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111.
Oku H Futamori N Masuda K Shimabukuro Y Omine T Iwasaki H 《Bioscience, biotechnology, and biochemistry》2003,67(10):2106-2114
It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway. 相似文献
112.
The effects of protease inhibitors on the production of recombinant protein were investigated using a recombinant baculovirus containing GFPuv-human beta 1,3-N-acetylglucosaminyltransferase 2 (beta 3GnT2) connected to the prepromelittin signal sequence. The addition of leupeptin as a cysteine protease inhibitor at 2.5 microg/ml improved intra- and extracellular beta 3GnT activities 5- and 3-fold, respectively, compared to those without addition, which was due to a suppression of protease activity. With the leupeptin addition only four degraded molecular bands lower than 32 kDa appeared, but 9 degraded molecular bands between 29 kDa and 41 kDa existed without addition. In contrast, pepstatin A as a carboxyl protease inhibitor had no influence on the improvement of beta 3GnT production, judging from SDS-PAGE. Moreover, when 50 microM carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), known as a proteasome inhibitor, was used in combination with the leupeptin, a ladder of low molecular mass bands of fusion protein was diminished. The intracellular beta 3GnT activity increased 9-fold, to as high as that without addition of two kinds of protease, but the extracellular activity was not different from that with the addition of only leupeptin. These findings indicate that the decrease in cell viability causes the decrease in the secretion rate of intracellular fusion protein, resulting the accumulation of the full-length of fusion protein. 相似文献
113.
114.
Totani K Kubota T Kuroda T Murata T Hidari KI Suzuki T Suzuki Y Kobayashi K Ashida H Yamamoto K Usui T 《Glycobiology》2003,13(5):315-326
Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers. 相似文献
115.
Katoh M Egashira K Kataoka C Usui M Koyanagi M Kitamoto S Ohmachi Y Takeshita A Narita H 《American journal of physiology. Heart and circulatory physiology》2001,280(5):H2306-H2312
We previously reported that chronic inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) induces vascular inflammation at week 1 and produces subsequent arteriosclerosis at week 4 and that cotreatment with an angiotensin-converting enzyme (ACE) inhibitor prevents such changes. In the present study, we tested the hypothesis that treatment with an ACE inhibitor after development of vascular inflammation could inhibit arteriosclerosis in rats. Wistar-Kyoto rats were randomized to four groups: the control group received no drugs, the 4wL-NAME group received L-NAME (100 mg x kg(-1) x day(-1)) for 4 wk, the 1wL + 3wNT group received L-NAME for 1 wk and no treatment for the subsequent 3 wk, and the 1wL + 3wACEI group received L-NAME for 1 wk and the ACE inhibitor imidapril (20 mg x kg(-1) x day(-1)) for the subsequent 3 wk. After 4 wk, we observed significant arteriosclerosis of the coronary artery (medial thickening and fibrosis) and increased cardiac ACE activity in the 1wL + 3wNT group as well as in the 4wL-NAME group, but not in the 1wL + 3wACEI group. In a separate study, we examined apoptosis formation and found that posttreatment with imidapril (20 mg x kg(-1) x day(-1)) or an ANG II AT1-receptor antagonist, CS-866 (5 mg x kg(-1) x day(-1)), induced apoptosis (TdT-mediated nick end-labeling) in monocytes and myofibroblasts appearing in the inflammatory lesions associated with a clear degradation in the heart (DNA electrophoresis). In conclusion, treatment with the ACE inhibitor after 1 wk of L-NAME administration inhibited arteriosclerosis by inducing apoptosis in the cells with inflammatory lesions in this study, suggesting that increased ANG II activity inhibited apoptosis of the cells with inflammatory lesions and thus contributed to the development of arteriosclerosis. 相似文献
116.
