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601.
Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates, enabling the bacterium to survive in a continuously changing environment. These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market. Unfortunately, the secretion of heterologous proteins, originating from Gram-negative bacteria or from eukaryotes, is often severely hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed. Nevertheless, research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host. This review focuses on studies that aimed at optimizing B. subtilis as cell factory for commercially interesting heterologous proteins. 相似文献
602.
Zanen G Antelmann H Westers H Hecker M van Dijl JM Quax WJ 《Journal of bacteriology》2004,186(17):5956-5960
Bacillus subtilis contains three proteins of the signal recognition particle-GTPase family known as Ffh, FtsY, and FlhF. Here we show that FlhF is dispensable for protein secretion, unlike Ffh and FtsY. Although flhF is located in the fla/che operon, B. subtilis 168 flhF mutant cells assemble flagella and are motile. 相似文献
603.
van der Neut Kolfschoten M Dirven RJ Vos HL Tans G Rosing J Bertina RM 《The Journal of biological chemistry》2004,279(8):6567-6575
Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1=5.4 x 10(4) M(-1) s(-1)) and a slow phase (kx2=3.2 x 10(3) M(-1) s(-1)). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained approximately 80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV. 相似文献
604.
605.
Background
Capillary DNA sequencing machines allow the generation of vast amounts of data with little hands-on time. With this expansion of data generation, there is a growing need for automated data processing. Most available software solutions, however, still require user intervention or provide modules that need advanced informatics skills to allow implementation in pipelines. 相似文献606.
Postmenopausal diabetic women are at greater risk for heart disease compared with men of similar age and with other risk factors. We examined the hypothesis that 17beta-estradiol and trivalent chromium inhibit secretion of the pro-inflammatory cytokine interleukin (IL)-6 and oxidative stress in monocytes exposed to high glucose (HG). U937 human monocytes were cultured with HG (30 mM) with and without 17beta-estradiol (0-1000 nM) and chromium chloride (Cr(3+), 0-10 muM) at 37 degrees C for 24 h. Results show that 17beta-estradiol inhibits IL-6 and adhesion to endothelial cells (p <. 05) by HG-treated monocytes. Treatment with 17beta-estradiol+Cr(3+) required a significantly lower dose of estradiol-17beta compared with 17beta-estradiol alone for IL-6 inhibition. 17beta-Estradiol+Cr(3+) also inhibited lipid peroxidation and the adhesivity to human endothelial cells in HG-treated monocytes. Thus, 17beta-estradiol+Cr(3+) inhibits oxidative stress, IL-6 secretion, and monocytic adhesion to endothelial cells, risk factors in the development of heart disease. The female body requires E but studies on some patients indicate side effects with increased amounts of 17beta-estradiol-supplementation. The potential benefit of a lower estrogen dose in combination with chromium is novel and needs to be explored in postmenopausal diabetic women. 相似文献
607.
Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome. 总被引:9,自引:0,他引:9
Harold Tjalsma Haike Antelmann Jan D H Jongbloed Peter G Braun Elise Darmon Ronald Dorenbos Jean-Yves F Dubois Helga Westers Geeske Zanen Wim J Quax Oscar P Kuipers Sierd Bron Michael Hecker Jan Maarten van Dijl 《Microbiology and molecular biology reviews》2004,68(2):207-233
Secretory proteins perform a variety of important "remote-control" functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which approximately 90 extracellular proteins were identified. Analysis of these proteins disclosed various "secrets of the secretome," such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only approximately 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance. 相似文献
608.
Rogier W Sanders Martijn M Dankers Els Busser Michael Caffrey John P Moore Ben Berkhout 《Retrovirology》2004,1(1):1-11
Background
The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W)-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clément, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816–48826, 2002). In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae.Results
Our data clearly shows that integrity of N-terminal α-helix I (17–33) and α-helix III (53–83) is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides.Conclusions
Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes α-helix I (17–33) and α-helix III (53–83). Furthermore, the mechanism(s) underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells. 相似文献609.
van Bennekum AM Wei S Gamble MV Vogel S Piantedosi R Gottesman M Episkopou V Blaner WS 《The Journal of biological chemistry》2001,276(2):1107-1113
Transthyretin (TTR) acts physiologically in the transport of retinol in the circulation. We previously reported the generation and partial characterization of TTR-deficient (TTR(-)) mice. TTR(-) mice have very low circulating levels of retinol and its specific transport protein, retinol-binding protein (RBP). We have examined the biochemical basis for the low plasma retinol-RBP levels. Cultured primary hepatocytes isolated from wild type (WT) and TTR(-) mice accumulated RBP in their media to an identical degree, suggesting that RBP was being secreted from the hepatocytes at the same rate. In vivo experiments support this conclusion. For the first 11 h after complete nephrectomy, the levels retinol and RBP rose in the circulations of WT and TTR(-) mice at nearly identical rates. However, human retinol-RBP injected intravenously was more rapidly cleared from the circulation (t(12) = 0.5 h for TTR(-) versus t(12) >6 h for WT) and accumulated faster in the kidneys of TTR(-) compared with WT mice. The rate of infiltration of the retinol-RBP complex from the circulation to tissue interstitial fluids was identical in both strains. Taken together, these data indicate that low circulating retinol-RBP levels in TTR(-) mice arise from increased renal filtration of the retinol-RBP complex. 相似文献
610.