that in their mothers, and the fetus took less time to balance the enhanced osmolality and sodium concentrations. The results suggest that there may exist additional mechanisms in utero at near-term in protecting fetuses from hypertonic challenge. In addition, molecular results in the present study provide new data on fetal angiotensinogen gene expressed differently in the liver and brain under the same condition of prenatal salt loading, indicating osmotic signals of intracellular dehydration related to an acute increase in angiotensinogen mRNA in the fetal liver, and subsequent decrease in angiotensinogen mRNA levels in the fetal brain.
Keywords: Osmoregulation; Fetal development; RAS;

Peptide YY (PYY) is a gastrointestinal hormone, localized in enteroendocrine L-cells. Its hydrolyzed form PYY(3-36) is a satiety factor. The aim of this study was to identify if intestinal PYY enteroendocrine cells or content correlate with the diet-induced obese (DIO) or diet-resistant (DR) phenotypes. We also examined intestinal sensitivity to PYY and PYY(3-36) in DIO and DR rats. Animals were maintained on a medium-high fat diet and split into DIO and DR groups based on weight gain. PYY immunoreactive cells were unaltered in DIO intestine and stomach compared to DR rats. PYY content and circulating levels were also unchanged in DIO rats. Intestinal PYY and PYY(3-36) responses were enhanced in fasted rats, and equipotent in both DIO and DR jejunum. We conclude that PYY cell number, tissue content and peripheral sensitivity are maintained in DIO rats. Our data suggests that neither PYY nor PYY(3-36) contribute to the maintenance of either the DIO or DR phenotype, and that peripheral resistance to PYY and PYY(3-36) does not accompany DIO.
Keywords: Small intestine; Diet-induced obesity; Peptide YY; L-cell;

Further structure–activity studies of lactam derivatives of MT-II and SHU-9119: Their activity and selectivity at human melanocortin receptors 3, 4, and 5 by Paolo Grieco; Minying Cai; Guoxia Han; Dev Trivedi; Pietro Campiglia; Ettore Novellino; Victor J. Hruby (1191-1196).
Keywords: Melanocortin receptors; Melanotropins; Structure–activity relationships; Cyclic melanotropins; Receptor selective melanotropin antagonists;

Pharmacokinetics of trefoil peptides and their stability in gastrointestinal contents by Stine Kjellev; Else Marie Vestergaard; Ebba Nexø; Peter Thygesen; Maria S. Eghøj; Palle B. Jeppesen; Lars Thim; Nis Borbye Pedersen; Steen Seier Poulsen (1197-1206).
Trefoil factor family (TFF) peptides are considered promising for therapeutic use in gastrointestinal diseases, and there is a need to explore the fate of injected TFF and the stability of the peptides in the gastrointestinal tract. We studied the pharmacokinetics of intravenously (i.v.) administered hTFF2 in mice and rats and of hTFF3 administered i.v., intramuscularly, intraperitoneally, and subcutaneously in mice, and estimated by ELISA the decay of the peptides added to rat and human gastrointestinal contents. We found that i.v. injected hTFF2 and hTFF3 were cleared from the circulation within 2–3 h, exhibiting comparable pharmacokinetic profiles. In contents from the rat stomach, hTFF levels remained unchanged for up to 6 days. In the small and large intestine of rats, the hTFF levels decreased markedly after 4 and 1 h, respectively. In small intestinal contents from humans, the levels remained stable for more than 24 h. We conclude that systemically administered hTFF2 and hTFF3 are rapidly eliminated from the circulation and that the stability of hTFF2 and hTFF3 in GI contents appeared higher in the gastric and small intestinal milieu than in the large intestine and feces, suggesting a higher stability toward gastric acid and digestive enzymes than toward microbial degradation.
Keywords: Trefoil factor family; TFF2; TFF3; Pharmacokinetics; Clearance; Stability;

