Enzymatic Characterization and Coenzyme Specificity Conversion of a Novel Dimeric Malate Dehydrogenase from Bacillus subtilis.

Malate is an important material to various industrials and clinical applications. Bacillus subtilis is a widely used biocatalyst tool for chemical production. However, the specific enzymatic properties of malate dehydrogenase from Bacillus subtilis (BsMDH) remain largely unknown. In the present study, BsMDH was cloned, recombinantly expressed and purified to test its enzymatic properties. The molecular weight of single unit of BsMDH was 34,869.7 Da. Matrix-Assisted Laser-Desorption Ionization-Time-of-Flight Mass Spectrometry and gel filtration analysis indicated that the recombinant BsMDH could form dimers. The kcat/Km values of oxaloacetate and NADH were higher than those of malate and NAD+, respectively, indicating a better catalysis in the direction of malate synthesis than the reverse. Furthermore, six BsMDH mutants were constructed with the substitution of amino acids at the coenzyme binding site. Among them, BsMDH-T7 showed a greatly higher affinity and catalysis efficiency to NADPH than NADH with the degree of alteration of 2039, suggesting the shift of the coenzyme dependence from NADH to NADPH. In addition, BsMDH-T7 showed a relatively lower Km value, but a higher kcat and kcat/Km than NADPH-dependent MDHs from Thermus flavus and Corynebacterium glutamicum. Overall, these results indicated that BsMDH and BsMDH-T7 mutant might be promising enzymes for malate production.

Association between PTGER4 polymorphisms and inflammatory bowel disease risk in Caucasian: A meta-analysis.

BACKGROUND: The results from previous studies on association between prostaglandin E receptor 4 (PTGER4) polymorphisms and inflammatory bowel disease (IBD) risk in Caucasian were conflict. The present study aimed to investigate the genetic association by conducting a meta-analysis. METHODS: Systematic literature search was conducted through Wiley Online Library, Chinese National Knowledge Infrastructure (CNKI), and PubMed databases. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to investigate the associations between rs4613763 T/C, 17234657T/G polymorphisms, and IBD risk in Caucasian. RESULTS: Twenty case-control studies consisting of 18,495 Crohn disease (CD) patients and 4203 ulcerative colitis (UC) patients, as well as 26,063 controls were included in this meta-analysis. The rs4613763T/C polymorphism had obvious influence on CD, UC risk in Caucasian. However, rs17234657T/G polymorphism had obvious influence on CD but not UC in Caucasian. CONCLUSION: This meta-analysis suggested that both the rs4613763 T/C, rs17234657T/G polymorphisms had obvious influence on risk of CD in Caucasian. In addition, rs4613763 T/C, polymorphism had obvious influence on risk of UC in Caucasian.

Prenatal caffeine exposure induced high susceptibility to metabolic syndrome in adult female offspring rats and its underlying mechanisms.

Our previous studies have demonstrated that prenatal caffeine exposure (PCE) induced an intrauterine programming of hypothalamic-pituitary-adrenal axis (HPAA)-associated neuroendocrine metabolism in 3-month-old offspring rats. In this study, we aimed to confirm this programming disorder and high susceptibility to metabolic syndrome (MS) in 10-month-old female PCE offspring with postnatal catch-up growth. We found that PCE female offspring rats showed decreased bodyweight but a higher rate of weight gain after birth. Moreover, in the offspring, basal hyperinsulinemia and insulin resistance were observed before unpredictable chronic stress (UCS), but serum total cholesterol (TCH) levels and triglyceride/high-density lipoprotein-cholesterol (TG/HDL-C), TCH/HDL-C and low-density lipoprotein-cholesterol/HDL-C (LDL-C/HDL-C) ratio changes were increased after UCS, accompanied by morphological damage of the related tissues. These results suggested that PCE adult female offspring rats were highly susceptible to MS, which is related to HPAA-associated neuroendocrine-metabolic programming disorder.

[Chinese parents' attitudes toward and their satisfaction with circumcision for 6－14 years old children].

OBJECTIVE: To investigate the attitudes of the parents toward circumcision for 6－14 years old children and their satisfaction with the results. METHODS: We performed circumcision in the Department of Urology of Zhongda Hospital for 220 children aged 6－14 years from 220 families between January 2010 and August 2016, including 70 cases of traditional and 150 cases of Shang Ring circumcision. We conducted telephone follow-ups among the parents of the patients concerning the decision-maker, reasons and regret for circumcision, acceptance of a second operation, source of information, satisfaction with surgical results, and reasons for dissatisfaction. RESULTS: Most decisions for circumcision were made by the father, chiefly for health and hygiene. Their main sources of information on circumcision were Internet and friends. The parents of 29 patients were dissatisfied for long recovery or peri- and post-operative pain, including 19 cases (27.1%， 19/70) of traditional and 10 cases (6.7%， 10/150) of Shang Ring circumcision, with statistically significant differences between the two groups (P <0.05). CONCLUSIONS: Most parents were satisfied with circumcision, and the main reasons for dissatisfaction were long recovery and pain. The rate of satisfaction with Shang Ring circumcision was higher than that with traditional circumcision. Shang Ring circumcision is recommended for children aged 6－14 years old.

[Urethroplasty for male urethral stricture: Application and outcomes].

