Peptides (v.32, #8)

Toward understanding the role of leptin and leptin receptor antagonism in preclinical models of rheumatoid arthritis by Laszlo Otvos; Wen-Hai Shao; Anne S. Vanniasinghe; Michael A. Amon; Marianna Csilla Holub; Ilona Kovalszky; John D. Wade; Michelle Doll; Philip L. Cohen; Nicholas Manolios; Eva Surmacz (1567-1574).
• Leptin and a leptin receptor agonist peptide constitutively activate the NF-κB pathway in peripheral blood mononuclear cells, and the leptin receptor antagonist peptide Allo-aca reverses the process. • In a mouse serum transfer rheumatoid arthritis model, Allo-aca added subcutaneously partially inhibits the proinflammatory effects of leptin. • In a rat adjuvant-induced rheumatoid arthritis, Allo-aca reverses all inflammatory symptoms in early stages, but is only partially effective in a more advanced disease.A potential link between obesity, circulating leptin levels and autoimmune disease symptoms suggests that targeting the leptin receptor (ObR) might be a viable novel strategy to combat rheumatoid arthritis (RA). However, studies in animal models and evaluation of clinical cases did not provide clear view on leptin's involvement in RA. To validate ObR as RA target, we used our peptide-based ObR agonists and antagonist in different in vitro and in vivo models of the disease. In human peripheral blood mononuclear cells, leptin and its agonist fragment, desI2-E1/Aca, moderately induced constitutive activation of a major proinflammatory transcription factor, NF-κB, while the ObR antagonist peptide Allo-aca inhibited the process. Leptin administration itself did not induce arthritis in rats, but worsened the clinical condition of mice given K/BxN serum transfer arthritis. Simultaneous administration of Allo-aca reduced leptin-dependent increase in disease severity by more than 50%, but the antagonist was ineffective when injected with a 3-day delay. In rats inflicted with mild adjuvant-induced arthritis, both leptin and Allo-aca reduced the extent of joint swelling and the number of arthritic joints. In a more aggressive disease stage, Allo-aca decreased the number of arthritic joints in a dose-dependent manner but did not affect other arthritis markers. In summary, leptin exerts diverse effects on RA depending on the experimental model. This might reflect the heterogeneous character of RA, which is differently impacted by leptin and is unmasked by ObR antagonism. Nevertheless, the results suggest that ObR antagonists might become useful therapeutics in leptin-sensitive early stages of RA.
Keywords: Adjuvant-induced arthritis; Antagonist; Inflammation; K/BxN serum arthritis; Leptin; NF-κB pathway; Peptides;

Effects of increased hypothalamic leptin gene expression on ovariectomy-induced bone loss in rats by M.A. Jackson; U.T. Iwaniec; R.T. Turner; T.J. Wronski; S.P. Kalra (1575-1580).
► Peripheral leptin was reported to attenuate estrogen deficiency-induced bone loss. ► Hypothalamic administration of leptin was reported to be antiosteogenic. ► We evaluated bone in ovx rats with increased hypothalamic leptin gene expression. ► Ovx resulted in severe osteopenia. ► Increasing hypothalamic leptin gene expression had no additional effect on bone.Estrogen deficiency results in accelerated bone turnover with a net increase in bone resorption. Subcutaneous administration of leptin attenuates bone loss in ovariectomized (ovx) rats by reducing bone resorption. However, in addition to its direct beneficial effects, leptin has been reported to have indirect (central nervous system-mediated) antiosteogenic effects on bone, which may limit the efficacy of elevated serum leptin to prevent estrogen deficiency-associated bone loss. The present study evaluated the long-term effects of increased hypothalamic leptin transgene expression, using recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy, on bone mass, architecture, and cellular endpoints in sexually mature ovx Sprague-Dawley rats. Ovx rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained for 10 weeks. Additional controls consisted of ovary-intact rats and ovx rats pair-fed to rAAV-Lep rats. Lumbar vertebrae were analyzed by micro-computed tomography and tibiae by histomorphometry. Cancellous bone volume was lower and osteoclast perimeter, osteoblast perimeter, and bone marrow adipocyte density were greater in ovx rats compared to ovary-intact controls. In contrast, differences among ovx groups were not detected for any endpoint evaluated. In conclusion, whereas estrogen deficiency resulted in marked cancellous osteopenia, increased bone turnover and marrow adiposity, increasing hypothalamic leptin transgene expression in ovx rats had neither detrimental nor beneficial effects on bone mass, architecture, or cellular endpoints. These findings demonstrate that the antiresorptive effects of subcutaneous leptin administration in ovx rats are mediated through leptin targets in the periphery.
Keywords: Bone marrow adiposity; White adipose tissue; μCT; Histomorphometry; Osteoclasts; Osteoblasts;

The feeding responses evoked by cholecystokinin are mediated by vagus and splanchnic nerves by Thelma A.L. Brown; Martha C. Washington; Shannon A. Metcalf; Ayman I. Sayegh (1581-1586).
► Effects of VGX and CMGX on the feeding responses by CCK-8 and 33 were tested. ► Reduction of meal size (MS) by CCK-8 and 33 is mediated by the vagus. ► Prolongation of IMI by CCK-33 is mediated by celiaco-mesenteric ganglia. ► Both neuronal components are required in mediating feeding responses by CCK.Total or selective branch vagotomy attenuates the reduction of cumulative food intake by cholecystokinin (CCK)-8 and CCK-33 respectively. However, the role of the sympathetic innervation of the gut and the role of the vagus nerve in feeding responses, which include meal size (MS) and intermeal interval (IMI), evoked by CCK-8 and CCK-33 have not been evaluated. Here, we tested the effects of total subdiaphragmatic vagotomy (VGX) and celiaco-mesenteric ganglionectomy (CMGX) on the previous feeding responses by CCK-8 and CCK-33 (0, 1, 3, and 5 nmol/kg given intraperitoneally). We found (1) that both peptides reduced meal size and CCK-8 (5 nmol) and CCK-33 (1 and 3 nmol) prolonged IMI, (2) that VGX attenuated the reduction of MS but failed to attenuate the prolongation of IMI by both peptides and (3) that CMGX attenuated the reduction of meal size by CCK-8 and the prolongation of IMI by both peptides. Therefore, the feeding responses evoked by CCK-8 require intact vagus and splanchnic nerves: the reduction of MS by CCK-33 requires an intact vagus nerve, and the prolongation of IMI requires the splanchnic nerve. These findings demonstrate the differential peripheral neuronal mediation of the feeding responses evoked by CCK-8 and CCK-33.
Keywords: Meal size; Intermeal interval; Vagus nerve; Splanchnic nerve; Spinal afferents;