Regulation of food intake in the goldfish by interaction between ghrelin and orexin by Tohru Miura; Keisuke Maruyama; Sei-Ichi Shimakura; Hiroyuki Kaiya; Minoru Uchiyama; Kenji Kangawa; Seiji Shioda; Kouhei Matsuda (1207-1213).
Intracerebroventricular (ICV) administration of ghrelin, orexin and neuropeptide Y (NPY) stimulates food intake in goldfish. Orexin and NPY interact with each other in the regulation of feeding, while ghrelin-induced feeding has also shown to be mediated by NPY in the goldfish model. To investigate the interaction between ghrelin and orexin, we examined the effects of a selective orexin receptor-1 antagonist, SB334867, and a growth hormone secretagogue-receptor antagonist, [D-Lys3]-GHRP-6, on ghrelin- and orexin-A-induced feeding. Ghrelin-induced food intake was completely inhibited for 1 h following ICV preinjection of SB334867, while [D-Lys3]-GHRP-6 attenuated orexin-A stimulated feeding. Furthermore, ICV administration of ghrelin or orexin-A at a dose sufficient to stimulate food intake increased the expression of each other's mRNA in the diencephalon. These results indicate that, in goldfish, ghrelin and orexin-A have interacting orexigenic effects in the central nervous system. This is the first report that orexin-A-induced feeding is mediated by the ghrelin signaling in any animal model.
Keywords: Goldfish; Ghrelin; Orexin; [D-Lys3]-GHRP-6; SB334867; Orexigenic action;

Antiepileptic effects of ghrelin on pentylenetetrazole-induced seizures in rats by Basra Deniz Obay; Ezel Tasdemir; Cemil Tümer; Hakkı Murat Bilgin; Abdurrahman Sermet (1214-1219).
It is well known that neuropeptide Y (NPY) and gamma-aminobutyric acid (GABA) exert antiepileptic effects in animal models. It has recently been shown that ghrelin neurons increase the activities of GABA and NPY in the brain. Therefore it can be said that ghrelin is an antiepileptic agent. In this study we aimed to investigate the antiepileptic effect of ghrelin in an acute experimental epilepsy model in pentylenetetrazole (PTZ) injected rats. Adult male Wistar albino rats were divided into a control group and four experimental groups with seven rats in each group. In order to generate epileptic seizures, PTZ (50 mg/kg) was injected intraperitoneally. The experimental groups received intraperitoneal injections of ghrelin at doses of 20, 40, 60 and 80 μg/kg 30 min before PTZ injection. After PTZ injection, the latencies were separated into three components: first myoclonic jerk, generalized clonic seizures and tonic generalized extension. The injection of 50 mg/kg PTZ-induced epileptic seizures in the control group. The onset times of the three characteristic behavioral changes were significantly delayed and the duration of tonic generalized extension was diminished by dose-dependent ghrelin administration. Our results demonstrated that ghrelin suppresses the onset time of PTZ-induced seizures. In the light of our current knowledge, it seems that ghrelin may be considered as an antiepileptic drug.
Keywords: Epilepsy; Ghrelin; Pentylenetetrazole (PTZ);

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (NOP receptor), has been demonstrated to be involved in many physiological and pathological functions including pain modulation. It was reported that electroacupuncture (EA) had a potent analgesic effect on inflammatory pain by activating various endogenous transmitters such as the opioid peptides. In the present study, we investigated the effect of EA on peripheral inflammatory pain and the expression of N/OFQ and the NOP receptor in the spinal dorsal horn of rats, using a behavioral test, RT-PCR, immunohistochemistry and Western blot analysis techniques. The results showed: (1) EA had an accumulative analgesic effect on chronic inflammatory pain; (2) in the superficial layers of the spinal dorsal horn, the level of mRNA of the precursor protein for N/OFQ (preproN/OFQ, ppN/OFQ) was increased and the N/OFQ immunoreactivity was decreased after peripheral inflammation, and could be significantly increased by EA treatment; (3) both mRNA and protein levels of the NOP receptor in the spinal dorsal horn were significantly increased after chronic inflammatory pain and could be further enhanced by EA treatment. The present data demonstrated that EA could activate the endogenous N/OFQ–NOP receptor system, and this might underlie the effectiveness of EA in the treatment of inflammatory pain.
Keywords: Peripheral inflammatory pain; Electroacupuncture; Spinal cord; N/OFQ; NOP receptor;

