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31.
Chunyu Cao Hisao Kurazono Shinji Yamasaki Keiko Kashiwagi Kazuei Igarashi Yoshifumi Takeda 《Microbiology and immunology》1994,38(6):441-447
The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed. 相似文献
32.
Yoshihito Shirai Masaaki Yamaguchi Atsuko Kobayashi Akihiro Nishi Hisao Nakamura Hiroki Murakami 《Cytotechnology》1994,14(2):129-146
The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to ajust the pH level when using medium-circulating-fermenters. 相似文献
33.
Zenjiro Osawa Tsubura Morota Kenichi Hatanaka Toshihiro Akaike Kei Matsuzaki Hideki Nakashima Naoki Yamamoto Eiichiro Suzuki Hiroshi Miyano Tohru Mimura Yutaro Kaneko 《Carbohydrate polymers》1993,21(4):283-288
Sulfopropyl curdlan was synthesized, its structure was determined, and the anti-HIV activity was compared with that of standard curdlan sulfates obtained with piperidine N-sulfonic acid in dimethyl sulfoxide. It was shown that sulfopropyl curdlan exhibits weaker anti-HIV activity than curdlan sulfate. Curdlan sulfates were synthesized with a SO3-pyridine complex in a heterogeneous phase. It was shown from 13C-NMR spectra of acetylated curdlan sulfates that they had a different substituent distribution from standard curdlan sulfate. The cytotoxicity of the curdlan sulfates was attributed to their heterogeneous structure. 相似文献
34.
35.
Kawarabayasi Yutaka; Tanaka Ayako; Ohara Osamu; Arakawa Taku; Oka Masanori; Kato Hisako; Morita Miyo; Fujisawa Hisao 《DNA research》1994,1(6):289-296
To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained. 相似文献
36.
Zaw Lin Shinji Yamasaki Hisao Kurazono Mari Ohmura Tadahiro Karasawa Takahiro Inoue Seisuke Sakamoto Takahiro Suganami Tomohiro Takeoka Yoshihiro Taniguchi Yoshifumi Takeda 《Microbiology and immunology》1993,37(6):451-459
We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada. 相似文献
37.
Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae. 相似文献
38.
Noritsugu Yabe Kouji Komiya Tetsuya Takezono Hisao Matsui 《In vitro cellular & developmental biology. Animal》1993,29(10):795-806
Summary Lysozyme at 1 to 100μg/ml of exposure levels augmented or inhibited proliferative response of human peripheral blood lymphocytes stimulated with
interleukin-2 (IL-2). This contradictory effect of lysozyme depended on IL-2 concentration, activating state of lymphocytes,
addition time of lysozyme, and serum existence. Lymphocytes increased their IL-2-mediated proliferating ability in response
to lysozyme when stimulated with less than suboptimal concentration of IL-2. Lymphocyte activation with anti-CD3 antibody
changed the augmented proliferative response into the inhibited response by lysozyme addition whereas elimination of MHC class
II molecule-expressing cells augmented the response. Addition of lysozyme within 1 h after IL-2 exposure was most effective
in promoting the proliferation whereas additions after 16 to 24 h were ineffective or inhibitory. Addition after longer than
24 h inversely restored the proliferative response. Serum seemed to retard lysozyme action because either sequential serum
addition 1 h after exposure of IL-2 and lysozyme to cells or exposure of IL-2 and serum after pretreatment of cells with lysozyme
changed the proliferative responsiveness from inhibition into augmentation. Thus lysozyme may regulate lymphocyte proliferation
responding to a magnitude of antigenic stimuli and to the progression of cellular events that periodically occur. 相似文献
39.
Hisao Yamaguchi Keiko Hosokawa Zheng-Lin Jiang Akira Takahashi Toshitaka Ikehara Hiroshi Miyamoto 《Journal of cellular biochemistry》1993,53(1):13-20
Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [3H]thymidine. The progression was arrested in a chemically defined medium in which K+ was replaced by Rb+ (Rb-CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb-CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K?-depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase. 相似文献
40.
Koichi Matsuzaki Mikio Kan Wallace L. McKeehan 《In vitro cellular & developmental biology. Animal》1996,32(6):345-360
Summary Two transmembrane serine-threonine kinases (type I and II receptors), a membrane-anchored proteoglycan (type III), and a homodimeric
ligand participate in the transforming growth factor beta type on (TGFβ1) signal transduction complex. The expression of recombinant
receptors in insect cells co-infected with up to three recombinant baculoviruses was employed to study interactions among
the ectodomains of the three types of receptors and the TGFβ1 ligand in absence of uncontrollable extrinsic factors in mammalian
cells. Multi-subunit complexes were assembled in intact cells and purified on glutathione-conjugated beads for analysis by
tagging one of the subunits with glutathione S-transferase (GST). Intrinsic ligand-independent interactions were observed
among receptor subunits as follows: type III–III type I–I, type III-I, and type II-I. The homeotypic complex of type II–II
receptors and the heterotypic type III-II interaction was ligand dependent. The type I, but not the type III, subunit displaced
about 50% of the type II component in either ligand-dependent homomeric type II-type II complexes or heteromeric type III-type
II complexes to form type II-I or type III-II-I oligomers, respectively. The type II subunit displaced type I subunits in
oligomers of the type I subunit. Specificity of type I receptors may result from differential affinity for the type II receptor
rather than specificity for ligand. A monomeric subunit of the TGFβ1 ligand bound concurrently to type III and type II or
type III and type I receptors, but failed to concurrently bind to the type II and type I subunits. The binding of TGFβ1 to
the type I kinase subunit appears to require an intact disulfide-linked ligand dimer in the absence of a type III subunit.
The combined results suggest a pentameric TGFβ signal transduction complex in which one unit each of the type III, type II,
and type I components is assembled around the two subunits of the dimeric TGFβ1 ligand. An immobilized GST-tagged subunit
of the receptor complex was utilized to assemble multi-subunit complexesin vitro and to study the phosphorylation events among subunits in the absence of extrinsic cell-derived kinases. The results revealed
that (a) a low level of ligand-independent autophosphorylation occurs in the type I kinase; (b) a high level of autophosphorylation
occurs in the type II kinase; (c) both the type III and type I subunits aretrans-phosphorylated by the type II subunit; and (d) the presence of both type I and II kinases complexed with the type III subunit
and dimeric TGFβ1 ligand in a pentameric complex causes maximum phosphorylation of all three receptor subunits. 相似文献