Schistosoma mansoni neuropeptide amidating enzymes

Schistosoma mansoni neuropeptide amidating enzymes

Abstracts / Regulatory Peptides 164 (2010) 26–34 Discussion: Thus desHis1Pro4Glu9glucagon-amide is more effective than its fatty acid derivatized cou...

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Abstracts / Regulatory Peptides 164 (2010) 26–34

Discussion: Thus desHis1Pro4Glu9glucagon-amide is more effective than its fatty acid derivatized counterpart peptide at antagonising glucagon induced increases in plasma glucose and insulin. doi:10.1016/j.regpep.2010.07.077

S23 Evaluation of role of islet cell paracrine interactions in regulation of insulin secretion using a new heterotypic pseudoislet model C. Kellya,b, H. Guo Parkea,b, J.T. McCluskeya, P.R. Flatta, H.N. McClenaghana a The SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK b School of Medicine, Dentistry and Biomedical Science, Queen's University of Belfast, Northern Ireland, UK Introduction: Islets of Langerhans are anatomically complex microorgans comprised of heterogenous cell types that secrete insulin, glucagon or somatostatin. However, the importance of cellular interactions in overall regulation of insulin secretion and islet function is unclear. Aim: This study evaluated a heterotypic pseudoislet model containing clonal insulin-(MIN6), glucagon-(αTC1.9) and somatostatin-(TGP52) secreting cells, and compared these pseudoislets with traditional cultured monolayer preparations. Methods: Heterotypic pseudoislets formed in culture were sectioned and stained for insulin. Cellular viability (MTT and LDH) and proliferation (BrdU ELISA) were measured in monolayer and pseudoislet cultures. Cellular hormone content and functional insulin release in response to a variety of stimuli was measured by RIA. Differential expression of adhesion molecules was assessed by RTPCR and Western blot. Results: Heterotypic pseudoislets appeared anatomically correct with a central insulin stained core. Pseudoislet cells displayed reduced proliferation coupled with an increase in cytotoxicity (P < 0.001), which likely contributes to their static size in culture. While MIN6 and TGP52 cells expressed E-cadherin and showed sustained or improved hormone content when configured as pseudoislets, αTC1.9 lacked E-cadherin and contained less glucagon following pseudoislet formation. MIN6 and αTC1.9 cells expressed connexin36, while TGP52 cells expressed connexin43 only. Insulin release from heterotypic pseudoislets was equal (in response to arginine, alanine and GLP-1) or greater (P < 0.01, extracellular calcium) than MIN6 pseudoislets. However, release from heterotypic monolayers was significantly reduced (P < 0.001) compared with MIN6 monolayers. Discussion: Heterotypic pseudoislets display enhanced functionality indicating the utility of this model to study interactions between insulin, glucagon and somatostatin secreting cells. doi:10.1016/j.regpep.2010.07.078

S24 A novel analogue of oxyntomodulin with improved glucose-lowering, insulinotropic and anorexigenic actions B.D. Kerr, P.R. Flatt, V.A. Gault The SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK Introduction: Oxyntomodulin (Oxm) is a 37-aa peptide which has been shown to exhibit a range of beneficial actions for alleviation of obesity-diabetes. However, possible exploitation of Oxm-based therapies has been hindered due to proteolytic degradation by dipeptidylpeptidase-IV (DPP-IV).

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Aim: To assess the glucose-lowering, insulinotropic and anorexigenic actions of novel chemically modified analogues of Oxm. Methods: Synthetic Oxm, (d-Ser2)Oxm and (d-Ser2)Oxm[mPEGPAL] were assessed for in vitro enzyme stability, cAMP activity and insulin secretion. In vivo actions of Oxm analogues on glucose homeostasis, insulin secretion, food intake, body weight and lipid markers were examined in obese diabetic (ob/ob) mice. Results: (d-Ser2)Oxm and (d-Ser2)Oxm[mPEG-PAL] exhibited enhanced DPP-IV resistance. Oxm, (d-Ser2)Oxm and (d-Ser2)Oxm [mPEG-PAL] stimulated in vitro cAMP production and insulin secretion in a concentration-dependent manner with similar potency in BRIN-BD11 cells. Acute administration of (d-Ser2)Oxm[mPEG-PAL] or (d-Ser2)Oxm reduced plasma glucose (1.5-fold; P < 0.01) and food intake (1.4-fold; P < 0.01), while plasma insulin levels were elevated (1.8-fold; P < 0.001). Once-daily administration of (d-Ser2)Oxm [mPEG-PAL] for 14 days to ob/ob mice decreased food intake (1.5fold; P < 0.001), bodyweight (1.5-fold; P < 0.01), plasma glucose (1.5fold; P < 0.01) and increased plasma insulin (1.5-fold; P < 0.01). Furthermore, daily (d-Ser2)Oxm[mPEG-PAL] administration improved glucose tolerance (1.6-fold; P < 0.001) and increased glucose-mediated insulin secretion (1.8-fold; P < 0.01), pancreatic insulin content (1.5-fold; P < 0.001), and circulating adiponectin (1.4-fold; P < 0.01). It decreased both visfatin (1.3-fold; P < 0.01) and triglyceride levels (1.4-fold; P < 0.01). Discussion: The effectiveness of (d-Ser2)Oxm[mPEG-PAL] in improving glucose homeostasis, insulin secretion, satiety, bodyweight and markers of fat metabolism suggests significant promise for Oxm-based therapies in obesity-diabetes. doi:10.1016/j.regpep.2010.07.079