Altered anxiety-related behavior in nociceptin/orphanin FQ receptor gene knockout mice by Elaine C. Gavioli; Anna Rizzi; Giuliano Marzola; Silvia Zucchini; Domenico Regoli; Girolamo Calo’ (1229-1239).
Studies showed that nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) agonists produce anxiolytic-like actions, while little is known about the effects of blockade of NOP receptor signaling in anxiety. To this aim, we investigated the behavioral phenotype of NOP receptor gene knockout mice (NOP−/−) in different assays. In the elevated plus-maze and light-dark box, NOP−/− mice displayed increased anxiety-related behavior. In the novelty-suppressed feeding behavior and elevated T-maze, NOP−/− mice showed anxiolytic-like phenotype, while no differences were found in the open-field, hole-board, marble-burying, and stress-induced hyperthermia. Altogether, these findings suggest that the N/OFQ-NOP receptor system modulates anxiety-related behavior in a complex manner.
Keywords: Nociceptin/orphanin FQ; NOP receptor; Anxiety disorders; Behavioral tests; Knockout mice phenotype;

In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor by Anna Rizzi; Barbara Spagnolo; Richard D. Wainford; Carmela Fischetti; Remo Guerrini; Giuliano Marzola; Anna Baldisserotto; Severo Salvadori; Domenico Regoli; Daniel R. Kapusta; Girolamo Calo (1240-1251).
[(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC50 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP−/− mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1–100 pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10 pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP−/− mice. Equi-effective high doses of UFP-112 (0.1 nmol) and N/OFQ (10 nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16 h. N/OFQ produced a clear inhibitory effect which lasted for 60 min while UFP-112 elicited longer lasting effects (>6 h). In conscious rats, UFP-112 (0.1 and 10 nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.
Keywords: Nociceptin/orphanin FQ; NOP receptor; UFP-112; Isolated tissues; Tail withdrawal assay; Locomotor activity; Food intake; Cardiovascular and renal functions;

In a previous study, we found that calcitonin gene-related peptide (CGRP) could be induced by proinflammatory factor IL-1β in A549 human type II alveolar (AEII) epithelial cells. We investigated the mechanism of IL-1β-induced CGRP secretion and found that the PKC-p38 MAPK-NF-κB pathway was involved. In the present study, we found that IL-1β stimulation induced c-Jun N-terminal kinase (JNK) activity within 15 min in A549 cells. In investigating whether JNK was involved in IL-1β-induced CGRP secretion, the JNK II inhibitor SP600125 was used and it significantly attenuated IL-1β-induced CGRP secretion and c-Jun activity, which was elevated after IL-1β stimulation from mRNA to protein level. EMSA results showed the activation of activator protein 1 (AP-1) after 2-h IL-1β stimulation, and the JNK II inhibitor blocked c-Jun and AP-1 activity. Bioinformatic analysis showed five predicted AP-1 binding sites on the promoter of β-CGRP; deletion analysis identified an AP-1 consensus site at −643 bp relative to the initiation site, which mediates the β-CGRP gene transcription in response to IL-1β. These data suggest that besides the PKC-p38 MAPK-NF-κB pathway, the JNK-AP-1 pathway is involved in IL-1β-induced CGRP secretion in A549 human type II alveolar epithelial cells, and a 643-bp site upstream of the transcription start site on the promoter of β-CGRP is the AP-1 response element.
Keywords: CGRP; IL-1β; AEII cell; JNK; AP-1;

Widespread and ample peptide overlapping between HCV and Homo sapiens proteomes by Anthony Kusalik; Miķelis Bickis; Christopher Lewis; Ying Li; Guglielmo Lucchese; Francesco M. Marincola; Darja Kanduc (1260-1267).
Alignment of protein sequences is fundamental in analyzing homology, evolutionary events and functional relationships. Searching for the epitopic peptide platform underlying hepatitis C virus (HCV) infection and autoimmune phenomena, we have used sequence–sequence peptide matching to compare the HCV polyprotein sequence to the human proteome. The following results were obtained: (1) pentamers from HCV polyprotein have a widespread and high level of similarity to a large number of human proteins (19,605 human proteins, that is 57.6% of the human proteome); (2) remarkable similarity between the two proteomes persists even using longer peptide motifs as probes for identity scanning; (3) only a limited number of HCV pentameric fragments have no similarity to the human host, so representing molecular sequence signatures of the virus. We conclude that the widespread sharing of numerous perfect exact matches between HCV and human proteomes might explain HCV persistence in humans.
Keywords: Similarity analysis; Sequence–sequence peptide matching; Viral versus human proteome overlapping; HCV-related autoimmunity;

