All posts in Raw News

Publication date: Available online 24 January 2014 Source:Peptides

Author(s): Gregory A.F. Douglas , Rebecca McGirr , Carlie L. Charlton , Dov B. Kagan , Lisa M. Hoffman , Leonard G. Luyt , Savita Dhanvantari

Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R) are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr³(octanoyl), Lys¹⁹(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys¹⁹(Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissue.

Publication date: Available online 17 January 2014 Source:Peptides

Author(s): Graham E. Jackson , Riedaa Gamieldien , Grace Mugumbate , Gerd Gäde

Melme-CC (pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp amide) and Declu-CC (pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn amide) are members of the insect adipokinetic hormone family with very different activities in the locust bioassay. The conformations of both peptides were determined in water and in a phospholipid (DPC) micelle solution using nuclear magnetic resonance (NMR) restrained molecular dynamics simulations. In water, Melme-CC has one dominant conformation while in DPC solution it has two preferred confirmations. In water, Declu-CC has two conformations but in DPC solution it has one preferred conformation, which is similar to one of the water conformations. All the conformations have type IV β-turn between residues 4 and 7. The binding of the two peptides to the DPC micelle is different. Melme-CC does not bind strongly to the surface and is oriented with the β-turn facing the surface. Declu-CC interacts more strongly with the β-turn facing away from the surface. Both termini having hydrophobic interactions with the surface. In Declu-CC the side chain of Asp⁷ projects away from the chain while in Melme-CC the Asn⁷ side chain is folded inside the chain. The different orientation of these side chains may account for the much higher biological activity of Declu-CC in mobilizing lipids in the locust compared to the poor biological effect of Melme-CC in this bioassay. Receptor binding of Declu-CC was tested using a model AKH receptor from Anopheles gambiae. A free energy of binding of −38.5kJmol⁻¹ was found.

Graphical abstract

Publication date: January 2014 Source:Peptides, Volume 51

Author(s): Shunsuke Shimizu , Hiroyuki Kaiya , Kouhei Matsuda

Ghrelin is a potent orexigenic peptide implicated in appetite regulation in rodents. However, except for teleost fish, the involvement of ghrelin in the regulation of feeding in non-mammalian vertebrates has not been well studied. Anuran amphibian larvae feed and grow during the pre- and prometamorphic stages, but, thereafter they stop feeding as the metamorphic climax approaches. Therefore, orexigenic factors seem to play important roles in growing larvae. In the present study, we examined the effect of intraperitoneal (IP) or intracerebroventricular (ICV) administration of synthetic bullfrog ghrelin (n-octanoylated 28-amino acid form) on food intake in larvae at the prometamorphic stages. Cumulative food intake was significantly increased by IP (8 and 16pmol/g body weight (BW)) or ICV (0.5 and 1pmol/g BW) administration of ghrelin during a 15-min observation period. The orexigenic action of ghrelin at 8pmol/g BW (IP) or at 0.5pmol/g BW (ICV) was blocked by treatment with a growth hormone secretagogue-receptor antagonist, [D-Lys³]GHRP-6 at 80pmol/g BW (IP) or at 5pmol/g BW (ICV). We then investigated the effect of feeding status on expression levels of the ghrelin transcript in the hypothalamus and gastrointestinal tract. Ghrelin mRNA levels in both were decreased 15 and 60min after feeding. These results indicate that ghrelin acts as an orexigenic factor in bullfrog larvae.

Publication date: February 2014 Source:Peptides, Volume 52

Author(s): Ke-ke Qi , Jie Wu , Jing Wan , Xiao-ming Men , Zi-wei Xu

The rapid degradation of porcine glucagon-like peptide-2 (pGLP-2) by the enzyme dipeptidyl peptidase-IV (DPP-IV) is the main impediment in the development of pGLP-2 as a potential therapeutic agent for intestinal dysfunction and damage. In this study, one mono-modified Lys³⁰-polyethylene glycol (PEG)-pGLP-2 was prepared using mPEG-succinimidyl propionate. To determine the optimized condition for PEGylation, the reactions were monitored by RP-HPLC and MALDI-TOF-MS. Stability was tested in purified DPP-IV in vitro. In vivo, the protective effects for colonic injury were measured in dextran sulfate sodium (DSS)-induced colitis in mice. The monoPEGylated products reached the maximum yield at 4:1 ratio of mPEG5k-SPA to pGLP-2. An effective method of successfully separating PEGylated pGLP-2 from mPEG-SPA5kD using CM Sepharose Fast Flow resin was established. The half-life of Lys³⁰-PEG-pGLP-2 was 16-fold longer than that of pGLP-2 in DPP-IV. The DSS mice exhibited marked weight loss), which was significantly reduced by Lys³⁰-PEG-pGLP-2 therapy. DSS treatment significantly increased colonic damage score, which was significantly reduced by administration of Lys³⁰-PEG-pGLP-2 in DSS-mice. DSS-induced colitis clearly induced Myeloperoxidase activity in the colon, which was significantly reduced by treatments with 3% DSS-pGLP-2 or 3% DSS-PEG-pGLP-2. These results showed that site-specific Lys³⁰-PEG-GLP-2 was resistant to degradation and reduced the severity of colonic injury in murine colitis. The enhanced biological potency of this product highlighted its potential as a therapeutic agent for intestinal diseases.