Gastric relaxation induced by glucagon-like peptide-2 in mice fed a high-fat diet or fasted by Alessandra Rotondo; Antonella Amato; Sara Baldassano; Laura Lentini; Flavia Mulè (1587-1592).
► GLP-2R expression in stomach from fasted, standard or high-fat diet-fed mice was examined. ► GLP-2 efficacy in inducing gastric relaxation was determined in differently fed mice. ► In murine gastric preparations, the nutritional state influences GLP-2R expression. ► GLP-2R down- or up-regulation is associated with changes of GLP-2 gastric relaxant efficacy.Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive gut hormone that increases the intestinal absorption. Exogenous GLP-2 also induces gastric fundus relaxation with possible implications for emptying rate or feeling of satiety. GLP-2 actions are mediated by GLP-2 receptor (GLP-2R), located on enteric neurons and myofibroblasts in murine gastrointestinal tract. Because it is not known whether changes in the endogenous GLP-2R levels occur in different nutritional states, we examined the GLP-2R gene and protein expression in gastric fundus from standard diet (STD)-fed, 12-h and 24-h fasted and re-fed, or high-fat diet (HFD)-fed mice and we analyzed the mechanical responses to exogenous GLP-2 in the stomach from different groups of animals. GLP-2 expression was examined using real-time reverse-transcription polymerase chain reaction and western blotting. The gastric mechanical activity from whole-organ was recorded in vitro as changes of intraluminal pressure. GLP-2R expression in fundic region from 12-h or 24-h fasted mice was reduced in comparison with STD-fed animals and returned to control values in re-fed mice, while it was increased in HFD-fed mice. The exogenous GLP-2 efficacy in inducing gastric relaxation, normalized to isoproterenol response, was decreased in stomach from fasted mice and it was increased in stomach from HFD-fed mice in comparison with STD-fed mice. In conclusion, the nutritional state influences GLP-2R expression in murine gastric preparations. The changes in the GLP-2R expression are associated with modifications of GLP-2 gastric relaxant efficacy. This could represent an adaptive response to reduced or increased nutrient intake.
Keywords: GLP-2; GLP-2 receptor expression; Fasting; Obesity;

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method by David J. Roberts; Pascale Vertongen; Magali Waelbroeck (1593-1599).
Display Omitted► SCAM of the first glucagon receptor extracellular loop (197–224). ► TM2 continues to position 202, TM3 from 215 ► Most side chains important for active glucagon conformation. ► S217 and D218 contact glucagon, V221 very close to the binding site. ► Receptor activation increases the accessibility of L198C mutant cysteine.Glucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor–glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO2-biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short β stretch (positions 203–209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.
Keywords: Glucagon receptor; Glucagon; Glucagon binding site; Substituted Cysteine Accessibility Method (SCAM); Mutagenesis (site directed);

► GRP reduces food intake by an incompletely known mechanism. ► We quantified enteric and DVC neurons positive for Fos-LI by GRP. ► GRP-10, -27 and -29 increased Fos-LI in the stomach and area postrema. ► The large forms increased Fos in the myenteric plexus of the duodenum. ► Only GRP-29 increased Fos in the submucosal plexus of the duodenum.We and others have shown that gastrin-releasing peptide (GRP) reduces food intake. In this study, we determined the activation of the gastrointestinal and dorsal vagal complex (DVC) neurons by various forms of GRP to determine the pathway involved in this reduction. We found the following: (1) GRP-10, -27 and -29 (2.1 nmol/kg, i.p.) increased the Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of the stomach and the area postrema (AP) of the DVC; (2) GRP-27 and GRP-29 increased the Fos-LI in the myenteric plexus of the duodenum; and (3) only GRP-29 increased the Fos-LI in the submucosal plexus of the duodenum. In conclusion, GRP may reduce food intake by activating the area postrema. The enteric neurons may have a potential role in this reduction through the direct activation of the AP or exerting local gut actions, such as the stimulation of gut motility or secretions.
Keywords: Fos; Bombesin; Submucosal plexus; Myenteric plexus; Area postrema;

Ghrelin postsynaptically depolarizes dorsal raphe neurons in rats in vitro by Masaki Ogaya; Juhyon Kim; Kazuo Sasaki (1606-1616).
▶ Most dorsal raphe neurons postsynaptically depolarized by ghrelin. ▶ Ionic mechanisms include increase in nonselective cationic conductance and decrease in K+ conductance. ▶ Most depolarized neurons were putative serotonergic neurons. ▶ Dorsal raphe neurons, especially serotonin-containing neurons, might be involved in ghrelin CNS effects.Ghrelin promotes growth hormone (GH) secretion and feeding. Recent studies further showed that ghrelin displayed a defending effect against the depressive-like symptoms and affected sleep in animals and humans. Serotonergic system is considered to be implicated in feeding, depression and other mood disorders, and sleep. The dorsal raphe nucleus (DRN) utilizes serotonin (5-HT) as its major neurotransmitter and expresses GH secretagogue receptors (GHS-Rs). Therefore, the present study was carried out to examine the electrophysiological effect of ghrelin on rat DRN neurons in vitro and determine the ionic mechanism involved. Whole-cell recording revealed that ghrelin depolarized DRN neurons dose-dependently in tetrodotoxin-containing artificial cerebrospinal fluid (TTX ACSF). Pretreatment with [d-Lys3]-GHRP-6, a selective antagonist for GHS-Rs, antagonized the ghrelin-induced depolarization. The depolarization was significantly reduced in a low-Na+ TTX ACSF and in a high-K+ TTX ACSF and was abolished in the combination of both ACSFs, suggesting that the ghrelin-induced depolarization is mediated by a dual ionic mechanism including an increase in nonselective cationic conductance and a decrease in K+ conductance. The experiments on the reversal potential also supported an involvement of the dual ionic mechanism in the ghrelin-induced depolarization. On the basis of their electrophysiological and pharmacological properties, approximately 80% of DRN neurons were classified as putative 5-HT-containing neurons and ghrelin depolarized 75% of them. These results suggest that DRN neurons, especially 5-HT-containing neurons, might be involved in the neural mechanisms through which ghrelin participates in the development and/or regulation of feeding behavior, sleep-wake states and depressive-like symptoms.
Keywords: Ghrelin; Dorsal raphe nucleus; Serotonin; Nonselective cationic conductance; K+ conductance; Anxiolytic effect; Feeding; Sleep;