Peptide defenses of the Cascades frog Rana cascadae: implications for the evolutionary history of frogs of the Amerana species group by J. Michael Conlon; Ahmed al-Dhaheri; Eissa al-Mutawa; Rokaya al-Kharrge; Eman Ahmed; Jolanta Kolodziejek; Norbert Nowotny; Per F. Nielsen; Carlos Davidson (1268-1274).
The Cascades frog Rana cascadae belongs to the Amerana (or Rana boylii) group that includes six additional species from western North America (R. aurora, R. boylii, R. draytonii, R. luteiventris, R. muscosa, and R. pretiosa). R. cascadae is particularly susceptible to pathogenic microorganisms in the environment and populations have declined precipitously in parts of its range so that the protection afforded by dermal antimicrobial peptides may be crucial to survival of the species. Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of six peptides with differential cytolytic activities that were present in high abundance. Structural characterization showed that they belonged to the ranatuerin-2 (one peptide), brevinin-1 (one peptide), and temporin (four peptides) families. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAGKLLETLKCKITGC) and brevinin-1CSa (FLPILAGLAAKIVPKLFCLATKKC) showed broad spectrum antibacterial activity (MIC ≤ 32 μM against Escherichia coli and Staphylococcus aureus) but only brevinin-1CSa was strongly hemolytic against human erythrocytes (LC50  = 5 μM). The taxonomy of ranid frogs is currently in a considerable state of flux. The ranatuerin-2 gene is expressed in all members of the Amerana group studied to-date and cladistic analysis based upon a comparison of the amino acid sequences of this peptide indicates that R. cascadae, R. muscosa and R. aurora form a clade that is distinct from one containing R. draytonii, R. boylii, and R. luteiventris. This conclusion is consistent with previous analyses based upon comparisons of the nucleotide sequences of mitochondrial genes.
Keywords: Peptidomics; Antimicrobial; Ranatuerin-2; Brevinin-1; Temporin; Frog skin;

The complex array of bradykinin-related peptides (BRPs) in the peptidome of pickerel frog (Rana palustris) skin secretion is the product of transcriptional economy by Cian Michael McCrudden; Mei Zhou; Tianbao Chen; Martin O’Rourke; Brian Walker; David Hirst; Chris Shaw (1275-1281).
Previous peptidomic analyses of the defensive skin secretion from the North American pickerel frog, Rana palustris, have established the presence of canonical bradykinin and multiple bradykinin-related peptides (BRPs). As a consequence of the multiplicity of peptides identified and their diverse primary structures, it was speculated that they must represent the products of expression of multiple genes. Here, we present unequivocal evidence that the majority of BRPs (11/13) identified in skin secretion by the peptidomic approach can be generated by differential site-specific protease cleavage from a single common precursor of 321 amino acid residues, named skin kininogen 1, whose primary structure was deduced from cloned skin secretion-derived cDNA. The organization of skin kininogen 1 consists of a hydrophobic signal peptide followed by eight non-identical domains each encoding a single copy of either canonical bradykinin or a BRP. Two additional splice variants, encoding precursors of 233 (skin kininogen 2) or 189 amino acid residues (skin kininogen 3), were also cloned and were found to lack BRP-encoding domains 5 and 6 or 4, 5 and 6, respectively. Thus, generation of peptidome diversity in amphibian defensive skin secretions can be achieved in part by differential protease cleavage of relatively large and multiple-encoding domain precursors reflecting a high degree of transcriptional economy.
Keywords: Amphibian; Cloning; Precursor; Kininogen; Peptide;

Neuropeptide precursors in Tribolium castaneum by Andinet Amare; Jonathan V. Sweedler (1282-1291).
Neuropeptides and neurohormones are among the more diverse and functionally important classes of cell-to-cell signaling molecules involved in animal development and behavior. Less is known about the hormones and neuropeptides of the red flour beetle, Tribolium castaneum, than many other insects. However, the genomic information becoming available from this organism presents an opportunity to identify multiple neuropeptide and hormone genes, and hence their associated protein precursors. Using similarity-based prediction, we report new neuropeptides and hormone precursors from T. castaneum, bringing the number of annotated precursors to 37. We identified one prohormone (SVDPIDGDLIG-containing) having little similarity to other insect prohormones. The conversion of the protein precursors into bioactive peptides requires a suite of processing enzymes and a number of enzymatic steps; using the web-based NeuroPred application and similarity-based bioinformatics approaches, we predict 132 likely peptides that may result from the enzymatic processing of these gene products.
Keywords: Neuropeptides; NeuroPred; Prohormone; Tribolium;