Usui I Haruta T Iwata M Takano A Uno T Kawahara J Ueno E Sasaoka T Kobayashi M 《Biochemical and biophysical research communications》2000,275(1):115-120
In the early phase of adipocyte differentiation, transient increase of DNA synthesis, called clonal expansion, and transient hyperphosphorylation of retinoblastoma protein (Rb) are observed. We investigated the role of these phenomena in insulin-induced adipocyte differentiation of 3T3-L1 cells. Insulin-induced clonal expansion, Rb phosphorylation and adipocyte differentiation were all inhibited by the PI 3-kinase inhibitors and rapamycin, but not the MEK inhibitor, whereas the MEK inhibitor, but not PI 3-kinase inhibitors or rapamycin, decreased c-fos induction. We conclude that insulin induces hyperphosphorylation of Rb via PI 3-kinase and mTOR dependent pathway, which promotes clonal expansion and adipocyte differentiation of 3T3-L1 cells. 相似文献
117.
Hirofumi Usui Rintaro Inoue Osamu Tanabe Yasumasa Nishito Masahiro Shimizu Hideyuki Hayashi Hiroyuki Kagamiyama Masao Takeda 《FEBS letters》1998,430(3)
Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B″ (δ) subunit, was phosphorylated at serine residues of B″ in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 μM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B″, respectively. The Km value of A-kinase for CAB″ was 0.17±0.01 μM in the presence of OA. The major in vitro phosphorylation sites of B″ were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B″ did not dissociate B″ from CA, and stimulated the molecular activity of CAB″ toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a. 相似文献
118.
A clear parallelism was demonstrated between the efficiency as substrate of the substituted oligopeptides corresponding to the carboxy-terminal (C-terminal) sequence of the precursor D1 protein (pD1) in the in vitro enzymatic assay and their competitive inhibitory capacity toward the proteolytic C-terminal processing of the full-length pD1 integrated in the intact photosystem II complex embedded in the thylakoid membrane of Scenedesmus obliquus LF-1 mutant, as shown e.g. by the influence of L343A, A345G and A345V substitutions and the effect of C-terminal fragments. This suggests that the basic mechanism for substrate recognition by the processing protease elucidated in the enzymatic analysis using synthetic oligopeptides is also effective in vivo, although it can sometimes be difficult to detect the consequence of amino acid substitution in the integrated systems. 相似文献
119.
Yumiko Nakagaito Motonobu Satoh Haruhiko Kuno Toshi Iwama Masao Takeuchi Akira Hakura Touho Yoshida 《In vitro cellular & developmental biology. Animal》1998,34(7):585-592
Summary We have established a multipotent clonal cell line, named MEB5, from embryonic mouse forebrains after the infection of a retrovirus
carrying E7 oncogene of human papillomavirus type 16. MEB5 cells proliferated in serum-free, epidermal growth factor (EGF)-supplemented
medium. They expressed markers for neural precursor cells (nestin, A2B5, and RC1) and did not express markers for neurons
(class III β-tubulin), astrocytes (glial fibrillary acidic protein), and oligodendrocytes (galactocerebroside). MEB5 cells
were stably maintained in an undifferentiated state with a diploid karyotype in the presence of EGF. When they were deprived
of EGF, about 50% of the cells died due apoptosis within 24 h. The remaining cells differentiated into neurons, astrocytes,
or oligodendrocytes within 2 wk. The newly developed cells with neuronal morphology were immunoreactive for γ-aminobutyric
acid and exhibited neuronal electrophysiological properties. When MEB5 cells were treated with leukemia inhibitory for 7 d,
they were induced to differentiate exclusively into astrocytes. These results inducate that MEB5 is a cell line with characteristics
of EGF-dependent, multipotent neural precursor cells. This cell line should provide a good model system to study the mechanisms
of survival, proliferation, and differentiation of the multipotent precursor cells in the central nervous system. 相似文献
120.
Po-sung Chu Hirotoshi Ebinuma Nobuhiro Nakamoto Kazuo Sugiyama Shingo Usui Yuko Wakayama Nobuhito Taniki Akihiro Yamaguchi Shunsuke Shiba Yoshiyuki Yamagishi Takaji Wakita Toshifumi Hibi Hidetsugu Saito Takanori Kanai 《PloS one》2015,10(5)
Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3− NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2. 相似文献