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31.
Two molecularly cloned coisolates of human immunodeficiency virus type 1 (HIV-1) have been found to exhibit different phenotypes of viral expression, either rapid and cytopathic (N1T-A virus) or delayed and noncytopathic (N1T-E virus [X. Ma, K. Sakai, F. Sinangil, E. Golub, and D. J. Volsky, Virology 176:184-194, 1990]). To identify the viral genetic elements responsible for these phenotypes, we prepared reciprocal recombinants in different regions of N1T-A and N1T-E viral genomes. Infectivity experiments with the recombinant viruses revealed that the rapid/cytopathic (N1T-A-like) phenotype assorted cleanly with the V1f-coding region and Vif expression. The smallest HIV-1 DNA region that conferred the complete phenotypic switch was a 284-bp NdeI-StuI fragment within the vif open reading frame. Nucleotide sequence analysis revealed a 35-bp deletion starting at nucleotide 218 in the N1T-E vif gene. A 23-kDa Vif protein was detected by immunoblotting using Vif-specific antiserum in extracts of cells infected with N1T-A but not N1T-E virus. No detectable vif protein was found in association with sedimented particles of either virus. Cotransfection of a eucaryotic vif expression plasmid with N1T-E DNA complemented the N1T-E defect; rapid/cytopathic infection similar to that in N1T-A-transfected cells was observed. We conclude that Vif controls the rate, and consequently the cytopathic outcome, of HIV-1 infection. 相似文献
32.
N Sakai J Tamaoki K Kobayashi T Kanemura K Isono K Takeyama S Takeuchi T Takizawa 《Regulatory peptides》1991,34(1):33-41
We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP. 相似文献
33.
Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF. 总被引:53,自引:15,他引:38
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Y Gotoh K Moriyama S Matsuda E Okumura T Kishimoto H Kawasaki K Suzuki I Yahara H Sakai E Nishida 《The EMBO journal》1991,10(9):2661-2668
MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization. 相似文献
34.
35.
M Hoshi T Akiyama Y Shinohara Y Miyata H Ogawara E Nishida H Sakai 《European journal of biochemistry》1988,174(2):225-230
It has previously been demonstrated that microtubule-associated protein 2 (MAP2) is a good substrate for the purified protein kinase C [Tsuyama, S., Bramblett, G. T., Huang, K.-P. & Flavin, M. (1986) J. Biol. Chem. 261, 4110-4116; Akiyama, T., Nishida, E., Ishida, J., Saji, N., Ogawara, H., Hoshi, M., Miyata, Y. & Sakai, H. (1986) J. Biol. Chem. 261, 15648-15651]. We have shown here that phosphorylation of MAP2, catalyzed by protein kinase C, reduces the ability to induce tubulin polymerization. MAP2 is divided into two domains by digestion with alpha-chymotrypsin; the microtubule-binding and the non-binding (projection) domains. The limited chymotryptic digestion following phosphorylation of MAP2 by protein kinase C has shown that both the domains of MAP2 were phosphorylated by protein kinase C, 50-60% of the incorporated phosphates being detected in the microtubule-binding domain. Polypeptide fragments, containing the microtubule-binding domain of MAP2, were purified by DEAE-cellulose column chromatography after chymotryptic digestion of MAP2. The purified microtubule-binding fragments were competent to polymerize tubulin, and served as good substrates for protein kinase C. The phosphorylation of the microtubule-binding fragments by protein kinase C reduced their ability to induce tubulin polymerization. These results suggest that the ability of MAP2 to induce tubulin polymerization is inhibited by phosphorylation of the microtubule-binding domain catalyzed by protein kinase C. 相似文献
36.
Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells 总被引:3,自引:0,他引:3
Y Miyata M Hoshi S Koyasu T Kadowaki M Kasuga I Yahara E Nishida H Sakai 《Experimental cell research》1988,178(1):73-83
Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors. 相似文献
37.
In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells. 相似文献
38.
p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme 总被引:3,自引:0,他引:3
p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay. 相似文献
39.
Microtubule-binding domain of tau proteins 总被引:12,自引:0,他引:12
H Aizawa H Kawasaki H Murofushi S Kotani K Suzuki H Sakai 《The Journal of biological chemistry》1988,263(16):7703-7707
Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain. 相似文献
40.
T Akiyama E Nishida J Ishida N Saji H Ogawara M Hoshi Y Miyata H Sakai 《The Journal of biological chemistry》1986,261(33):15648-15651
We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction. 相似文献