Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10^?9, 1×10^?8, 1×10^?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10^?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10^?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10^?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10^?8 and 10^?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.     

Production of a thermostable metal-tolerant laccase from Trametes versicolor and its application in dye decolorization

Production of laccase using a submerged culture of Trametes versicolor sdu-4 was optimized using a central composite design of the Response Surface Methodology. Optimized conditions gave a laccase yield of 4,213 U/L which was approximately three times of that in basal medium. The laccase was purified to homogeneity using a three-step process. The overall yield of the purification was 58%, with a purification fold of 11.4 and a specific activity of 1320.7 U/mg protein. The molecular mass of the laccase was 60 kDa. The optimum pH values of the enzyme were 2.2, 3.7, and 7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme had adaptability to a broad pH range and high temperature and wsa stable at pH 3.0 ∼ 10.0. The half-life of this laccase at 70°C was 2.2 h. Methyl red, 2-bromophenol, and 4-bromophenol were oxidized by the purified laccase in the absence of mediators. Purified laccase was effective in the decolorization of several dyes and was not inhibited by Cu²⁺, Mn²⁺, Zn²⁺, Na⁺, K⁺, Mg²⁺, Ba²⁺, and Ca²⁺ at 5 mM. These excellent characteristics made it a highly attractive candidate for industrial use.     

Insulin receptor signaling activated by penta-<Emphasis Type="Italic">O</Emphasis>-galloyl-α-<Emphasis Type="SmallCaps">d</Emphasis>-glucopyranose induces p53 and apoptosis in cancer cells

p53 is essential for cell cycle arrest and apoptosis induction while insulin receptor (IR) signaling is important for cell metabolism and proliferation and found upregulated in cancers. While IR has recently been found to be involved in apoptosis, p53 induction or apoptosis mediated through IR signaling activation has never been documented. Here, we report that the IR signaling pathway, particularly the IR-MEK pathway, mediates biological and biochemical changes in p53 and apoptosis in tumor cells. Specifically, natural compound penta-O-galloyl-α-d-glucopyranose (α-PGG), a previously characterized IR signaling activator, induced apoptosis in RKO cells without significantly affecting its normal counterpart FHC cells. α-PGG induced apoptosis in RKO cells through p53, Bax and caspase 3. Importantly, α-PGG’s ability to elevate p53 was diminished by IR inhibitor and IR-siRNA, suggesting a non-conventional role of IR as being involved in p53 induction. Further studies revealed that α-PGG activated MEK, a downstream signaling factor of IR. Blocking MEK significantly suppressed α-PGG-induced p53 and Bax elevation. All these results suggested that α-PGG induced p53, Bax, and apoptosis through the IR-MEK signaling pathway. The unique activity of α-PGG, a novel IR phosphorylation and apoptosis inducer, may offer a new therapeutic strategy for eliciting apoptotic signal and inhibiting cancer growth.     

Prediction of novel pre-microRNAs with high accuracy through boosting and SVM

High-throughput deep-sequencing technology has generated an unprecedented number of expressed short sequence reads, presenting not only an opportunity but also a challenge for prediction of novel microRNAs. To verify the existence of candidate microRNAs, we have to show that these short sequences can be processed from candidate pre-microRNAs. However, it is laborious and time consuming to verify these using existing experimental techniques. Therefore, here, we describe a new method, miRD, which is constructed using two feature selection strategies based on support vector machines (SVMs) and boosting method. It is a high-efficiency tool for novel pre-microRNA prediction with accuracy up to 94.0% among different species. AVAILABILITY: miRD is implemented in PHP/PERL+MySQL+R and can be freely accessed at http://mcg.ustc.edu.cn/rpg/mird/mird.php.     

RASP: rapid modeling of protein side chain conformations

MOTIVATION: Modeling of side chain conformations constitutes an indispensable effort in protein structure modeling, protein-protein docking and protein design. Thanks to an intensive attention to this field, many of the existing programs can achieve reasonably good and comparable prediction accuracy. Moreover, in our previous work on CIS-RR, we argued that the prediction with few atomic clashes can complement the current existing methods for subsequent analysis and refinement of protein structures. However, these recent efforts to enhance the quality of predicted side chains have been accompanied by a significant increase of computational cost. RESULTS: In this study, by mainly focusing on improving the speed of side chain conformation prediction, we present a RApid Side-chain Predictor, called RASP. To achieve a much faster speed with a comparable accuracy to the best existing methods, we not only employ the clash elimination strategy of CIS-RR, but also carefully optimize energy terms and integrate different search algorithms. In comprehensive benchmark testings, RASP is over one order of magnitude faster (~ 40 times over CIS-RR) than the recently developed methods, while achieving comparable or even better accuracy.     

Cloning and characterization of the <Emphasis Type="Italic">Tne</Emphasis>DI restriction–modification system of <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis>

A putative Type II restriction–modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF′ and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.     

Protective effect of intermedin on myocardial cell in a rat model of severe acute pancreatitis

Severe acute pancreatitis (SAP) is a common disease with a poor prognosis. Heart failure is one cause of SAP patient death. Intermedin (IMD) is a potent endogenous cardio-protective substance. Administration of exogenous IMD showed beneficial effects in cardiovascular diseases. The aim of this study was to investigate the myocardial damage in SAP and to determine the therapeutic potential of IMD for SAP. Using an SAP rat model, we examined endogenous IMD expression following SAP induction, and determined the effect of IMD on myocardial function, histological morphology, apoptosis-related gene expression, and prognosis. Our results indicated that the cardiac function and histological structure were significantly disrupted in SAP rats. Infusion of exogenous IMD significantly preserved cardiac function and ameliorated myocardial damage. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) revealed that myocardial apoptosis was extensively present in SAP rats, and IMD infusion led to increased expression of the prosurvival factor Bcl-2, but decreased pro-apoptotic factors Bax and caspase-3. In addition, IMD infusion also reversed the change of IMD receptor systems in SAP rat heart tissue. Furthermore, we found that IMD infusion greatly decreased mortality of SAP rats. In conclusion, administration of SAP produced therapeutic effects in SAP through modulating apoptotic and pro-survival gene expression, inhibiting myocardial apoptosis, preserving cardiac function, and a useful therapeutic agent for SAP, and provides us an insight for a clinical trial of IMD for treating human severe acute pancreatitis.     

Genetic Linkage Mapping and Analysis of Muscle Fiber-Related QTLs in Common Carp (Cyprinus carpio L.)

A genetic linkage map of common carp (Cyprinus carpio L.) was constructed using Type I and Type II microsatellite markers and a pseudo-testcross mapping strategy. The microsatellite markers were isolated from microsatellite-enriched genomic libraries and tested for their segregation in a full-sib mapping panel containing 92 individuals. A total of 161 microsatellite loci were mapped into 54 linkage groups. The total lengths of the female, male and consensus maps were 2,000, 946, and 1,852?cM, with an average marker spacing of approximately 13, 7, and 11?cM, respectively. Muscle fiber-related traits, including muscle fiber cross-section area and muscle fiber density, were mapped to the genetic map. Three QTLs for muscle fiber cross-section area and two QTLs for muscle fiber density were identified when considering both significant and suggestive QTL effects. The QTLs with largest effects for muscle fiber cross-section area and muscle fiber density were 21.9% and 18.9%, and they were located in LG3, respectively.