Proregion of Acanthoscelides obtectus cysteine proteinase: A novel peptide with enhanced selectivity toward endogenous enzymes by F.B. Silva; A.C.S. Monteiro; R.P. Del Sarto; B.M. Marra; S.C. Dias; E.L.Z. Figueira; G.R. Oliveira; T.L. Rocha; D.S.L. Souza; M.C.M. da Silva; O.L. Franco; M.F. Grossi-de-Sa (1292-1298).
Acanthoscelides obtectus is a devastating storage insect pest capable of causing severe bean crop losses. In order to maintain their own development, insect pest larvae feed continuously, synthesizing efficient digestive enzymes. Among them, cysteine proteinases (CPs) are commonly produced as inactive precursors (procysteines), requiring a cleavage of the peptide proregion to become active. The proregion fits tightly into the active site of procysteines, efficiently preventing their activity. In this report, a CP cDNA (cpao) was isolated from A. obtectus midgut larvae. In silico studies indicated that the complete CP sequence contains a hydrophobic signal peptide, a prodomain and a conserved catalytic region. Moreover, the encoding cDNA contains 963 bp translating into a 321 residue protein, CPAo, which was expressed in E. coli, fused with thioredoxin. Enzymatic assays using the recombinant protein revealed that the enzyme was catalytically active, being able to cleave the synthetic substrate Z-Phe-Arg-7-AMC. Additionally, this report also focuses the cpao propeptide (PCPAo) subcloning and expression. The expressed propeptide efficiently inhibited CPAo, as well as digestive CP of other bean bruchids. Little or no activity was found against proteolytic enzymes of two other coleopterans: Rhyzopertha dominica and Anthonomus grandis. The data reported here indicate the possibility of endogenous propeptides as a novel strategy on bruchids control, which could be applicable to bean improvement programs.
Keywords: Acanthoscelides obtectus; Cysteine proteinase; Propeptide; Plant defense;

The lipopeptides Pal–Lys–Lys–NH2 and Pal–Lys–Lys soaking alone and in combination with intraperitoneal vancomycin prevent vascular graft biofilm in a subcutaneous rat pouch model of staphylococcal infection by Oscar Cirioni; Andrea Giacometti; Roberto Ghiselli; Wojciech Kamysz; Carmela Silvestri; Fiorenza Orlando; Federico Mocchegiani; Agnese Della Vittoria; Elzbieta Kamysz; Vittorio Saba; Giorgio Scalise (1299-1303).
Staphylococcal infections are often associated with the use of implantable medical devices. Such infections are difficult to treat because of biofilm resistance to antibiotics and are common causes of morbidity and mortality. Graft infections were established in the back subcutaneous tissue of adult male Wistar rats by implantation of Dacron prostheses followed by topical inoculation with 2 × 107 colony-forming units of bacterial strains. The study included a control group, a contaminated group that did not receive any antibiotic prophylaxis and five contaminated groups that received intraperitoneal vancomycin, Pal–Lys–Lys–NH2 and Pal–Lys–Lys-soacked graft, and vancomycin plus Pal–Lys–Lys–NH2 or Pal–Lys–Lys-soacked graft, respectively. The infection was evaluated by using sonication and quantitative agar culture. Moreover, an in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms was performed to elucidate the same activity. When tested alone, vancomycin and lipopeptides showed comparable efficacies. All combinations showed efficacies significantly higher than that of each single compound. Vancomycin combined to Pal–Lys–Lys–NH2 exerted the strongest anti-staphylococcal efficacies. The in vitro studies showed, that MIC and MBC values for vancomycin were lower in presence of lipopeptides. They reduce the bacterial load and to enhance the effect of vancomycin in the prevention of vascular graft staphylococcal infections.
Keywords: Lipopeptides; Antimicrobial peptides; Vascular graft infection; Bacterial biofilm;

