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Wang S.,Northeast Normal University | Wang S.,Shenzhen University | Liu G.,Shenzhen University | Yu J.,Shenzhen Key Laboratory of Marine Biotechnology and Ecology | And 3 more authors.
Enzyme and Microbial Technology

The cellulase and xylanase genes of filamentous Trichoderma fungi exist under carbon catabolite repression mediated by the regulator carbon catabolite repressor (CREI). Our objective was to find the role of CREI in a cellulase-hyperproducing mutant of Trichoderma koningii, and address whether enzyme production can be further improved by silencing the cre1 gene. cre1 partially silenced strains were constructed to improve enzyme production in T. koningii YC01, a cellulase-hyperproducing mutant. Silencing of cre1 resulted in derepression of cellulase gene expression in glucose-based cultivation. The cre1 interference strain C313 produced 2.1-, 1.4-, 0.8-, and 0.8-fold higher amounts of filter paper activity, β-1,4-exoglucanase activity (ρ-nitrophenyl-β-. d-cellobioside as substrate), β-1,4-endoglucanase activity (sodium carboxymethyl cellulose as substrate), and xylanase activity, respectively, than the control strain, suggesting that silencing of cre1 resulted in enhanced enzyme production capability. In addition, downregulation of cre1 resulted in elevated expression of another regulator of xylanase and cellulase expression, xyr1, indicating that CREI also acted as a repressor of xyr1 transcription in T. koningii under inducing conditions. These results show that RNAi is a feasible method for analyzing the regulatory mechanisms of gene expression and improving xylanase and cellulase productivity in T. koningii. © 2013 Elsevier Inc.. Source

Wang S.,Shenzhen University | Wang S.,Northeast Normal University | Liu G.,Shenzhen University | Wang J.,Shenzhen University | And 3 more authors.
Journal of Industrial Microbiology and Biotechnology

To investigate whether enzyme production can be enhanced in the Trichoderma reesei industrial hyperproducer strain RUT C30 by manipulation of cellulase regulation, the positive regulator Xyr1 was constitutively expressed under the control of the strong T. reesei pdc promoter, resulting in significantly enhanced cellulase activity in the transformant during growth on cellulose. In addition, constitutive expression of xyr1 combined with downregulation of the negative regulator encoding gene ace1 further increased cellulase and xylanase activities. Compared with RUT C30, the resulting transformant exhibited 103, 114, and 134 % greater total secreted protein levels, filter paper activity, and CMCase activity, respectively. Surprisingly, strong increases in xyr1 basal expression levels resulted in very high levels of CMCase activity during growth on glucose. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression, and suggest an attractive new single-step approach for increasing total cellulase productivity in T. reesei. © 2013 Society for Industrial Microbiology and Biotechnology. Source

Chen P.,Shenzhen University | Wang L.,Shenzhen Key Laboratory of Marine Biotechnology and Ecology | Li N.,Shenzhen University | Liu Q.,Shenzhen Key Laboratory of Marine Biotechnology and Ecology | Ni J.,Shenzhen University

Selenium is an important trace mineral necessary for human health. Clinical trials have shown potential inhibitory effects of selenium in advanced or aggressive prostate cancer. However, its mechanism of action remains unclear. This study investigated the mechanism of action of sodium selenite in human prostate cancer PC-3 cells using proteomics. CCK-8 assays were used to detect cell viability and the inhibitory rate. Cell apoptosis was detected by annexin V-FITC and propidium iodide double staining using flow cytometry. Selenite inhibited the growth of PC-3 cells causing them to display morphological changes typical of apoptosis. The rate of cell apoptosis also increased. Proteomics identified a variety of differentially expressed proteins in PC-3 cells exposed to selenite. Eighteen protein spots were identified by MALDI-TOF mass spectrometry. These proteins were separated into those involved in redox balance, protein degradation and cellular energy metabolism. Three differently expressed proteins (SOD1, Stathmin and Erp29) were chosen for Western blot verification, together with several apoptosis-related proteins. Western blot analyses showed that selenite-induced apoptosis was accompanied by activation of caspase-8 and specific proteolytic cleavage of PARP. This led to an increase in the pro-apoptotic protein Bax, and to a decrease in the anti-apoptotic protein Bcl-2 and in hypoxia inducible factor-1α. Increased ROS generation and decreased mitochondrial membrane potential were consistent with reduced expression of antioxidative proteins identified by comparative proteomics. We therefore propose that sodium selenite induces the apoptosis of PC-3 cells mainly through the mitochondrial pathway, but also via ER stress and HIF-1α mediated pathways. © 2013 The Royal Society of Chemistry. Source

Du X.,Shenzhen University | Li H.,Shenzhen Key Laboratory of Marine Biotechnology and Ecology | Wang Z.,Shenzhen University | Qiu S.,Shenzhen Key Laboratory of Marine Biotechnology and Ecology | And 2 more authors.

Aggregation and cytotoxicity of the amyloid-β (Aβ) peptide with transition metal ions in neuronal cells have been suggested to be involved in the progression of Alzheimer's disease (AD). A therapeutic strategy to combat this incurable disease is to design chemical agents to target metal-Aβ species. Selenoproteins are a group of special proteins that contain the 21st amino acid Sec in their sequence. Due to the presence of Sec, studies of this group of proteins are basically focused on their roles in regulating redox potential and scavenging reactive oxygen species. Here, we reported that the His-rich domain of selenoprotein P (SelP-H) and the Sec-to-Cys mutant selenoprotein M (SelM′) are capable of binding transition metal ions and modulating the Zn2+-mediated Aβ aggregation, ROS production and neurotoxicity. SelM′ (U48C) and SelP-H were found to coordinate 0.5 and 2 molar equivalents of Zn2+/Cd2+ with micromolar and submicromolar affinities, respectively. Metal binding induced the structural changes in SelP-H and SelM′ according to the circular dichorism spectra. Zn2+ binding to Aβ42 almost completely suppressed Aβ42 fibrillization, which could be significantly restored by SelP-H and SelM′, as observed by thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM). Interestingly, both SelP-H and SelM′ inhibited Zn2+-Aβ42-induced neurotoxicity and the intracellular ROS production in living cells. These studies suggest that SelP and SelM may play certain roles in regulating redox balance as well as metal homeostasis. © 2013 The Royal Society of Chemistry. Source

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