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981.
Iwatsuki K Ikeda Y Ohashi K Nakamura K Kai C 《Microbes and infection / Institut Pasteur》1999,1(12):987-991
We produced a B95a lymphoid cell line persistently infected with canine distemper virus (CDV), in which virus-specific antigens were present in nearly 100% of cells without causing cytopathic effect. The virus recovered from this cell line was able to infect fresh B95a cells persistently, indicating that a persistent CDV was established. 相似文献
982.
H Ohashi S Hashizume H Murakami K Aihara K Shinohara H Omura 《Cell biology international reports》1986,10(2):77-83
A 6-thioguanine-resistant human B-lymphoblastoid cell line, HO-323, was isolated for making human-human hybridomas with high efficiency. Fusions with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients and lymphocytes isolated from lymph nodes of lung and breast cancer patients yield constantly more than one hybridoma clones per 10(5) HO-323 cells plated. HO323 cells also fused with lymphocytes from normal peripheral blood to give hybridomas in the same efficiency. The HO-323 cells were diploid with 46 chromosomes and non-secretors of immunoglobulins. This parent cells doubled every 15 hr and could proliferate in serum-containing medium, even if they were plated at low cell density of less than 10(3) cells/ml. The cells could grow in serum-free medium as well as in serum-containing medium, and the resultant human-human hybridomas could also grow in the same media. 相似文献
983.
Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus. 总被引:2,自引:2,他引:2 下载免费PDF全文
H Ohashi Y Katsuta T Hashizume S N Abe H Kajiura H Hattori T Kamei M Yano 《Applied microbiology》1988,54(11):2603-2607
Penicillin G acylase was purified from the cultured filtrate of Arthrobacter viscosus 8895GU and was found to consist of two distinct subunits with apparent molecular weights of 24,000 (alpha) and 60,000 (beta). The partial N-terminal amino acid sequences of the alpha and beta subunits were determined with a protein gas phase sequencer, and a 29-base oligonucleotide corresponding to the partial amino acid sequence of the alpha subunit was synthesized. An Escherichia coli transformant having the penicillin G acylase gene was isolated from an A. viscosus gene library by hybridization with the 29-base probe. The resulting positive clone was further screened by the Serratia marcescens overlay technique. E. coli carrying a plasmid designated pHYM-1 was found to produce penicillin G acylase in the cells. This plasmid had an 8.0-kilobase pair DNA fragment inserted in the EcoRI site of pACYC184. 相似文献
984.
Yamada T Ohashi Y Ohshima M Inui H Shiota N Ohkawa H Ohkawa Y 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,104(2-3):308-314
A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide
tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order
to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were
obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA
corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 μmol/pot), whereas no accumulation of the corresponding
mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves
by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron
and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides
to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1
cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed
a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides.
These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study
demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred
chemical-inducible herbicide tolerance on potato.
Received: 15 March 2001 / Accepted: 14 June 2001 相似文献
985.
Yoshikatsu Eto Hiroshi Kawame Yoriyasu Hasegawa Toya Ohashi Hiroyuki Ida Toshiharu Tokoro 《Molecular and cellular biochemistry》1993,119(1-2):179-184
The characterization of mutations in Japanese patients with lipidosis, particularly in metachromatic leukodystrophy (MLD) and Gaucher disease has been studied in detail. Metachromatic leukodystrophy is characterized by an accumulation of sulfatide in nervous tissues and kidney due to a deficiency of arylsulfatase A (ASA). We analyzed the presence of three known mutant arylsulfatase A alleles in Japanese patients with MLD. Among 10 patients of Japanese patients with MLD, we found that allele 445A mutation has moderately high incidence and also homozygosity of this mutation results in the late infantile form. Allele 2381T was not found in Japanese patients. Furthermore, we found novel mutation which is G- to A mutation at the 1070 nucleotide of the ASA gene (designated 1070 A) in Japanese patients with juvenile onset. This mutation results in a amino acid substitution of Gly245 by Arg and found in heterozygote form. Our studies of molecular analysis in 10 Japanese patients with MLD indicate that Japanese MLD patients have unique characteristics of ASA mutations compared with those of Caucasian patients. On the other hand, Gaucher disease is the most prevalent sphingolipidosis, characterized by an accumulation of glucocerebroside in macrophage derived cells due to a deficiency of lysosomal hydrolase glucocerebrosidase. To study the molecular basis of Gaucher disease in Japanese patients, we analyzed the presence of the two known mutations (6433C and 3548A) in the glucocerebrosidase gene of 15 patients with Gaucher disease. We found that the 6433C and 3548A mutations occur in all subtypes of Japanese patients with Gaucher disease. Most frequent mutations among them was the 6433C mutation, 40% of 30 chromosomes, whereas the novel mutation of the 3548A found in Japanese patients with neuronopathic Gaucher disease was found in 20% (6 out of 30 chromosomes). The characteristics of these mutations in Japanese patients with Gaucher disease is different from those of Caucasian populations reported previously. 相似文献
986.
