[ ABSTRACT] AIM:To observe the effect of high glucose on the protein expression of calreticulin ( CRT) and its association with cell apoptosis and mitochondrial dysfunction in the cardiomyocytes.METHODS: AC-16 cardiomyocytes were randomly divided into normal glucose group, high glucose group, high glucose+CRT siRNA group and isotonic con-trol group.The cell apoptotic rate, reactive oxygen species (ROS), mitochondrial membrane potential level, respiratory enzyme activity, and protein expression of CRT were observed.RESULTS: Compared with the cardiomyocytes in normal glucose group, the apoptotic rate and ROS production of cardiomyocytes increased in high glucose group, accompanying with the decreases in the mitochondrial membrane potential level and enzyme activitiy of the respiratory chain.The protein expression of CRT was significantly increased in high glucose group.However, compared with high glucose group, high glucose+CRT siRNA decreased the expression of CRT and attenuated the damage of mitochondria, but CRT siRNA did not reduce the ROS level in cardiomyocytes.CONCLUSION:High glucose brings about CRT over-expression to induce mito-chondrial injury, thus increasing myocardial apoptosis.

Objective To explore the changes of cardiac structure and function in streptozotocin (STZ)-induced diabetic cardiomyopathy (DCM)rats so as to detect the level of angiotensinⅡ (AngⅡ)in blood and the myocardium and illuminate the role of AngⅡ in DCM.Methods We randomly divided 30 SD rats into control group and DCM group (which received a single injection of streptozotocin,65 mg/kg).The changes of cardiac structure and function were observed by ultrasonic cardiogram at the end of 16 weeks after injection.Then the changes in the myocardium were also analyzed by using hemotoxylin and eosin staining and Sirius red staining.The level of AngⅡ in blood was tested by radioimmunoassay.The expression of AngⅡ in the myocardium was detected by immunofluorescence. Results Compared with those of the controls,the hearts were dilated in DCM rats accompanied with cardiac dysfunction.There were significant increases in LVEDd,LVESd,LVEDv,and LVESv but decrease in LVEF (P <0.05).There were arrangement disorder and hypertrophic cardiomyocytes in DCM.Then the fibrosis area of DCM was significantly increased compared with that of the controls.The level of AngⅡ in blood and myocardium was significantly higher in DCM than in controls.At the same time,Pearson analysis revealed that the level of AngⅡ in blood was positively related to the collagen content of myocardium (r =0.907,P <0.05).Conclusion Our study provides experimental evidence that AngⅡ plays an important role in myocardial fibrosis of DCM.

Objective To investigate the distribution and drug resistance of pathogens in intensive care unit (ICU) so as to provide scientific basis for antibiotic adoption and the prevention and control of nosocomial infections. Methods The various specimens collected from the patients admitted into ICU in the First People's Hospital of Shunde Affiliated to the South Medical University from January 2007 to December 2014 were used to isolate the pathogens that might cause nosocomial infections and retrospectively analyze their clinical distribution and drug resistance. Kirby-Bauer paper diffusion and minimal inhibitory concentration (MIC) methods were applied to test the drug sensitivity, and according to National Committee for Clinical Laboratory Standards/Clinical and Laboratory Standards Institute (NCCLS/CLSI) standard, the results were identified.Results The sputum was the major specimen source in ICU, accounting for 68.8%, followed by urine (12.4%) and blood (6.8%). All together 557 pathogens in ICU causing nosocomial infections were isolated of which there were 377 gram-negative (G-) bacilli (67.7%), 103 gram-positive (G+) cocci (18.5%), and 77 fungi (13.8%). Among G- bacilli, the top three wereAcinetobacter baumannii (34.5%), Klebsiella pneumonia (17.8%), andPseudomonas aeruginosa (13.0%). Beside carbapenem, the drug resistance rates of Acinetobacterbaumannii to other antibiotics were more than 40%. The main G+ coccus causing nosocomial infection wasSaphylococcus aureus (36.9%) in ICU. The drug resistance rates ofSaphylococcus aureus to penicillin, gentamicin and erythromycin were higher than 50%. In 77 fungus strains,Candida albicans was ranked the first, accounting for 41.6%.Conclusion The main infection site in ICU is primarily respiratory tract, the G- bacilli are the predominate pathogens, and the drug resistance to antibiotics found in this report is serious, so clinically, the antibiotics should be properly used to avoid the occurrence of pathogenic strain with drug tolerance.