Sequence characterization and expression patterns of two defensin-like antimicrobial peptides from the tick Haemaphysalis longicornis by Jinlin Zhou; Min Liao; Mami Ueda; Haiyan Gong; Xuenan Xuan; Kozo Fujisaki (1304-1310).
Two cDNAs encoding defensin-like antimicrobial peptides were cloned and sequenced from the tick Haemaphysalis longicornis. The full-length cDNA of Hlgut-defensin (H. longicornis midgut defensin) is 333 bp, encoding an expected protein with 73 amino acids. The full-length cDNA of Hlsal-defensin (H. longicornis salivary gland defensin) is 382 bp, encoding an expected protein with 81 amino acids. The antibacterial activities of the synthetic peptides based on the Hlgut-defensin and Hlsal-defensin sequences were tested against a variety of Gram-positive and Gram-negative bacteria. Using real-time PCR, the tissue-specific expression of two defensin-like peptides were determined and it was also found that the gene transcripts of Hlgut-defensin and Hlsal-defensin were significantly induced by a lipopolysaccharide (LPS) injection.
Keywords: Haemaphysalis longicornis; Antimicrobial peptide; Expression patterns; Sequence characterization;

Anredera cordifolia (Ten.) Steenis, or the synonymous name of Boussingaultia baselloides or Boussingaultia gracilis var. pseudobaselloides, is a South American species of ornamental succulent vine, commonly known as the madeira-vine. The fresh leaves of madeira-vine are frequently used as vegetables. A. cordifolia is an evergreen climber that grows from fleshy rhizomes. The rhizome contained one major (23 kDa) protein band under non-reducing condition in the SDS-PAGE. The first 15 amino acids in the N-terminal region of the major protein band (23 kDa), named tentatively ancordin, were KDDLLVLDIGGNPVV which were highly homologous to sequences of winged bean seed protein ws-1, Medicago truncatula proteinase inhibitor, soybean trypsin inhibitor, and sporamin. By using activity stains, the ancordin showed trypsin inhibitory activity in the SDS-PAGE gel which was found not only in rhizomes but also in aerial tubers, but few in fresh leaves. The crude extracts from rhizomes of madeira-vine were directly loaded onto trypsin–Sepharose 4B affinity column. After washing with 100 mM Tris–HCl buffer (pH 7.9) containing 100 mM NaCl, the ancordin was eluted directly by 0.2 M KC1–HC1 buffer (pH 2.0). In calculation, the purified protein exhibited 0.0428 μg trypsin inhibition/μg ancordin (corresponding to 0.53 unit of TPCK-treated trypsin inhibited/μg ancordin). The purified ancordin was used to evaluate the nitric oxide productions in RAW264.7 cells in the presence of polymyxin B (poly B, 50 μg/ml) to eliminate the lipopolysaccharide (LPS) contaminations. It was found that ancordin (1.25–5 μg/ml) could dose-dependently (R  = 0.954) stimulate the nitric oxide (NO) productions (expressed as nitrite concentrations) in RAW264.7 cells without significant cytotoxicity, and kept the similar effects in NO production in 6.25 μg/ml ancordin.
Keywords: Anredera cordifolia; Madeira-vine; Nitric oxide; Rhizome; Trypsin inhibitory activity;

Adipokines and the blood-brain barrier by Weihong Pan; Abba J. Kastin (1317-1330).
Just as the blood-brain barrier (BBB) is not a static barrier, the adipocytes are not inert storage depots. Adipokines are peptides or polypeptides produced by white adipose tissue; they play important roles in normal physiology as well as in the metabolic syndrome. Adipokines secreted into the circulation can interact with the BBB and exert potent CNS effects. The specific transport systems for two important adipokines, leptin and tumor necrosis factor alpha, have been characterized during the past decade. By contrast, transforming growth factor beta-1 and adiponectin do not show specific permeation across the BBB, but modulate endothelial functions. Still others, like interleukin-6, may reach the brain but are rapidly degraded. This review summarizes current knowledge and recent findings of the rapidly growing family of adipokines and their interactions with the BBB.
Keywords: Adipocytes; Adipokines; Cytokines; Peptides; Polypeptides; Blood-brain barrier; Leptin; TNFα; TGF-β; IL-6; Adiponectin; Angiotensinogen; PAI-1; Relaxin; Resistin; ASP; RBP;