S25 Schistosoma mansoni neuropeptide amidating enzymes Louise E. Atkinsona, Paul McVeigha, Nikki J. Marksa, Michael J. Kimberb, Tim A. Dayb, Betty A. Eipperc, Richard E. Mainsc, Aaron G. Maulea a Molecular Biosciences-Parasitology, School of Biological Sciences, Queen's University Belfast, UK b Department of Biomedical Sciences, Iowa State University, USA c University of Connecticut Health Centre, Connecticut, USA Introduction: The majority of biologically active neuropeptides display a C-terminal amide (NH2), generated by the sequential action of two neuropeptide processing enzymes, peptidylglycine alphahydroxylating monooxygenase (PHM) and peptidylglycine alphaamidating lyase (PAL). In vertebrates, PHM and PAL are expressed as separate domains of the bifunctional protein peptidylglycine alphaamidating monooxygenase (PAM), while a number of invertebrates display various different arrangements of monofunctional enzymes. Previous work has characterised Schistosoma mansoni PHM (SmPHM), suggesting that SmPHM and S. mansoni PAL (SmPAL) are expressed as separate monofunctional proteins. Aims: This study aimed to use biochemical methods and postgenomic tools to validate SmPAL and SmPHM candidature as drug targets. Methods: Functional expression of SmPAL in hEK cells, in situ hybridisation of SmPAL transcript, and RNA interference (RNAi) of SmPHM and SmPAL. Results: In situ hybridization demonstrated that in adult schistosomes, SmPAL mRNA (Sm-pal-1) is expressed in the central nervous system, consistent with its role in the amidation of neuropeptides in S. mansoni. Heterologous expression showed that SmPAL is a catalytically active, efficiently secreted amidating enzyme, with functional characteristics analogous to other eukaryotic amidating enzymes. Unfortunately,

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Abstracts / Regulatory Peptides 164 (2010) 26–34

RNA interference (RNAi) of Sm-phm-1 and Sm-pal-1 was inconsistent and did not associate with any observable aberrant phenotype. Discussion: The fundamental role and biological importance of SmPAL in neuropeptide maturation, and key structural differences from the mammalian host enzyme, make it an appealing drug target candidate. doi:10.1016/j.regpep.2010.07.080

S26 Construction of cDNA libraries from trifluoroacetic acid-solvated amphibian skin secretions: molecular cloning of multiple bombinin-like peptide precursor transcripts from a library of yellow-bellied toad (Bombina variegata) secretion B. Bai, L. Wang, M. Zhou, T. Chen, C. Shaw Molecular Therapeutics Research, School of Pharmacy, Queens University Belfast, Northern Ireland, UK Amphibian skin secretions are renowned as complex mixtures of bioactive peptides many of which are analogues of endogenous regulatory peptides. While skin secretions can be obtained noninvasively for peptidome analysis, parallel studies on the granular gland transcriptome required specimen sacrifice. The aim of the present study was to analyse archived skin secretions to determine the robustness of bioactive peptide precursor-encoding polyadenylated mRNAs in an attempt to extract maximum molecular information from rare samples. A range of solvated skin secretion samples were examined after lyophilisation for their potential to generate viable and comprehensive cDNA libraries based upon polyadenylated mRNA capture and amplification/cloning using appropriate commercial kits. Here we present unequivocal data that the granular gland transcriptome persists in a PCR amenable format even after storage for as long as 12 years in 0.1%(v/v) aqueous trifluoroacetic acid (TFA). We used a pooled skin secretion sample (2 ml) from the yellow-bellied toad, Bombina variegata (n =14), containing the equivalent of 5 mg/ml of lyophilised skin secretion, that had been used in part for peptide isolation purposes in 1998 and had been stored at −20 °C since that time. In the first cloning experiment, 12 different bombinin-like peptide precursor cDNAs were cloned encoding 17 different bombinins, the majority of which were novel. Subsequently, bombesin and bradykininrelated peptide precursor transcripts have been cloned successfully. These data illustrate the unexpected stability/longevity of the transcriptome in these secretions — a finding with implications for both this field of research and for the wider field of molecular biology. doi:10.1016/j.regpep.2010.07.081