Publication date: January 2014 Source:Peptides, Volume 51

Author(s): Gao Sang , Jian-Min Du , Yong-Yi Chen , Yang-Bo Chen , Jun-Xian Chen , Yong-Can Chen

Copeptin reflects the individual stress level, and is correlated with outcomes of critical illness. This study was designed to evaluate its relationship with disease severity, local complications, organ failure and mortality of severe acute pancreatitis (SAP). Seventy-eight SAP patients and 78 sex- and age-matched healthy individuals were recruited. Plasma samples were obtained on admission from SAP patients and at study entry from healthy individuals. Copeptin concentration was determined using enzyme-linked immunosorbent assay. Plasma copeptin level was obviously higher in patients than in healthy individuals, was identified as an independent predictor of local complications, organ failure and in-hospital mortality, was highly associated with traditional predictors of disease severity and mortality including the Acute Physiology and Chronic Health Care Evaluation II score, Ranson score, multiple organ dysfunction score, sequential organ failure assessment score, and predicted local complications, organ failure, and in-hospital mortality of SAP patients with high areas under receiver operating characteristic curve. Furthermore, its predictive value was similar to the traditional predictors’. However, it could not improve these traditional predictors’ predictive values. Therefore, increased plasma copeptin level is associated with disease severity and identified as a novel prognostic marker of local complications, organ failure and mortality after SAP.

Publication date: February 2014 Source:Peptides, Volume 52

Author(s): Xiu Qiu , Jian-Rong He , Ming-Guang Zhao , Ya-Shu Kuang , Shu-Qin Xu , Hui-Zhu Zhang , Shun-Ping Hu , Jun Chen , Hui-Min Xia

Adropin is a recently identified peptide and participates in the regulation of energy homeostasis and vascular function. The aim of this study was to examine the relationships between human cord blood adropin levels and fetal growth. A total of 159 newborns [preterm delivery (PTD), n =72; term delivery, n =87] were recruited. Adropin levels in cord blood were determined using enzyme-linked immunosorbent assay kits. Clinical information on fetal growth was collected. Adropin levels in PTD babies (median, 2028; 25th–75th, 1413–2484pg/ml) were lower than those in term delivery babies (median, 2305; 25th–75th, 1960–2684pg/ml, P =0.01). Birth weight and length z score, Ponderal index, placental length, breadth, thickness, surface area, volume and density were not significantly correlated to adropin concentrations in term delivery group. However, we found adropin concentrations were significantly correlated to gestational age at birth (Spearman's correlation coefficient=0.35, P <0.01) and placental weight (Spearman's correlation coefficient=0.24, P =0.04) in PTD group. We also found that boys had lower adropin levels than girls in PTD group (P =0.01). When the analysis was extended to the whole group (PTD and term deliveries combined), the results were similar to those for PTD group alone. After adjusting for maternal age and newborn's sex, every 100pg/ml increase of adropin concentration was significantly associated with a decreased risk of PTD (odds ratio, 0.95; 95% confidence interval, 0.91–0.99). Our study showed that cord blood adropin levels were positively correlated with gestational age and placental weight but not with other fetal growth parameters.

Publication date: Available online 22 January 2014 Source:Peptides

Author(s): Shilpi Mahajan , Min Liao , Paris Barkan , Kazuhiro Takahashi , Aditi Bhargava

Urocortins (Ucn1-3), members of the corticotropin-releasing factor (CRF) family of neuropeptides, are emerging as potent immunomodulators. Localized, cellular expression of Ucn1 and Ucn2, but not Ucn3, has been demonstrated during inflammation. Here, we investigated the role of Ucn3 in a rat model of Crohn's colitis and the relative contribution of CRF receptors (CRF1 and CRF2) in regulating Ucn3 expression at baseline and during inflammation. Ucn3 mRNA and peptide were ubiquitously expressed throughout the GI tract in naïve rats. Ucn3 immunoreactivity was seen in epithelial cells and myenteric neurons. On day 1 of colitis, Ucn3 mRNA levels decreased by 80% and did not recover to baseline even by day 9. Next, we ascertained pro or anti-inflammatory actions of Ucn3 during colitis. Surprisingly, unlike observed anti-inflammatory actions of Ucn1, exogenous Ucn3 did not alter histopathological outcomes during colitis and neither did it alter levels of pro-inflammatory cytokines IL-6 and TNF-α. At baseline, colon-specific knockdown of CRF1, but not CRF2 decreased Ucn3 mRNA by 78%, whereas during colitis, Ucn3 mRNA levels increased after CRF1 knockdown. In cultured cells, co-expression of CRF1 +CRF2 attenuated Ucn3-stimulated intracellular Ca²⁺ peak by 48% as compared to cells expressing CRF2 alone. Phosphorylation of p38 kinase increased by 250% during colitis and was significantly attenuated after Ucn3 administration. Thus, our results suggest that a balanced and coordinated expression of CRF receptors is required for proper regulation of Ucn3 at baseline and during inflammation.