Short amyloid-beta immunogens show strong immunogenicity and avoid stimulating pro-inflammatory pathways in bone marrow-derived dendritic cells from C57BL/6J mice in vitro by Jian-Quan Shi; Jun Chen; Bian-Rong Wang; Yin-Wei Zhu; Yan Xu; Jun Wang; Hang Xiao; Jing-Ping Shi; Ying-Dong Zhang; Jun Xu (1617-1625).
• Short Aβ peptides increase migration/endocytosis of immature DCs. • Short Aβ peptides increase MHC II molecule expression and alloreactive T cell activation in TNF-α-matured DCs. • Short Aβ peptides exhibit decreased production of Th1/Th2 cytokines and pro-inflammatory cytokines.Amyloid beta peptide 1–15 (Aβ1–15) and its derivatives have attracted the attention of the scientific community as candidate vaccines for Alzheimer's disease (AD) immunotherapy. Recent studies suggested that Aβ1–42 modulated the immune system by inducing pro-inflammatory dendritic cells (DCs) with reduced antigen-presenting function. However, it remains elusive how Aβ1–15 impacts DCs function. We therefore investigated the modulation by short Aβ peptides of DCs from C57Bl/6J mice. Two new immunogens, a tandem repeat of two-lysine-linked Aβ1–15 sequences with or without an addition of a RGD motif, were tested. Chemotaxis, endocytosis, antigen presenting function and producing cytokines were measured. Both peptides increased migration/endocytosis of immature DCs and MHC II molecule expression/alloreactive T cell activation in TNF-α-matured DCs. In addition, they exhibited decreased production of Th1/Th2 cytokines and pro-inflammatory cytokines. Overall, the two peptides demonstrated strong immunogenicity but did not stimulate pro-inflammatory pathways. These results support the use of short Aβ immunogens in AD immunotherapy.
Keywords: Alzheimer's disease; Immunotherapy; Aβ1–15; Th1 cytokines; Anti-inflammatory cytokines; Immunogenicity;

Neuropeptide Y modulates functions of inflammatory cells in the rat: Distinct role for Y1, Y2 and Y5 receptors by Katarina Mitić; Stanislava Stanojević; Nataša Kuštrimović; Vesna Vujić; Mirjana Dimitrijević (1626-1633).
► Inflammatory cells express Y1, Y2 and Y5 receptor subtypes. ► NPY in vivo suppresses inflammatory cells functions via Y2/Y5 receptors. ► NPY in vitro stimulates leukocytes functions via Y1 receptor. ► Anti-inflammatory effect of NPY depends on Y receptors interplay and DP4 activity.Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.
Keywords: Granulocytes; Inflammation; Monocytes; Neuropeptide Y; Rat; Y receptor subtypes;

Apelin alleviates diabetes-associated endoplasmic reticulum stress in the pancreas of Akita mice by Hong Chen; Chenghong Zheng; Xin Zhang; Jianshuang Li; Jiong Li; Ling Zheng; Kun Huang (1634-1639).
► We examine the effects of adipokine apelin treatment on ER stress in the pancreas of Akita mice. ► Apelin treatment down-regulates IRE1α and PERK pathways. ► Apelin shows beneficial effects on the pancreatic islet mass and insulin content.Apelin, a newly identified bioactive adipokine, has been found to play important roles in multiple diseases, including diabetes, hypertension and cardiovascular diseases with unclear molecular mechanisms. The present study aimed to investigate the effects of apelin on endoplasmic reticulum (ER) stress in the pancreas of Akita mice, a well-established type 1 diabetic model. Apelin-13 (400 pmol/kg) was injected from tail vein for 10 weeks. The physiological characters of experimental animals were evaluated, pancreatic islet morphology and insulin content were assessed by immunohistochemistry, and ER stress markers in the pancreas were examined by Western blots. Our results indicate apelin treatment significantly ameliorates diabetes-induced reduction in pancreatic islet mass and insulin content. Further studies suggested apelin treatment alleviates ER stress by inhibiting the diabetes induced up-regulation of PERK and IRE1α and chaperones (GRP78, calnexin and Hsp70) levels in Akita mice. We also demonstrated that apelin treatment normalizes the diabetes induced alteration of AKT and ERK activations in the pancreas of Akita mice. Taken together, these results suggest a novel physiological role of apelin in alleviating ER stress in the pancreas of type 1 diabetes.
Keywords: Apelin; Akita mouse; ER stress; Diabetes mellitus; Pancreas;

Mimotopes of mutalysin-II from Lachesis muta snake venom induce hemorrhage inhibitory antibodies upon vaccination of rabbits by R.A. Machado de Avila; S. Stransky; M. Velloso; P. Castanheira; F.S. Schneider; E. Kalapothakis; E.F. Sanchez; C. Nguyen; F. Molina; C. Granier; C. Chávez-Olórtegui (1640-1646).
► Investigation of reactivity of a monoclonal antibody against continuous peptides from mutalysin-II. ► Identification of mutalysin-II peptide mimotopes using phage-display approach. ► Neutralization of hemorrhagic activity induced by Lachesis muta snake venom by peptide mimotope antibodies. ► Synthetic immunogens may be promising candidates for alternative therapeutic serum development.Mutalysin-II (mut-II) from Lachesis muta snake venom is an endopeptidase with hemorrhagic activity. A mAb against mutalysin-II that neutralized the hemorrhagic effect was produced previously. To identify the mAb epitopes, sets of 15-mer overlapping peptides covering the mut-II amino acid sequence were synthesized using the SPOT method and tested but failed to react with the mAb. Using a phage-display approach seventeen clones reactive with mAb were identified. Additional immunoassays with the peptides and mAb identified the QCTMDQGRLRCR, TCATDQGRLRCT, HCFHDQGRVRCA, HCTMDQGRLRCR and SCMLDQGRSRCR sequences as possible epitopes. Immunization of rabbits with these peptides induced antibodies that recognize mut-II and protected against the hemorrhagic effects of Lachesis venom.
Keywords: Lachesis muta venom; Antivenom; Phage-display; Synthetic peptides; Mimotope; Monoclonal antibody;

