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Biomedical subjects

D Ayusawa

Publications and source records attributed to D Ayusawa.

99 records · Page 6Linked to original sources
PubMed1981

Resistance to methotrexate in thymidylate synthetase-deficient mutants of cultured mouse mammary tumor FM3A cells.

Growth inhibition by methotrexate (MTX) of a cultured mouse mammary carcinoma FM3A line and its mutants deficient in thymidine kinase and thymidylate synthetase was studied under a variety of conditions. In medium containing 10 microM thymidine, the thymidylate synthetase-deficient mutants were slightly more resistant to MTX than the wild-type line and the thymidine kinase-deficient mutant. The addition of both 10 microM thymidine and 50 microM hypoxanthine to the medium completely eliminated the inhibition by MTX of the wild-type and thymidylate synthetase-deficient mutants but had no effect on inhibition of the thymidine kinase-deficient mutant. Thus, addition of either thymidine or purine alone was not sufficient to protect FM3A cells against the growth inhibitory effect of MTX. In contrast, addition of a small amount of 5-methyltetrahydrofolate to medium containing 10 microM thymidine caused an increase of several orders of magnitude in the resistance of thymidylate synthetase-deficient mutants to MTX but did not affect that of the wild-type line. Wild-type cells became almost as resistant to MTX as did the mutant cells by addition of 1 microM 5-fluorodeoxyuridine as well as a small amount of 5-methyltetrahydrofolate with thymidine. These results show directly that thymidylate synthetase is essential in determining the cytotoxicity of MTX by modulating the intracellular tetrahydrofolate pool.

Animals↗
PubMed1981

Effect of tunicamycin on production by mouse fibroblast L929 cells of the factor-stimulating differentiation of mouse myeloid leukemic cells and the colony-stimulating factor.

Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor), which is suggested to be a glycoprotein. On the other hand, growth and differentiation of normal precursor cells of macrophages and granulocytes can be stimulated by a glycoprotein termed colony-stimulating factor (CSF). Mouse fibroblast L929 cells were found to produce both the D-factor and CSF. The properties of the D-factor and CSF and the roles of carbohydrates in the molecules of these factors were examined using tunicamycin, a specific inhibitor of asparaginase-linked glycosylation. Although both the D-factor and CSF were produced by L-cells in usual medium containing fetal calf serum, production of D-factor, but not CSF, was reduced by omission of serum from the medium. The activity of the D-factor was slightly decreased by treating the L-cells with tunicamycin (0.5 microgram/ml) in the presence of 2% fetal calf serum, without any decrease in CSF activity. Conditioned medium of L-cells incubated with or without tunicamycin was fractionated by gel filtration on a Sephadex G-200 column. Normal D-factor appeared as a single peak with an apparent molecular weight of 67,000. D-factor produced in the presence of tunicamycin had an apparent molecular weight of 25,000. On the other hand, most of the CSF was eluted in the void volume, even when it was produced in the presence of tunicamycin. The D-factor produced in the presence of tunicamycin was more sensitive than normal D-factor was to trypsin or heat treatment at 70 degrees. The CSF produced in the presence of tunicamycin was resistant to these treatments. These results indicate that the D-factor is distinct from CSF. Furthermore, the results suggest that the D-factor produced by L-cells is also a glycoprotein and that, although carbohydrate is not essential for production or activity of the D-factor, it contributes to stabilizing the protein portion of D-factor.

Animals↗
PubMed1980

Single-step selection of mouse FM3A cell mutants defective in thymidylate synthetase.

A tritium-suicide method for isolating thymidine auxotrophic mutants is described. Mutagenized mouse FM3A cells were cultured in medium containing [3H] deoxyuridine. Most of the surviving clones examined showed a phenotype of absolute thymidine auxotrophy. This phenotype is very stable and was found to be genetically recessive in cell-cell hybridization experiments. The growth of these variant clones was not supported by various pyrimidine nucleosides other than thymidine. The activity of thymidylate synthetase in crude extracts of these clones was less than 1% of that of FM3A cells. These results strongly indicate that the thymidine auxotrophic phenotype resulted from a genetic defect in thymidylate synthetase.

