Hepatitis C virus nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane associated protein and a potent causative factor of ER stress. Here we reported that unfolded protein response (UPR) can be activated by HCV NS4B through inducing both XBP1 mRNA splicing and ATF6 cleavage in human hepatic cells. Flow cytometric analysis revealed that HCV NS4B stimulates the production of reactive oxygen species (ROS) by perturbing intracellular Ca(2+) homeostasis. Luciferase assay showed that HCV NS4B also activates the multifunctional transcription factor, NF-kappaB, in a dose-dependent manner through Ca(2+) signaling and ROS. Further immunoblot analysis showed that HCV NS4B promotes NF-kappaB translocation into the nucleus via protein-tyrosine kinase (PTK) mediated phosphorylation and subsequent degradation of IkappaBalpha. These studies provide an important insight into the implication of NS4B in HCV life cycle and HCV-associated liver disease by affecting host intracellular signal transduction pathways.
In order to improve the productivity of vitamin B(12) by Pseudomonas denitrificans carried out in a 120-m(3) fermenter, the effect of pH on vitamin B(12) biosynthesis was investigated. Results obtained from shake flask experiments showed that the feeding of carbon source (beet molasses or glucose) and methyl-group donor (betaine or choline chloride) significantly influenced the pH and the biosynthesis of vitamin B(12). In contrast to beet molasses or choline chloride, using glucose as a feed medium and betaine as a methyl-group donor, pH could be maintained at a stable range. As a result, higher vitamin B(12) production was achieved. Accordingly, an effective and simplified pH-stat control strategy was established for the fermentation of vitamin B(12) in a 120-m(3) industrial fermenter. When the new pH control strategy was applied, pH was stably kept in the range of 7.15-7.30 during fermentation. Thus, 214.3 microg/mL of vitamin B(12) was achieved.
The aerobic Pseudomonas denitrificans is widely used for industrial and commercial vitamin B12 fermentation, due to its higher productivity compared to the anaerobic vitamin B12-producing microorganisms. This paper aimed to develop a cost-effective fermentation medium for industrial vitamin B12 production by P. denitrificans in 120,000-l fermenter. It was found that maltose syrup (a low-cost syrup from corn starch by means of enzymatic or acid hydrolysis) and corn steep liquor (CSL, a by-product of starch industry) were greatly applicable to vitamin B12 production by P. denitrificans. Under the optimal fermentation medium performed by response surface methodology, 198.27 ± 4.60 mg/l of vitamin B12 yield was obtained in 120,000-l fermenter, which was close to the fermentation with the refined sucrose (198.80 mg/l) and was obviously higher than that obtained under beet molasses utilization (181.75 mg/l). Therefore, maltose syrups and CSL were the efficient and economical substrates for industrial vitamin B12 fermentation by P. denitrificans.
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