► PAC1hop, null and hip confer cAMP generation upon PACAP treatment in NG108-15 cells. ► PAC1hop and null confer elevation of [Ca2+]i, PAC1hop more efficaciously. ► Gene induction proceeds through PAC1hop-specific combinatorial or cAMP signaling. ► Gene induction through cAMP is mediated by PKA-dependent or -independent signaling.Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. Heterologous expression of the three major splice variants of the rat PAC1 receptor, PAC1hop, null and hip, in neural NG108-15 cells conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca2+]i occurred only in PAC1hop-, and to a lesser extent in PAC1null-expressing cells. Induction of vasoactive intestinal polypeptide (VIP) and stanniocalcin 1 (STC1), two genes potentially involved in PACAP's homeostatic responses, was examined as a function of the expressed PAC1 variant. VIP induction was greatest in PAC1hop-expressing cells, suggesting that a maximal transcriptional response requires combinatorial signaling through both cAMP and Ca2+. STC1 induction was similar for all three receptor splice variants and was mimicked by the adenylate cyclase activator forskolin, indicating that cAMP elevation is sufficient to induce STC1. The degree of activation of two different second messenger pathways appears to determine the transcriptional response, suggesting that cellular responses to stressors are fine-tuned through differential receptor isoform expression. Signaling to the VIP gene proceeded through cAMP and protein kinase A (PKA) in these cells, independently of the MAP kinase ERK1/2. STC1 gene induction by PACAP was dependent on cAMP and ERK1/2, independently of PKA. Differential gene induction via different cAMP dependent signaling pathways potentially provides further targets for the design of treatments for stress-associated disorders.
Keywords: PACAP; PAC1 receptor; cAMP; Calcium; Gene induction; Nervous system;

The action of a synthetic derivative of Met5-enkephalin-Arg6-Phe7 on behavioral and endocrine responses by Krisztina Csabafi; Miklós Jászberényi; Zsolt Bagosi; Géza Tóth; Mária Wollemann; Gyula Telegdy (1656-1660).
• The neuroendocrine actions of a synthetic derivate of MERF were investigated. • We examined the effect on corticosterone secretion and open field behavior. • Tyr-d-Ala-Gly-Phe-d-Nle-Arg-Phe (DADN) activated the HPA axis and stimulated the horizontal and vertical locomotion in the open field experiment. • Predominantly μ-receptors mediate the endocrine and behavioral actions of DADN.The neuroendocrine and behavioral effects of Tyr-d-Ala-Gly-Phe-d-Nle-Arg-Phe (DADN), a more stable derivative of the endogenous opiate Met-enkephalin related peptide Met5-enkephalin-Arg6-Phe7 were investigated in mice. The behavioral experiments consisted of monitoring the horizontal (square crossing) and vertical (rearing) locomotion in the open field system. To evaluate the effect of the heptapeptide on the hypothalamo–pituitary–adrenal (HPA) axis, the plasma corticosterone level was measured. DADN induced dose-dependent increases in locomotion and rearing 30 min after intracerebroventricular injection and also elicited marked activation of the hormonal stress response. To elucidate the receptors involved in the mediation of these actions, animals were pretreated with the nonselective opioid antagonist naloxone, the selective κ-receptor antagonist nor-binaltorphimine or the μ1-receptor blocker naloxonazine. Both the HPA activation and the behavioral responses were diminished by the preadministration of naloxone. Nor-binaltorphimine did not display a significant effect, while naloxonazine completely abolished the hyperactivity and the corticosterone elevation elicited by the analog. These findings suggest that μ-receptors predominate in the mediation of the neuroendocrine actions of DADN, while κ-receptors do not play a significant role.
Keywords: Met5-enkephalin-Arg6-Phe7; Open field; Opioid receptors; Hypothalamic–pituitary–adrenal axis;

► Peripheral administration of substance P (1–7) inhibits the expression of morphine tolerance in the rat. ► SP(1–7)-morphine treated rats display altered levels of dynorphin B in brain areas involved in opioid tolerance and dependence. ► SP(1–7)-morphine treated rats display altered levels of nociceptin in brain areas involved in opioid tolerance and dependence.The N-terminal substance P fragment SP1–7 is known to modulate hyperalgesia and opioid withdrawal in animal models. This study examined the effects of intraperitoneal (i.p.) injections of SP1–7 on chronic morphine tolerance and on the levels of dynorphin B (DYN B) and nociceptin/orphanin FQ (N/OFQ) in various brain areas of male Sprague–Dawley rats. Morphine tolerance was induced by subcutaneous injections of the opioid (10 mg/kg) twice daily for 7 days. SP1–7 injected i.p. (185 nmol/kg) 30 min prior to morphine reduced the development of morphine tolerance. Immunoreactive (ir) DYN B and N/OFQ peptide levels were measured in several areas of the central nervous system. Levels of ir DYN B in rats treated with SP1–7 and morphine were decreased in the nucleus accumbens, substantia nigra and ventral tegmental area and increased in the frontal cortex. The ir N/OFQ levels were increased in the periaqueductal gray and decreased in the nucleus accumbens. Since the concentration profiles of the two peptides were altered by SP1–7 in the areas that are implicated in the modulation of opioid tolerance and analgesia, it is suggested that DYN B and N/OFQ systems may be involved in the effects of SP1–7 on opioid tolerance.
Keywords: Dynorphin B (DYN B); Morphine; Nociceptin/orphanin FQ (N/OFQ); Opioid tolerance; Rat brain; Substance P (SP); SP1–7;

Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by prazosin by Albert Sattin; Albert Eugene Pekary; James Blood (1666-1676).
► Prazosin, an α1-adrenoceptor blocker, is an effect treatment for post-traumatic stress disorder. • Prazosin modulates release of TRH and TRH-like peptides in rat brain and peripheral tissues. ► TRH and TRH-like peptides have neuroprotective, antidepressant, and anxiolytic effects. ► These peptides may mediate some of the therapeutic effects of prozosin.Hyperresponsiveness to norepinephrine contributes to post-traumatic stress disorder (PTSD). Prazosin, a brain-active blocker of α1-adrenoceptors, originally used for the treatment of hypertension, has been reported to alleviate trauma nightmares, sleep disturbance and improve global clinical status in war veterans with PTSD. Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) may play a role in the pathophysiology and treatment of neuropsychiatric disorders such as major depression, and PTSD (an anxiety disorder). To investigate whether TRH or TRH-like peptides (pGlu-X-Pro-NH2, where “X” can be any amino acid residue) participate in the therapeutic effects of prazosin, male rats were injected with prazosin and these peptides then measured in brain and endocrine tissues. Prazosin stimulated TRH and TRH-like peptide release in those tissues with high α1-adrenoceptor levels suggesting that these peptides may play a role in the therapeutic effects of prazosin.
Keywords: Post-traumatic stress disorder; Thyrotropin releasing hormone; Limbic system;

Bombesin receptor subtype-3 agonists stimulate the growth of lung cancer cells and increase EGF receptor tyrosine phosphorylation by Terry W. Moody; Veronica Sancho; Alessia di Florio; Bernardo Nuche-Berenguer; Samuel Mantey; Robert T. Jensen (1677-1684).
• BRS-3 agonists stimulate the proliferation of lung cancer cells. BRS-3 ant. increased the cytotoxicity of gefitinib. • BRS-3 agonists increased the tyrosine phosphorylation of EGF receptor in lung cancer cells. • The EGF receptor tyrosine phosphorylation causes by addition of BRS-3 agonists to lung cancer cells occurs in a Src-, MMP- and oxygen-dependent manner.The effects of bombesin receptor subtype-3 (BRS-3) agonists were investigated on lung cancer cells. The BRS-3 agonist (DTyr6, (Ala11, Phe13, Nle14) bombesin6–14 (BA1), but not gastrin releasing peptide (GRP) or neuromedin B (NMB) increased significantly the clonal growth of NCI-H1299 cells stably transfected with BRS-3 (NCI-H1299-BRS-3). Also, BA1 addition to NCI-H727 or NCI-H1299-BRS-3 cells caused Tyr1068 phosphorylation of the epidermal growth factor receptor (EGFR). Similarly, (DTyr6, R-Apa11, Phe13, Nle14) bombesin6–14 (BA2) and (DTyr6, R-Apa11, 4-Cl,Phe13, Nle14) bombesin6–14 (BA3) but not gastrin releasing peptide (GRP) or neuromedin B (NMB) caused EGFR transactivation in NCI-H1299-BRS-3 cells. BA1-induced EGFR or ERK tyrosine phosphorylation was not inhibited by addition of BW2258U89 (BB2R antagonist) or PD168368 (BB1R antagonist) but was blocked by (DNal-Cys-Tyr-DTrp-Lys-Val-Cys-Nal)NH2 (BRS-3 ant.). The BRS-3 ant. reduced clonal growth of NCI-H1299-BRS-3 cells. BA1, BA2, BA3 and BRS-3 ant. inhibit specific 125I-BA1 binding to NCI-H1299-BRS-3 cells with an IC50 values of 1.1, 21, 15 and 750 nM, respectively. The ability of BRS-3 to regulate EGFR transactivation in NCI-H1299-BRS-3 cells was reduced by AG1478 or gefitinib (EGFR tyrosine kinase inhibitors), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant), Tiron (superoxide scavenger) and DPI (NADPH oxidase inhibitor). These results demonstrate that BRS-3 agonists may stimulate lung cancer growth as a result of EGFR transactivation and that the transactivation is regulated by BRS-3 in a Src-, reactive oxygen and matrix metalloprotease-dependent manner.
Keywords: Bombesin receptor subtype-3; Epidermal growth factor receptor; Tyrosine phosphorylation; Lung cancer; Proliferation;

Pharmacology and selectivity of various natural and synthetic bombesin related peptide agonists for human and rat bombesin receptors differs by Hirotsugu Uehara; Nieves González; Veronica Sancho; Samuel A. Mantey; Bernardo Nuche-Berenguer; Tapas Pradhan; David H. Coy; Robert T. Jensen (1685-1699).
• Pharmacology of 24 natural occurring/synthetic bombesin related agonists is compared for rat and human receptor bearing tissues. • Binding affinities/potencies and efficacy of cell activation are determined for all peptides. • 12 peptides had high affinity/potency for hGRP receptors, 8 for hNMB receptors, 1 for hBRS-3 receptor. • There was no correlation between affinities for hGRP and rat GRP receptors, but there was with h- and rat NMB receptors. • Results show the human and rat peptide agonist pharmacophore for human and rat GRP receptors differ markedly. • Results also report a number of high affinity/potency agonists that may be useful for human studies.The mammalian bombesin (Bn)-receptor family [gastrin-releasing peptide-receptor (GRPR-receptor), neuromedin B-receptor (NMB receptor)], their natural ligands, GRP/NMB, as well as the related orphan receptor, BRS-3, are widely distributed, and frequently overexpressed by tumors. There is increased interest in agonists for this receptor family to explore their roles in physiological/pathophysiological processes, and for receptor-imaging/cytotoxicity in tumors. However, there is minimal data on human pharmacology of Bn receptor agonists and most results are based on nonhuman receptor studies, particular rodent-receptors, which with other receptors frequently differ from human-receptors. To address this issue we compared hNMB-/GRP-receptor affinities and potencies/efficacies of cell activation (assessing phospholipase C activity) for 24 putative Bn-agonists (12 natural, 12 synthetic) in four different cells with these receptors, containing native receptors or receptors expressed at physiological densities, and compared the results to native rat GRP-receptor containing cells (AR42J-cells) or rat NMB receptor cells (C6-glioblastoma cells). There were close correlations (r  = 0.92–99, p  < 0.0001) between their affinities/potencies for the two hGRP- or hNMB-receptor cells. Twelve analogs had high affinities (≤1 nM) for hGRP receptor with 15 selective for it (greatest = GRP, NMC), eight had high affinity/potencies for hNMB receptors and four were selective for it. Only synthetic Bn analogs containing β-alanine11 had high affinity for hBRS-3, but also had high affinities/potencies for all GRP-/hNMB-receptor cells. There was no correlation between affinities for human GRP receptors and rat GRP receptors (r  = 0.131, p  = 0.54), but hNMB receptor results correlated with rat NMB receptor (r  = 0.71, p  < 0.0001). These results elucidate the human and rat GRP-receptor pharmacophore for agonists differs markedly, whereas they do not for NMB receptors, therefore potential GRP-receptor agonists for human studies (such as Bn receptor-imaging/cytotoxicity) must be assessed on human Bn receptors. The current study provides affinities/potencies on a large number of potential agonists that might be useful for human studies.
Keywords: Bombesin; Neuromedin B; Gastrin-releasing peptide; Bombesin receptor; Gastrin-releasing peptide receptor; BRS-3; Neuromedin B receptor; Satiety; Peptide mediated cytotoxicity; Tumor imaging; PRRT;