Carbonyl reductases catalyze carbonyl compounds to chiral alcohols that are important building blocks in fine chemical industry. To study carbonyl reductase from Pichia pastoris GS115 (ppcr), we discovered a new gene (ppcr) encoding an NADPH-dependent carbonyl reductase by genomic data mining. It was amplified by PCR from the genomic DNA, and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity. The optimum temperature was 37 degrees C and the optimum pH of PPCR was 6.0. PPCR was stable below 45 degrees C. The Km and k(cat) value of the enzyme for ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s, respectively. The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, a-ketoesters, beta-ketoesters and aryl ketones to give the corresponding alcohols with >97% ee with only a few exceptions, showing its application potential in the synthesis of chiral alcohols.
Alcohol Oxidoreductases ; biosynthesis ; chemistry ; genetics ; Amino Acid Sequence ; Biotechnology ; methods ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Pichia ; enzymology ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Stereoisomerism ; Substrate Specificity ; Temperature

Asymmetric reduction of bulky diaryl ketones is still one of the challenging tasks in biocatalysis. By genomic data mining, a putative carbonyl reductase gene pascr was found in Pichia pastoris GS115. pascr was cloned and over-expressed in Escherichia coli Rosseta2 (DE3). The recombinant enzyme was purified to homogeneity by Ni-NTA column and its catalytic properties were studied. PasCR strictly used NADPH as cofactor, gel filtration and SDS-PAGE analysis suggested that the native form of PasCR was a dimmer. PasCR exhibited the highest activity at 35 degrees C in phosphate buffer at pH 6.5. The enzyme catalyzed the reduction of some bulky diaryl ketones, such as 4-methylbenzophenone, 2-methylbenzophenone and 4-chlorobenzophenone, especially for 4-methylbenzophenone, the product S--alcohol was obtained with 85% ee.
Alcohol Oxidoreductases ; genetics ; Amino Acid Sequence ; Catalysis ; Cloning, Molecular ; Ketones ; chemistry ; Molecular Sequence Data ; Pichia ; enzymology ; genetics ; Stereoisomerism

OBJECTIVETo study the phosphorylation activity of mitochondrial signal transducer and activator of transcription 3 (STAT3) in the myocardium of rats with selenium deficiency and its association with myocardial injury.

METHODSThirty-six rats were randomized into normal control group (n=18) and selenium deficiency model group (n=18) for feeding with normal and low-selenium chow, respectively, for 20, 30 and 40 weeks. The cardiac function of the rats was evaluated by carotid artery intubation, and the damage of cardiac mitochondria was observed under electron microscopy. The cardiac mitochondria were extracted for assessing succinate dehydrogenase and cytochrome C oxidase activities, and the protein expressions of phosphorylated and total STAT3 were detected.

RESULTSCompared with the corresponding control groups, the rats in the model group showed significantly decreased cardiac function with obvious structural and functional damage of the cardiac mitochondria (P<0.05), which aggravated as the low-selenium feeding time extended (P<0.05). The rats in the model group also showed significantly decreased mitochondrial STAT3 activity (p-STAT3/STAT3) in the myocardium as the low-selenium feeding time prolonged (P<0.05). Pearson linear correlation analysis showed that the activity of cardiac mitochondrial STAT3 had positive correlations with the left ventricular systolic pressure, maximal increased rate of the left ventricular pressure, and the activities of succinate dehydrogenase and cytochrome C oxidase (P<0.01).

CONCLUSIONSelenium deficiency down-regulates the activity of mitochondrial STAT3 in rat heart to contribute to cardiac mitochondrial injury and the progression of heart failure.

Animals ; Diet ; Electron Transport Complex IV ; metabolism ; Female ; Heart Injuries ; metabolism ; Male ; Mitochondria, Heart ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; STAT3 Transcription Factor ; metabolism ; Selenium ; deficiency ; pharmacology ; Signal Transduction ; Succinate Dehydrogenase ; metabolism

OBJECTIVETo observe the effect of angiotensin II (Ang II) on calreticulin (CRT) expression and its association with mitochondrial dysfunction in cardiomyocytes.

METHODSPrimary neonatal rat cardiomyocytes were randomly divided into CRT siRNA group, control siRNA group, control group, Ang II+ CRT siRNA group, Ang II+ control siRNA group and Ang II group. The cell surface area, protein synthesis rate, mitochondrial membrane potential level, enzyme activities, and CRT expression were observed.