Urethroplasty is now the optimal option for the treatment of male urethral stricture. The rapid development of tissue substitution techniques over the past decades provides a great possibility of achieving good long-term outcomes of urethroplasty. Necessary techniques required for urethroplasty, such as those to present the position, length and severity of the stricture and precisely evaluate the quality of the transplanted tissue, are critical for the success of surgery. This review focuses on the application and outcomes of adoptable uretheroplasty for male urethral strictures in different anatomical positions.

The mechanism of adenosine-mediated activation of lncRNA MEG3 and its antitumor effects in human hepatoma cells.

Long non-coding RNA MEG3 is suggested to function as a tumor suppressor. However, the activation mechanism of MEG3 is still not well understood and data are not available on its role under adenosine-induced apoptosis. In this study, HepG2 cells were treated with adenosine or 5-Aza­cdR. Methylation status of MEG3 promoter was detected by methylation specific PCR (MSP) and MEG3 expression was determined by qRT-PCR. PcDNA3.1-MEG3 recombinant plasmid was constructed and transfected to hepatoma HepG2 and Huh7 cells. Cell growth, morphological changes, cell-cycle distribution and apoptosis were analyzed by MTT assay, fluorescence microscopy and flow cytometry. The mRNA and protein expression levels were detected by qRT-PCR and western blot analysis. MEG3 binding proteins were screened by the improved MS2 biotin tagged RNA affinity purification method. The co-expression network of MEG3 was generated by GO analysis and ILF3 was identified as MEG3 binding protein by RNA pulldown and western blot analysis. Both adenosine and 5-Aza-CdR increased MEG3 mRNA expression and the CpG island of MEG3 gene in HepG2 cells was typical hypermethylation. Ectopic expression of MEG3 inhibited hepatoma cell growth in a time-dependent manner, resulted in cell cycle arrest and induced apoptosis. Ectopic expression of MEG3 increased p53, caspase-3 mRNA and protein levels, decreased MDM2 and cyclin D1 mRNA and protein levels, as well as ILF3 protein expression in HepG2 cells. These findings are the first to identify that adenosine increases MEG3 expression by inhibition of DNA methylation and its antitumor effects is involved in MEG3 activation. ILF3 may participate in the anticancer regulation of MEG3 by interacting with MEG3.

Enhanced antitumor effects of adenoviral-mediated siRNA against GRP78 gene on adenosine-induced apoptosis in human hepatoma HepG2 cells.

Our previous studies show that adenosine-induced apoptosis is involved in endoplasmic reticulum stress in HepG2 cells. In this study, we have investigated whether knockdown of GRP78 by short hairpin RNA (shRNA) increases the cytotoxic effects of adenosine in HepG2 cells. The adenovirus vector-delivered shRNA targeting GRP78 (Ad-shGRP78) was constructed and transfected into HepG2 cells. RT-PCR assay was used to determine RNA interference efficiency. Effects of knockdown of GRP78 on adenosine-induced cell viabilities, cell-cycle distribution and apoptosis, as well as relative protein expressions were determined by flow cytometry and/or Western blot analysis. The intracellular Ca2+ concentration was detected by laser scanning confocal microscope. Mitochondrial membrane potential (ΔΨm) was measured by a fluorospectrophotometer. The results revealed that GRP78 mRNA was significantly downregulated by Ad-shGRP78 transfection. Knockdown of GRP78 enhanced HepG2 cell sensitivity to adenosine by modulating G0/G1 arrest and stimulating Bax, Bak, m-calpain, caspase-4 and CHOP protein levels. Knockdown of GRP78 worsened cytosolic Ca2+ overload and ΔΨm loss. Knockdown of caspase-4 by shRNA decreased caspase-3 mRNA expression and cell apoptosis. These findings indicate that GRP 78 plays a protective role in ER stress-induced apoptosis and show that the combination of chemotherapy drug and RNA interference adenoviruses provides a new treatment strategy against malignant tumors.

Apoptosis induced by adenosine involves endoplasmic reticulum stress in EC109 cells.

Apoptosis plays a critical role in the development and homeostasis of multicellular organisms, and endoplasmic reticulum stress (ERS) is one of the intrinsic apoptosis pathways. Previous studies have shown that adenosine induces apoptosis in several cancer cell lines. However, the molecular mechanism remains poorly understood. In this study, we explored whether adenosine triggers apoptosis of EC109 esophageal carcinoma (EC) cells by ERS. The MTT assay was used to determine cell proliferation; cell cycle detection (FCM) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine cell apoptosis. The subcellular distribution and expression of the ERS-related proteins GRP78, cleaved caspase-3, cleaved caspase-4, CHOP and NF-κB p65 were detected by western blot techniques. NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The MTT assay demonstrated that adenosine inhibited EC109 cell proliferation in a dose- and time-dependent manner. FCM and TUNEL assay verified that adenosine caused an apoptotic peak in cell cycle arrest and a higher percentage of apoptotic cells. Western blot analysis confirmed that the expression of GRP78, cleaved caspase-4, CHOP, NF-κB p65 and cleaved caspase-3 were upregulated in a dose-dependent manner after adenosine treatment. EMSA revealed that adenosine activated NF-κB p65. This is the first demonstration that adenosine inhibits cell proliferation, increases GRP78 and NF-κB p65 expression and induces apoptosis by CHOP and caspase-4 pathways. The ERS pathway is involved in adenosine-induced apoptosis in EC109 cells.