Role of vascular Kinin B1 and B2 receptors in endothelial nitric oxide metabolism by Rodrigo A. Loiola; Felipe C.G. Reis; Elisa M. Kawamoto; Cristoforo Scavone; Dulcinéia S. Abdalla; Liliam Fernandes; João Bosco Pesquero (1700-1705).
Display Omitted► Kinin B1 (inducible) and B2 (constitutive) receptors are located in vascular cells. ► Vascular reactivity and nitric oxide metabolism were studied in B1 −/− and B2 −/− mice. ► B1 −/− and B2 −/− present endothelial dysfunction and reduced NO levels. ► NO reduced availability is not related to vascular NO synthase deficiency. ► Kinin receptors may control oxidative processes involved in NO inactivation.Kinin B1 and B2 receptors play an essential role in inflammatory process and cardiovascular homeostasis. The present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B1 (B1 −/−) and B2 receptor (B2 −/−) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B1 −/− and B2 −/− in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B1 −/− and B2 −/−. In contrast, in the presence of l-arginine, Ca2+ and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B1 −/− and B2 −/−. The present study demonstrated that targeted deletion of B1 or B2 receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B1 −/− and B2 −/− vessels. The present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability.
Keywords: B1 receptor; B2 receptor; Knockout; Bradykinin; Vascular reactivity; Nitric oxide;

Involvement of the atrial natriuretic peptide in the reduction of arterial pressure induced by swimming but not by running training in hypertensive rats by Patrick W. Endlich; Luciana B. Firmes; Washington L.S. Gonçalves; Sonia A. Gouvea; Margareth R. Moysés; Nazaré S. Bissoli; Adelina M. Reis; Glaucia R. Abreu (1706-1712).
► Under resting conditions, hypertensive rats showed an increase in plasma ANP levels from chronic training. ► The swimming training similarly modified the expression of the natriuretic peptide receptor-type A and C in the kidney of SHR. ► The increase in the plasma ANP levels by swimming training decreased the degradation in the mesenteric fat tissue. ► The ANP could be involved in the reduction of blood pressure that was induced by swimming training in SHR but not by running training.The aim of this study was to compare, under resting conditions, the influence of chronic training in swimming or running on mean arterial pressure (MAP) and the involvement of the natriuretic peptide system in this response. Two-month-old male spontaneously hypertensive rats (SHR) were divided into three groups—sedentary (SD), swimming (SW) and running (RN)—and were trained for eight weeks under regimens of similar intensities. Atria tissue and plasma atrial natriuretic peptide (ANP) concentrations were measured by radioimmunoassay. ANP mRNA levels in the right and left atria as well as the natriuretic peptide receptors (NPR), NPR-A and NPR-C, mRNA levels in the kidney were determined by real-time PCR. Autoradiography was used to quantify NPR-A and NPR-C in mesenteric adipose tissue. Both training modalities, swimming and running, reduced the mean arterial pressure (MAP) of SHR. Swimming, but not running, training increased plasma levels of ANP compared to the sedentary group (P  < 0.05). Expression of ANP mRNA in the left atrium was reduced in the RN compared to the SD group (P  < 0.05). Expression of NPR-A and NPR-C in the kidneys of the SW group decreased significantly (P  < 0.05) compared to the SD group. Although swimming increased 125I-ANP binding to mesenteric adipose tissue, displacement by c-ANF was reduced, indicating a reduction of NPR-C. These results suggest that the MAP reduction induced by exercise in SHR differs in its mechanisms between the training modalities, as evidenced by the finding that increased levels of ANP were only observed after the swimming regimen.
Keywords: Exercise training; Natriuretic peptide receptors; Spontaneously hypertensive rats; Fat tissue;

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes by S. Del Ry; M. Cabiati; F. Vozzi; B. Battolla; C. Caselli; F. Forini; C. Segnani; T. Prescimone; D. Giannessi; L. Mattii (1713-1718).
• Cardiac cells as well as endothelial cells were able to produce CNP. • NPR-B mRNA expression was found in cardiac and endothelial cells. • Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination and by immunocytochemistry studies. • By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes.C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP = 0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.
Keywords: C-type natriuretic peptide; Cardiomyocytes; Gene expression; Immunostaining;

