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81.
Y Sakai S Kimura T Yamashita K Umetsu T Suzuki F Sendo 《Microbiology and immunology》1989,33(7):569-577
A sensitive thymocyte co-stimulator assay of IL-1 using a beta-D-galactoside specific lectin (allo A) obtained from the beetle (Allomyrina dichotoma) is reported here. Allo A stimulated [3H]thymidine uptake of mouse thymocytes in the presence of IL-1. The allo A assay was more sensitive than the PHA or PNA- thymocyte assay, especially at low doses of IL-1. Optimal conditions for the allo A assay were as follows: allo A, 2.5-5.0 micrograms/ml; whole thymocytes, 0.5-1.0 x 10(6) cells/well; incubation time, 72-96 hr. The assay is sensitive and convenient and can easily be performed in any laboratory. 相似文献
82.
A Okuda M Imagawa Y Maeda M Sakai M Muramatsu 《The Journal of biological chemistry》1989,264(28):16919-16926
83.
Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions 总被引:3,自引:0,他引:3
H Kimura K Sogawa Y Sakai Y Fujii-Kuriyama 《The Journal of biological chemistry》1989,264(4):2338-2342
Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen. 相似文献
84.
Purification and characterization of a Ca2+-dependent actin filament severing protein from bovine adrenal medulla 总被引:3,自引:0,他引:3
S Maekawa M Toriyama S Hisanaga N Yonezawa S Endo N Hirokawa H Sakai 《The Journal of biological chemistry》1989,264(13):7458-7465
We describe the purification of an actin regulatory protein from bovine adrenal medulla. This protein caused a dose-dependent decrease of the specific viscosity of actin solution within 30 s of its addition in a Ca2+-sensitive way. Sedimentation assays and the observation by electron microscopy showed that this effect was ascribable to the fragmentation of actin filaments. This protein apparently promoted nucleation of actin polymerization and increased the critical concentration of actin for polymerization nearly 5-fold, suggesting its binding to the barbed end of actin filaments. The inhibitory effect of this protein on the elongation of actin from the barbed end of the myosin subfragment S1-labeled actin seeds confirmed this suggestion. These properties are similar to those of gelsolin. However, the physicochemical properties of this protein having a single polypeptide chain with a molecular weight of 74,000, a Stokes radius of 3.9 nm, a sedimentation coefficient (s0(20),w) of 4.5 S, and an immunological characterization showed that this protein is different from gelsolin. 相似文献
85.
Cytoprotective effect of TRK-100, a prostacyclin analogue, against chemical injuries in cultured human vascular endothelial cells 总被引:1,自引:0,他引:1
We studied the cytoprotective effect of TRK-100, a chemically stable analogue of prostacyclin (PGI2), in the cultured human endothelial cells from umbilical vein. TRK-100 (10 and 100 nM) stimulated significantly proliferation of endothelial cells but did not affect PGI2 production in endothelial cells. Exposure of cultured endothelial cells to homocysteine (2.5 mM) or glucose (50 mM) caused concentration-dependent cytotoxicity, as evidenced by a decrease in number of viable cells. When endothelial cells were treated with TRK-100 simultaneously or prior to, but not after, exposure to injury substances, decreases in viable cell were significantly suppressed. The protective effect of TRK-100 against homocysteine-induced cytotoxicity also appeared in endothelial cells treated with acetylsalicylic acid, suggesting that endogenous PGI2 did not involve in the protective effect of TRK-100. 相似文献
86.
T. Sakai J. Imamura 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,80(3):421-427
Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile. 相似文献
87.
Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2 总被引:3,自引:0,他引:3
R Shibata A Adachi H Sakai A Ishimoto T Miura M Hayami 《Journal of medical primatology》1990,19(3-4):217-225
We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus. 相似文献
88.
R Takayanagi K Ohnaka Y Sakai R Nakao T Yanase M Haji T Inagami H Furuta D F Gou M Nakamuta 《Biochemical and biophysical research communications》1992,183(2):910-916
We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II greater than Ang III greater than Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis. 相似文献
89.
H Sakai S S Park Y Kikkawa 《Biochemical and biophysical research communications》1992,187(3):1262-1269
Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity. 相似文献
90.
J Nishihira T Ishibashi M Sakai S Nishi T Kumazaki Y Hatanaka 《Biochemical and biophysical research communications》1992,189(2):1243-1251
1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand. 相似文献