Mikio Yagi Shinichiro Kato Yoshiko Kobayashi Kazuo Kubo Shinichi Oyama Toshiyuki Shimizu Tsuyoshi Nishitoba Toshiyuki Isoe Kazuhide Nakamura Hideya Ohashi Nami Kobayashi Noriko Iinuma Tatsushi Osawa Rie Onose Hiroyuki Osada 《Experimental cell research》1997,234(2):285
This report describes the biological effects of our original compound, Ki6783 ((3,4-dimethoxy)-4-phenoxy-6,7-dimethoxyquinoline), a potent and selective inhibitor of platelet-derived growth factor (PDGF) receptor autophosphorylation. This compound strongly inhibited autophosphorylation of the PDGF β-receptor in cultured rat glomerular mesangial cells (MC) bearing this receptor (IC500.1 μM), although it did not inhibit autophosphorylation of other growth factor receptors even at 100 μM.In a cell-free kinase experiment, it showed selective inhibition of PDGF β-receptor tyrosine kinase. A kinetic study of the compound to this tyrosine kinase revealed a competitive mode of action to ATP. [3H]Thymidine incorporation and cell proliferation of MC were inhibited by Ki6783 in a dose-dependent manner after Ki6783 and PDGF-BB were added to the culture medium. Furthermore, this compound normalized the fibrotic cell shape of v-sis-transformed NIH3T3 cells, which grow in an autocrine manner via the PDGF receptor. These effects could be explained by the inhibition of intracellular signal transduction triggered by PDGF receptor autophosphorylation, in which activation of mitogen-activated protein kinase occurs. These results suggest that Ki6783 is one of the more potent and selective inhibitors of PDGF receptor autophosphorylation and that it may be useful in ameliorating cell abnormalities due to excess action of PDGF and its receptor systems in several diseases. 相似文献
987.
A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale. 相似文献
988.
Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes 总被引:20,自引:0,他引:20
Shimano H Yahagi N Amemiya-Kudo M Hasty AH Osuga J Tamura Y Shionoiri F Iizuka Y Ohashi K Harada K Gotoda T Ishibashi S Yamada N 《The Journal of biological chemistry》1999,274(50):35832-35839
To elucidate the physiological role of sterol regulatory element-binding protein-1 (SREBP-1), the hepatic mRNA levels of genes encoding various lipogenic enzymes were estimated in SREBP-1 gene knockout mice after a fasting-refeeding treatment, which is an established dietary manipulation for the induction of lipogenic enzymes. In the fasted state, the mRNA levels of all lipogenic enzymes were consistently low in both wild-type and SREBP-1(-/-) mice. However, the absence of SREBP-1 severely impaired the marked induction of hepatic mRNAs of fatty acid synthetic genes, such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase, that was observed upon refeeding in the wild-type mice. Furthermore, the refeeding responses of other lipogenic enzymes, glycerol-3-phosphate acyltransferase, ATP citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase, and S14 mRNAs, were completely abolished in SREBP-1(-/-) mice. In contrast, mRNA levels for cholesterol biosynthetic genes were elevated in the refed SREBP-1(-/-) livers accompanied by an increase in nuclear SREBP-2 protein. When fed a high carbohydrate diet for 14 days, the mRNA levels for these lipogenic enzymes were also strikingly lower in SREBP-1(-/-) mice than those in wild-type mice. These data demonstrate that SREBP-1 plays a crucial role in the induction of lipogenesis but not cholesterol biosynthesis in liver when excess energy by carbohydrates is consumed. 相似文献
989.
R Sawada K Ohashi H Anaguchi H Okazaki M Hattori N Minato M Naruto 《DNA and cell biology》1990,9(3):213-220
To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6. 相似文献
990.
Three DNA constructs, pETB-40, 41, and 42, encoding human big endothelin-1 (ET-1) preceded by the specific recognition sequence (Ile-Glu-Gly-Arg) for the activated blood coagulation factor Xa (FXa), fused in frame to the N-terminal portion of beta Gal, were expressed in Escherichia coli. The fusion proteins, pETB-40P, 41P, or 42P, consisted of the 55-, 51-, or 42-aa N-terminal peptide of beta Gal and the 38-aa of big ET-1, and had 1, 0, or 0 Cys residues and 5, 5, or 1 Arg residues in the N-terminal peptide of beta Gal, respectively. Enzymatic cleavage of the purified fusion proteins by FXa or trypsin allowed the recovery of authentic human big ET-1. The rates of conversion of pETB-40P, 41P, and 42P to big ET-1 by FXa digestion were 5.6, 11.2, and 30.0%, respectively. pETB-40P with a deletion of one Cys residue and four Arg residues in the N-terminal part was a better substrate than the other two for FXa or trypsin in the production of big ET-1. 相似文献