S27 Natriuretic peptide infusion in myocardial ischemia/reperfusion is associated with reduced myocardial damage and inversed endogenous release of natriuretic peptide in pigs B.S. Kousholta,b, J.R. Larsena, J.M. Hasenkama, J.P. Goetzeb a Departments of Cardiothoracic and Vascular Surgery, University of Aarhus, Denmark b Department of Clinical Biochemistry, University of Copenhagen, Denmark Introduction: Plasma measurement of cardiac natriuretic peptides has gained clinical use in diagnosis of heart disease. In cardiac disease, the peptides may possess cardioprotective properties by reducing local fibrosis. Aims: We examined the acute cardiac effects of infusion of B-type natriuretic peptide (BNP) and a C-type natriuretic analogue (CD-NP) in a porcine model of myocardial ischemia/reperfusion.

Methods: Anaesthetized pigs were subjected to 1 h of regional cardiac ischemia followed by 3 h of reperfusion. The systemic response was monitored by hemodynamic measurements. Blood samples were collected every hour. Cardiac damage was assessed by biochemical measurements of myocardial damage by porcine-specific immunoassays and PCR. Infarct size was determined by tetrazolium staining. Results: Pigs tolerated the BNP and CD-NP infusion well with a similar decrease in systemic blood pressure (~20 mmHg) and an increase in diuresis compared to controls. BNP infusion selectively reduced the cardiac release of troponin T by 30%, suggesting a major cardioprotective effect. Moreover, the endogenous natriuretic peptide release was completely inversed by the BNP infusion with a decrease in plasma proANP concentrations compared to an increase in both control and CD-NP infused animals. Discussion: For the first time, we show that BNP infusion in ischemia/reperfusion damage selectively reduces cardiac damage and inverses the cardiac response in endogenous release of natriuretic peptides. The data suggest that the cardioprotective effects are mediated mainly through the NPR-A receptor. Taken together, our results advocate for pursuing natriuretic peptide treatment in cardioprotection in cardiac ischemia and myocardial infarction. doi:10.1016/j.regpep.2010.07.082

S28 Inhibition of endoplasmic reticulum stress by intermedin1-53 protects against myocardiac ischemia/reperfusion injury through CRLR/RAMPs-Akt signalling pathway Yong Fen Qia,b, Xu Tenga, Jun Qiu Songa, Gai Gai Zhanga, Yan Caia, Chao Shu Tanga,b a Laboratory of Cardiovascular Bioactive Molecule, School of Basic Medical Sciences, Peking University, Beijing 100191, China b Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100191, China Introduction: Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. Our previous investigations have shown that IMD1–53 ameliorates impaired cardiac function and apoptosis of myocardial ischemia/reperfusion (I/R). However, the mechanism of which is unclear. Aims: To explore whether the cardioprotective effect of IMD is mediated by inhibiting myocardial endoplasmic reticulum stress (ERS). Methods: An in vivo model of rat heart was used with ischemia/ reperfusion. Cardiac function was monitored, and degree of myocardial injury, level of ERS markers, and number of apoptotic cardiomyocytes were determined. Results: Compared with control group, the I/R group showed significantly decreased cardiac function, seriously damaged myocardial tissue, increased number of apoptotic cells, and overexpression of protein of ERS markers. However, administration of IMD1–53 in vivo (20 nmol/kg) greatly limited the myocardial infarction size, attenuated myocardial injury and apoptosis, and decreased the expression of ERS markers: it decreased the protein levels of glucose-regulated protein78 (GRP78), GRP94, C/EBP homologous protein (CHOP), and reduced caspase-12 activation. Furthermore, in vitro, IMD1–53 directly inhibited the myocardial ERS response induced by tunicamycin or dithiothreitol in rat cardiac tissue. The effect of IMD1–53 was abolished by IMD receptor antagonize, IMD17–47, and PI3K inhibitor, LY294002. Discussion: Our data demonstrated that IMD1–53 ameliorated I/R injury through inhibiting ERS, and the effect of IMD1–53 was via CRLR/RAMPs-Akt signalling pathway. doi:10.1016/j.regpep.2010.07.083