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Hou X.,Key Laboratory for Molecular Enzymology and Engineering | Dong Y.,Key Laboratory for Molecular Enzymology and Engineering | Lin R.,Key Laboratory for Molecular Enzymology and Engineering | Yu L.,Key Laboratory for Molecular Enzymology and Engineering | And 8 more authors.
Gaodeng Xuexiao Huaxue Xuebao/Chemical Journal of Chinese Universities | Year: 2014

The codons of D-ANase gene from Alcaligenes A-6 were substituted by the codons abundant in E. coli., then the D-ANase gene was synthesized by the two-step method based on PCR technology. Synthetic gene and pET-32a vector were digested with Bgl II and Xho I, ligated by T4 DNA ligase. The ligation mixture transformed into E. coli BL21 (DE3) competent cell. Recombinant protein was detected by SDS-PAGE, Western-blot and activity assay. D-ANase can be expressed efficiently in E. coli and the expressed protein content can reach to 69.2% of the total bacterial protein content. In addition, the fermentation activity can achieve 96 U/mL. After the ultrasonic cell disruption, the recombinant protein was purified by Ni2+ affinity chromatography column. The specific activity of the purified recombinant enzyme was 1692.3 U/mg and the purity could be up to 95%. Furthermore a firm foundation was laid for the industrial use of the D-ANase.

Fang F.,Jilin University | Gong P.S.,Key Laboratory for Molecular Enzymology and Engineering | Zhao H.G.,Jilin University | Bi Y.J.,Academy of Military Medical science | And 3 more authors.
Biomedical and Environmental Sciences | Year: 2013

Objective To investigate whether apoptosis induced by low-dose radiation (LDR) is regulated by mitochondrial pathways in testicular cells. Methods Male mice were exposed to whole-body LDR, and changes in mitochondrial function and in expression of apoptotic factors were analyzed in the testicular cells as follows. Total nitric-oxide synthase (T-NOS) and Na+/K+ ATPase activities were biochemically assayed. Reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were determined by flow cytometry using fluorescent probes. Levels of mRNAs encoding cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) were quantified by real-time reverse-transcription PCR (RT-PCR). Expression of Cyt c, AIF, caspase-9, and caspase-3 at the protein level was assessed by western blotting and immunohistochemistry. Results LDR induced an increase in T-NOS activity and ROS levels, and a decrease in Na+/K+ ATPase activity and mitochondrial Δψm, in the testicular cells. The intensity of these effects increased with time after irradiation and with dose. The cells showed remarkable swelling and vacuolization of mitochondria, and displayed a time- and dose-dependent increase in the expression of Cyt c, AIF, procaspase-9, and procaspase-3. Activation of the two procaspases was confirmed by detection of the cleaved caspases. The changes in expression of the four apoptotic factors were mostly limited to spermatogonia and spermatocytes. Conclusion LDR can induce testicular cell apoptosis through mitochondrial signaling pathways. © 2013 The Editorial Board of Biomedical and Environmental Sciences.

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