Isolation and characterization of cytotoxic cyclotides from Viola philippica by Wenjun He; Lai Yue Chan; Guangzhi Zeng; Norelle L. Daly; David J. Craik; Ninghua Tan (1719-1723).
• Eight new cyclotides were characterized from the Viola philippica. • All cyclotides were evaluated for their cytotoxic activity. • The novel cyclotides enhance the sequence variation observed for cyclotides.Cyclotides are a large family of plant peptides characterized by a macrocyclic backbone and knotted arrangement of three disulfide bonds. This unique structure renders cyclotides exceptionally stable to thermal, chemical and enzymatic treatments. They exhibit a variety of bioactivities, including uterotonic, anti-HIV, cytotoxic and hemolytic activity and it is these properties that make cyclotides an interesting peptide scaffold for drug design. In this study, eight new cyclotides (Viphi A–H), along with eight known cyclotides, were isolated from Viola philippica, a plant from the Violaceae family. In addition, Viba 17 and Mram 8 were isolated for the first time as peptides. The sequences of these cyclotides were elucidated primarily by using a strategy involving reduction, enzymatic digestion and tandem mass spectroscopy sequencing. Several of the cyclotides showed cytotoxic activities against the cancer cell lines MM96L, HeLa and BGC-823. The novel cyclotides reported here: (1) enhance the known sequence variation observed for cyclotides; (2) extend the number of species known to contain cyclotides; (3) provide interesting structure–activity relationships that delineate residues important for cytotoxic activity. In addition, this study provides insights into the potential active ingredients of traditional Chinese medicines.
Keywords: Cyclotides; Cyclic peptides; Violaceae; Viola philippica; Cytotoxic activity; Viphi A–H;

Isolation and characterization of Neosartorya fischeri antifungal protein (NFAP) by Laura Kovács; Máté Virágh; Miklós Takó; Tamás Papp; Csaba Vágvölgyi; László Galgóczy (1724-1731).
• New antifungal protein (NFAP) and its encoding gene were isolated and characterized. • This small, basic and cysteine-rich protein is homologous to similar proteins. • NFAP exhibited growth inhibitory activity against filamentous fungi. • The protein maintained its activity within broad pH and temperature ranges.A novel 6.6 kDa antifungal peptide (NFAP) from the culture supernatant of the mold, Neosartorya fischeri (anamorf: Aspergillus fischerianus), and its encoding gene were isolated in this study. NFAP is a small, basic and cysteine-rich protein consisting of 57 amino acid residues. It shows 37.9–50% homology to similar proteins described in literature from Aspergillus clavatus, Aspergillus giganteus, Aspergillus niger, and Penicillium chrysogenum. The in silico presumed tertiary structure of NFAP, e.g. the presence of five antiparallel β-sheet connected with filaments, and stabilized by three disulfide bridges, is very similar to those of the defensin-like molecules. NFAP exhibited growth inhibitory action against filamentous fungi in a dose-dependent manner, and maintained high antifungal activity within broad pH and temperature ranges. Furthermore, it exhibited relevant resistance to proteolysis. All these characteristics make NFAP a promising candidate for further in vitro and in vivo investigations aiming at the development of new antifungal compounds.
Keywords: Defensin-like protein; Filamentous fungi; Protein isolation; Antifungal activity; Antifungal susceptibility;

Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species (ROS) and disruption of cell wall against Candida albicans by Indresh Kumar Maurya; Sarika Pathak; Monika Sharma; Hina Sanwal; Preeti Chaudhary; Santosh Tupe; Mukund Deshpande; Virander Singh Chauhan; Rajendra Prasad (1732-1740).
Both scanning (top panel) and transmission electron (bottom panel) microscopy show cell wall damage by VS peptide.Display Omitted► VS2 and VS3 peptides are amphipathic cationic, containing a non- protein amino acid α,β-didehydrophenylalanine. ► VS2 and VS3 peptides enter the Candida cells by permeabilizating the membrane. ► Damage to the Candida cell wall was shown by scanning and transmission electron microscopy. ► VS2 and VS3 peptides rapidly kill albicans and non-albicans species of Candida by cell wall disruption, ROS accumulation and necrosis. ► These peptides are synergistic to azoles.In the present work, we investigated the antifungal activity of two de novo designed, antimicrobial peptides VS2 and VS3, incorporating unnatural amino acid α,β-dehydrophenylalanine (ΔPhe). We observed that the low-hemolytic peptides could irreversibly inhibit the growth of various Candida species and multidrug resistance strains at MIC80 values ranging from 15.62 μM to 250 μM. Synergy experiments showed that MIC80 of the peptides was drastically reduced in combination with an antifungal drug fluconazole. The dye PI uptake assay was used to demonstrate peptide induced cell membrane permeabilization. Intracellular localization of the FITC-labeled peptides in Candida albicans was studied by confocal microscopy and FACS. Killing kinetics, PI uptake assay, and the intracellular presence of FITC-peptides suggested that growth inhibition is not solely a consequence of increased membrane permeabilization. We showed that entry of the peptide in Candida cells resulted in accumulation of reactive oxygen species (ROS) leading to cell necrosis. Morphological alteration in Candida cells caused by the peptides was visualized by electron microscopy. We propose that de novo designed VS2 and VS3 peptides have multiple detrimental effects on target fungi, which ultimately result in cell wall disruption and killing. Therefore, these peptides represent a good template for further design and development as antifungal agents.
Keywords: Candida albicans; Antifungal peptides; Cell wall; ROS;

Identification of an antifungal peptide from Trapa natans fruits with inhibitory effects on Candida tropicalis biofilm formation by Santi M. Mandal; Ludovico Migliolo; Octavio L. Franco; Ananta K. Ghosh (1741-1747).
Cartoon structure evidences a single coil with the disulfide bridge.Display Omitted► Tn-AFP1, a small peptide of 1230 Da was purified from the fruits of Trapa natans, showed antifungal activity against human pathogen, Candida tropicalis. ► The MIC and IC50 values were 32 μg/ml and 16 μg/ml against C. tropicalis. ► Sequence of the peptide was determined by tandem mass spectrometry and found to contain eleven amino acid residues, showed a single coil structure attached by a unique disulphide bond anticipated by in silico analysis. ► Tn-AFP1 downregulated MDR1 and ERG11 gene expression level and disrupted bioflim formation of C. tropicalis. ► In summary, this peptide could be used as a promising candidate for the development of more effective antimycotic compounds.Due to recent emergence of fungal pathogens resistant to current antifungal therapies, several studies have been focused on screening of plant peptides to find novel compounds having antifungal activities. Here, a novel antifungal plant peptide, with molecular mass of 1230 Da was purified from fruits of Trapa natans by reverse phase high performance liquid chromatography using 300SB-C18 column and named as Tn-AFP1. Determination of complete amino acid sequences of this peptide by tandem mass spectrometry showed to contain following eleven amino acid residues: LMCTHPLDCSN. Purified Tn-AFP1 showed the inhibition of Candida tropicalis growth in vitro and disrupted the biofilm formation in a concentration dependent manner. It also showed downregulation of MDR1 and ERG11 gene expression in real time-PCR analysis. In silico molecular modeling predicted the structure of Tn-AFP1 as a single coil attached by a unique disulfide bond. Characterization of Tn-AFP1 could contribute in designing novel derivative(s) of this peptide for the development of more effective antimycotic compounds.
Keywords: Antifungal peptide; Candida tropicalis; Fruits; Trapa natans; Tn-AFP1;

