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11.
Quantification of cell nuclei isolated from hepatocytes by cell lysis with nonionic detergent in citric acid 总被引:1,自引:0,他引:1
A method was developed for determining the number of nuclei of hepatocytes cultured on collagen gel using a nonionic detergent, Nikkol BO-10TX. The cells were recovered in a test tube after solubilizing the gel by incubating it with the detergent in 0.1 M citric acid and then centrifuging the mixture. Nuclei were isolated from the cells with the same detergent solution and collected by centrifugation. The numbers of nuclei in cultures, scored with a hemocytometer or an electronic particle counter, were proportional to the lactate dehydrogenase activities of the cells. This method was also applicable for scoring the number of nuclei of hepatocytes cultured on collagen-coated plastic. 相似文献
12.
Membrane-stabilizing effect of vitamin E: effect of alpha-tocopherol and its model compounds on fluidity of lecithin liposomes 总被引:2,自引:0,他引:2
The effects of vitamin E (alpha-tocopherol) and its model compounds on the fluidity of liposomes composed of dipalmitoylphosphatidylcholin (DPPC) and fatty acids were investigated by the measurement of the fluorescent polarization (P) using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plobe. Although all tocopherols decreased the fluidity of liposomes which was perturbed by the inclusion of an unsaturated fatty acid having more than one double bond, alpha-tocopherol was more effective than the others. The fluidity in arachidonic acid-containing liposomes was decreased most in the presence of alpha-tocopherol and was decreased considerably by the inclusion of model compounds having a side chain at least one isoprene unit or a long straight chain instead of isoprenoid side chain. However, the chromanol with methyl group instead of the above side chain, and phytol, having no chromanol moiety, had no effect. These results show that a structural requirement for a membrane stabilization is to be either the chromanol moiety with methyl groups born on its aromatic ring or a side chain of appropriate length; an isoprenoid side chain of full length or one containing 4'a- and 8'a-methyl groups is not necessarily needed. 相似文献
13.
Cloning in Saccharomyces cerevisiae of a cycloheximide resistance gene from the Candida maltosa genome which modifies ribosomes. 总被引:1,自引:1,他引:0
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We have previously shown that cycloheximide resistance can be induced in a strain of Candida maltosa by modifying ribosomes (M. Takagi, S. Kawai, Y. Takata, N. Tanaka, M. Sunairi, M. Miyazaki, and K. Yano, J. Gen. Appl. Microbiol. 31:267-275, 1985). The present paper describes the cloning of the gene involved in this resistance (designated RIM-C for ribosome modification by cycloheximide) by using a host-vector system of Saccharomyces cerevisiae. 相似文献
14.
Ryuji Fukuda Ryoji Yano Toshikazu Fukui Toshiharu Hase Akira Ishihama Hiroshi Matsubara 《Molecular & general genetics : MGG》1985,201(2):151-157
Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with 32P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP
stringent starvation protein
- PTH
phenylthiohydantoin 相似文献
15.
Increase of translatable mRNA for major microsomal proteins in n-alkane-grown Candida maltosa. 总被引:1,自引:1,他引:0
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In an n-alkane-assimilating Candida sp., transfer from glucose- to n-alkane-containing medium induced changes in the microsomal proteins, and several distinctive polypeptides were demonstrated in the solubilized microsomal fraction derived from n-alkane-grown cells. Long-term-labeling and pulse-labeling experiments in vivo demonstrated the synthesis of the specific microsomal polypeptides. The polypeptides were synthesized as in vitro translation products directed by polyadenylated RNA extracted from n-alkane-grown cells. Two major polypeptides were partially purified from the microsomal fraction from n-alkane-grown cells, and antiserum was prepared in a rabbit. Immunoprecipitation of these two polypeptides was accompanied by an increase in the amount of translatable mRNA. The molecular weights of the polypeptides derived from long-term-labeling, pulse-labeling and in vitro translation experiments appeared to be identical. 相似文献
16.
Purification and characterization of a tuberculin-active substance from Mycobacterium bovis BCG 总被引:1,自引:0,他引:1
A new tuberculin-active substance, designated TAS-1D3, has been purified from the extract of Mycobacterium bovis BCG by precipitation at pH 4.2, ethanol fractionation, and column chromatography involving CM-cellulose, QAE-Sephadex A-25, Sephadex G-100, and Sephadex G-75. TAS-1D3 was homogeneous in polyacrylamide gel electrophoresis and positive in both Coomassie brilliant blue and periodic acid-Shiff staining, suggesting that TAS-1D3 is a glycoprotein. The molecular weight of TAS-1D3 was estimated to be 26,000 by gel filtration. In amino acid analysis, TAS-1D3 was distinctive in having proline as a dominant amino acid, and in that it lacked basic amino acids, sulfur-containing amino acids and aromatic amino acids. Moreover, TAS-1D3 was almost devoid of absorption at around 280 nm. In guinea pigs sensitized with BCG vaccine, the tuberculin activity of TAS-1D3 was about forty times more potent than that of purified protein derivative (PPD). 相似文献
17.
Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells
Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells. 相似文献
18.
Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide 总被引:11,自引:0,他引:11
A Noro K Kimata Y Oike T Shinomura N Maeda S Yano N Takahashi S Suzuki 《The Journal of biological chemistry》1983,258(15):9323-9331
Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts. 相似文献
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