Publication date: February 2014 Source:Peptides, Volume 52

Author(s): Jianhua Zhu , Chenghong Zheng , Jie Chen , Jing Luo , Bintao Su , Yan Huang , Wen Su , Zixi Li , Tianpen Cui

Ghrelin exhibits its biological effect through binding to the growth hormone secretagogue 1a receptor (GHS-R1a). Recently, it has been reported that ghrelin has an anti-apoptotic effect in several cell types. However, the molecule mechanisms underlying the anti-apoptotic effect of ghrelin remain poorly understood. In this study, we investigated the intracellular mechanisms responsible for anti-apoptotic effect of ghrelin on human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with ghrelin inhibited high glucose-induced cell apoptosis. Ghrelin stimulated the rapid phosphorylation of mammalian target of rapamycin (mTOR), P70S6K and S6. The GHS-R1a-specific antagonist [D-Lys3]-GHRP-6 abolished the anti-apoptotic effect and inhibited the activation of mTOR, P70S6K, S6 induced by ghrelin. Pretreatment of cells with specific inhibitor of mTOR blocked the anti-apoptotic effect of ghrelin. In addition, ghrelin protected HUVECs against high glucose induced apoptosis by increasing Bcl-2/Bax ratio. Taken together, our results demonstrate that ghrelin produces a protective effect on HUVECs through activating GHS-R1a and mTOR/P70S6K signaling pathway mediates the effect of ghrelin. These observations suggest that ghrelin may act as a survival factor in preventing HUVECs apoptosis caused by high glucose.

Publication date: Available online 6 January 2014 Source:Peptides

Author(s): Zhen Liu , Yi Zhou , Shaojun Liu , Qiong Zhao , Junchang Feng , Shuangqing Lu , Gang Xiong , Dizhi Xie , Jianshe Zhang , Yun Liu

The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium which has been regarded as a mediator of protein absorption. Here, we cloned and characterized PepT1 genes from diploid (red crucian carp), triploid and tetraploid fish. Then, the PepT1 expression pattern in different tissues and embryogenesis were assayed. Meanwhile, using real-time PCR and western blotting, we showed the expression profiles of diets with different protein levels, protein sources and additives (sodium butyrate) in triploids. The cDNAs of the three different ploidy fishes have a high sequence similarity of PepT1 among vertebrates. PepT1 mRNA expression was also developmentally regulated and showed the strongest expression around the 2-cell and 4-cell stage in all three kinds of fishes. The maternal transcripts were first detected in eggs and dropped from blastula stage to muscle contraction stage. Tissue expression studies showed higher expression of PepT1 genes in the intestines of fishes compared with other tissues. In adults, triploids showed significantly higher expression levels of PepT1 in the intestines of the three kinds of ploidy fishes during breeding season and non-breeding season. In addition, high or low protein level diets both promote PepT1 expression in the intestine. We also confirmed that fish meal showed a significant increase in PepT1 expression than soybean meal in triploid intestines. Furthermore, sodium butyrate additives induce PepT1 expression that may be mediated by CDX2 and CREB. This research provides a new insight into protein absorption and its regulation in triploid fish.

Publication date: February 2014 Source:Peptides, Volume 52

Author(s): Laura G. Nisembaum , Nuria de Pedro , María J. Delgado , Aída Sánchez-Bretaño , Esther Isorna

Orexins are neuropeptides mainly known for regulating feeding behavior and sleep–wakefulness cycle in vertebrates. Daily variations of orexin-A expression have been reported in fish, with the highest levels preceding feeding time. However, it is unknown if such variations could be related with daily rhythms of clock genes, which form the molecular core of circadian oscillators. The aim of the present study was to identify the possible role of orexin as an input element of the goldfish circadian system. It was investigated the effects of orexin-A (10ng/gbw) intracerebroventricular injections on the expression of clock genes, NPY and ghrelin, as well as on daily locomotor activity rhythms. Goldfish held under 12L:12D photoperiod and injected at midday with orexin or saline, were sacrificed at 1 and 3h post-injection. The analysis of genes expression by qReal Time PCR showed an increment of Per genes in hypothalamus and foregut at 3h post-injection, but not in hindgut and liver. The gBmal1a expression remained unaltered in all the studied tissues. Orexin induced NPY in the hypothalamus and ghrelin in the foregut. Locomotor activity was studied in fish daily injected with orexin for several consecutive days under different experimental conditions. Orexin synchronized locomotor activity in goldfish maintained in 24L and fasting conditions. Present results support a cross-talking between orexin-A and other feeding regulators at central and peripheral level, and suggest, for the first time, a role of this peptide as an input of the circadian system in fish.