Antimicrobial and antibiofilm activity of pleurocidin against cariogenic microorganisms by Rui Tao; Zhongchun Tong; Yuan Lin; Yunpeng Xue; Wei Wang; Rong Kuang; Ping Wang; Yu Tian; Longxing Ni (1748-1754).
► MIC and MBC results showed that pleurocidin had different antimicrobial activities against the tested oral strains. ► Pleurocidin dissolved in saliva still showed antimicrobial effects against oral microorganisms. ► Pleurocidin showed a favorable killing effect against BioFlux flow biofilms in vitro. ► Pleurocidin caused a remarkable modification of cell membrane or overall shape.Dental caries is a common oral bacterial infectious disease of global concern. Prevention and treatment of caries requires control of the dental plaque formed by pathogens such as Streptococcus mutans and Streptococcus sobrinus. Pleurocidin, produced by Pleuronectes americanus, is an antimicrobial peptide that exerts broad-spectrum activity against pathogenic bacteria and fungi. Moreover, pleurocidin shows less hemolysis and is less toxic than other natural peptides. In the present study, we investigated whether pleurocidin is an effective antibiotic peptide against common cariogenic microorganisms and performed a preliminary study of the antimicrobial mechanism. We assayed minimal inhibitory concentration (MIC), minimal bactericide concentration (MBC) and bactericidal kinetics and performed a spot-on-lawn assay. The BioFlux system was used to generate bacterial biofilms under controllable flow. Fluorescence microscopy and confocal laser scanning microscopy (CLSM) were used to analyze and observe biofilms. Scanning electron microscopy was used to observe the bacterial membrane. MIC and MBC results showed that pleurocidin had different antimicrobial activities against the tested oral strains. Although components of saliva could affect antimicrobial activity, pleurocidin dissolved in saliva still showed antimicrobial effects against oral microorganisms. Furthermore, pleurocidin showed a favorable killing effect against BioFlux flow biofilms in vitro. Our findings suggest that pleurocidin has the potential to kill dental biofilms and prevent dental caries.
Keywords: Pleurocidin; Dental caries; MIC; Inhibitory zone; Shear force; Biofilm; SEM;

Bactericidal effect of Naja nigricollis toxin γ is related to its membrane-damaging activity by Li-Wen Chen; Pei-Hsiu Kao; Yaw-Syan Fu; Wan-Ping Hu; Long-Sen Chang (1755-1763).
► Toxin γ shows a similar inhibitory activity on the growth of Staphylococcus aureus and Escherichia coli. ► Bactericidal activity of toxin γ depends on its ability to induce membrane permeability. ► Lipopolysaccharide and lipoteichoic acid structurally suppress bactericidal effect of toxin γ.The aim of the present study is to investigate the causal relationship between membrane-damaging activity and bactericidal activity of Naja nigricollis toxin γ. Toxin γ showed a similar inhibitory activity on the growth of Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria). Antibacterial activity of toxin γ correlated positively with increase in membrane permeability of bacterial cells. Morphological examination showed that toxin γ disrupted the integrity of bacterial membrane. Toxin γ showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall increased bactericidal effect of toxin γ on E. coli and S. aureus, respectively. Although the potency of toxin γ on permeabilzing model membrane of E. coli and S. aureus was similar, the mode of interaction between toxin γ and model membrane of E. coli and S. aureus differed. Membrane-damaging activity of toxin γ was inhibited by either LPS or LTA. Nevertheless, LPS and LTA altered differently membrane-bound conformation of toxin γ. Taken together, our data suggest that bactericidal activity of toxin γ depends on its ability to induce membrane permeability, and that LPS and LTA structurally suppresses bactericidal effect of toxin γ.
Keywords: Snake venom; Cardiotoxin; Bactericidal effect; Membrane-damaging activity;

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs by Edwin G. Walsh; Sam Maher; Marc Devocelle; Peter J. O’Brien; Alan W. Baird; David J. Brayden (1764-1773).
High content analysis for cytoxicity of peptides on epithelial cells. Images of dye-loaded cells are acquired via multi-well automation by epifluorescence microscopy and are then subjected to segmentation analysis.Display Omitted► We describe a multi-parameter high-content analysis protocol for assessing peptide toxicity on epithelial cells. ► Selected deletion of amino acids of the bee venom, melittin, reduces its cytotoxicity. Loss of cytotoxicity was correlated with loss of epithelial permeation enhancement but not a reduction in antimicrobial activity.Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development. We describe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic α-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.
Keywords: Antimicrobial peptide; Cytotoxicity screening; High-content-analysis (HCA); Melittin; MTT assay;

On the local cardiac renin angiotensin system. Basic and clinical implications by Walmor C. De Mello; Edward D. Frohlich (1774-1779).
• Role of local cardiac renin angiotensin system on cardiac remodeling in animal models and in humans. • Angiotensin II and regulation of heart cell volume. • Activation of the cardiac renin angiotensin system; influence on impulse propagation and cardiac arrhythmias. • Clinical and basic implications of activation of the local cardiac renin angiotensin system.In the present review we reevaluated the experimental and clinical evidence that there is a local renin angiotensin system in the heart as well as the presence of a functional intracrine component which is activated during pathological conditions like heart failure and hypertension. The implications of these findings for cardiology were discussed. The novel finding that cell swelling impairs cell coupling and impulse propagation through activation of ionic channels with consequent generation of cardiac arrhythmias and the evidence that AT1 receptors are mechanosensors able to alter the heart function independently of Ang II were discussed. Particular attention was given to the role of salt loading on the activation of a local cardiac renin angiotensin and its consequences.
Keywords: Local renin; Angiotensin; Heart failure; Hypertrophy; Cell swelling; Arrhythmia;