RESULTSCompared with those in the control group, the cell surface area and protein synthesis rate were both increased and mitochondrial membrane potential level and enzyme activities decreased in Ang II groups. CRT expression was significantly down-regulated in Ang II+ CRT siRNA group with increased cell surface area, protein synthesis rate, mitochondrial membrane potential level and enzyme activities as compared with those in Ang II+ control siRNA group.

CONCLUSIONAng II up-regulates CRT expression to induce mitochondrial injury, which may be an important mechanism of myocardial hypertrophy.

Angiotensin II ; pharmacology ; Animals ; Calreticulin ; metabolism ; Cardiomegaly ; Cells, Cultured ; Membrane Potential, Mitochondrial ; Mitochondria ; pathology ; Myocytes, Cardiac ; pathology ; Protein Biosynthesis ; RNA, Small Interfering ; Rats

Objective:To observe the effect of selenium supplementation on 10-year survival rate of chronic Keshan disease (KD).Methods:The 10-year follow-up data of 302 patients with chronic KD at the KD surveillance sites in Shaanxi Province were collected from the Shaanxi Institute for Endemic Disease Control and Research and Xi'an Jiaotong University, 170 (56.3%) cases were given selenium supplementation (oral administration of sodium selenite tablet, once a week, 1 mg/time) until the end point of follow-up as selenium supplementation group, and the rest (132 cases) were non-selenium supplementation group. Cox proportional hazards models were used to identify the independent predictors for 10-year survival rate of chronic KD. Kaplan-Meier method was used to analyze the 10-year survival rate of patients with chronic KD during the follow-up period and the Log-rank test was used to compare the 10-year survival rate between groups.Results:The follow-up deadline was October 2019. During the follow-up period, a total of 199 patients (199/302, 65.9%) of chronic KD died, including 101 patients (101/170, 59.4%) in the selenium supplementation group and 98 patients (98/132, 74.2%) in the non-selenium supplementation group. In COX proportional hazards model, after adjustment for other baseline characteristics [age, sex, body mass index (BMI), family history of KD, smoking, blood pressure, heart rate, ECG abnormalities, initial cardiothoracic ratio, left ventricular ejection fraction (LVEF), and blood selenium content], selenium supplementation and combined use of angiotensin-converting enzyme inhibitor + β receptor blocker (ACEI + BBs) were protective factors for 10-year survival in patients with chronic KD (selenium supplementation: HR = 0.39, 95% CI: 0.28 - 0.53; ACEI + BBs: HR = 0.57, 95% CI: 0.39 - 0.84). The 10-year survival rate of chronic KD patients after selenium supplementation was significantly higher than that of non-selenium supplementation group (Log-rank test, P < 0.05). Conclusion:Selenium supplementation and combined use of ACEI + BBs in chronic KD patients, are associated with better survival during the 10-year follow-up.

D-psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)(8) TIM barrel fold with a Mn(2+) metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexose-bound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.
Binding Sites ; Biocatalysis ; Catalytic Domain ; Clostridium cellulolyticum ; enzymology ; Hexoses ; chemistry ; Manganese ; chemistry ; Protein Structure, Quaternary ; Racemases and Epimerases ; chemistry ; metabolism ; Substrate Specificity

Taiwanofungus camphoratus is a valuable and rare medicinal mushroom with various bioactivities, such as liver protection and anti-cancer. T. camphoratus can produce many arthroconidia at the end of submerged fermentation, but molecular mechanism underlying this submerged conidiation remains unknown. In this study, we found that Ca²⁺ concentration in culture medium significantly affected the arthroconidium production of T. camphoratus. Then, we identified two proteins (CaM and HSP90) involved in Ca²⁺/calmodulin signaling pathway and one protein (AbaA) involved in FluG-mediated conidiation pathway by two-dimensional electrophoresis analyses. Furthermore, we proposed a Ca²⁺/calmodulin- and FluG-mediated signaling pathway by bioinformatics analysis. By real-time quantitative PCR analyses of 23 key genes in the Ca²⁺/calmodulin- and FluG-mediated conidiation pathway, we found that expression levels of 7 genes (crz1, hsp90, flbB, brlA, abaA, wetA and fadA) showed significant responses to Ca²⁺ concentration in fermentation medium. Our research is beneficial for elucidating the underlying mechanism of submerged fermentation conidiation for T. camphoratus.