Animals↗
PubMed1975

Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular alpha-amylase and protease in a Bacillus subtilis mutant.

A mutant of Bacillus subtilis 6160 that had been isolated by its hyperproduction of alpha-amylase and protease lacked flagella and motility, and its content of autolytic enzyme(s) was reduced to one-third to one-fourth that of the parent. These phenotypic differences were completely co-transferred by the deoxyribonucleic acid (DNA) of the mutant when five DNA recipient strains of B. subtilis were transformed. The revertants, isolated by motility with a frequency of approximately 10(-7), recovered a normal level of autolytic activity and showed reduced productivity of alpha-amylase and protease. This point mutation allowed normal flagellin synthesis, spore formation, and rate of growth. The comparison of cell envelope of the mutant with that of the parent indicated that there was no significant difference except loss of flagella. Therefore the association at the cell surface of a group of extracellular proteins consisting of alpha-amylase, proteases, flagellin, and autolytic enzymes(s) seem to be coordinately regulated by the gene or seem to be affected coordinately by certain undetected alterations of the cell envelope.

Alkaline Phosphatase↗
PubMedDate not supplied

A specialized form of chromosomal DNA degradation induced by thymidylate stress in mouse FM3A cells.

Thymidylate synthase-negative mutant mouse cells starved of thymidine or their parental FM3A cells treated with 5-fluoro-2'-deoxyuridine produced DNA fragments ranging from 50 to 200 kilobase pairs with a peak at 100 kb in length as determined by pulsed-field agarose gel electrophoresis. Accumulation of the DNA fragments following such thymidylate stress was time-dependent but their size distribution did not change in either case. Regions of the chromosomal DNA breaks seemed to be restricted to those where DNA replication was in progress as shown by pulse-labeling of the DNA synthesis. Emetine, an inhibitor of protein synthesis, blocked the accumulation of the DNA fragments when present during thymidylate stress. Cell-free extracts prepared from the thymidylate-stressed cells derived by either of the above means were capable of degrading DNA in chromatins prepared from normally growing cells in vitro. The resulting DNA fragments were similar but with a somewhat broader size distribution compared to those produced in vivo. The broader distribution of the fragments produced in the in vitro reaction became closer to the pattern obtained in vivo when ATP and 4 deoxyribonucleotides were added to the reaction.

Adenosine Triphosphate↗
PubMedDate not supplied

Increased sensitivity of thymidine kinase-deficient (TK-) tumor cell lines to the cell growth inhibitory effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and related compounds.

Various established antiherpetic drugs, including 1-beta-D-arabinofuranosylthymine (araT), acyclovir (ACV), 9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG), 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and structurally related analogues thereof, i.e. (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC), (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), and the carbocyclic analogues of BVDU (C-BVDU), IVDU (C-IVDU) and BVDC (C-BVDC), were evaluated for their inhibitory effects on the growth of murine mammary carcinoma (FM3A/0), murine leukemia (L1210/0) and murine fibroblast (LM/0) cells and the thymidine kinase-deficient (TK-) sublines derived from the FM3A/0, L1210/0 and LM/0 cells. BVDU, IVDU and BVDC showed a markedly increased cytostatic activity against the TK- cell lines. To determine the biochemical mechanism of the increased cytostatic action of these compounds toward TK- cell lines, BVDU and IVDU were further evaluated for their inhibitory effects on pyrimidine nucleotide metabolism, in particular thymidylate synthetase activity, their incorporation into DNA and into trichloroacetic acid (TCA)-insoluble material, and their effects on DNA, RNA and protein synthesis in both TK+ and TK- cells. No marked differences were noted in the interaction of BVDU and IVDU with these potential targets between TK+ and TK- cell lines. Furthermore, neither FM3A/0 nor FM3A/TK- cells expressed a significant phosphorylating activity for (125I) IVDU. However, BVDU and IVDU specifically inhibited the incorporation of (1-14C) mannose and (1-14C) glucose into glycoproteins of FM3A/TK- and L1210/TK- cells. To what extent the inhibition of the incorporation of these monosaccharides into glycoproteins may contribute to the increased cytostatic effects of BVDU and IVDU on TK- cells remains to be determined.

Animals↗