Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, cobalamin deficiency was produced in vitro by the use of nitrous oxide, known to inactivate the vitamin. In 14 sets of experiments, normal human lymphocytes stimulated with phytohemagglutinin on day 0 were exposed to nitrous oxide and oxygen on day 2. MeCbl was delivered later to half of the cells. Untreated cells served as a control. On day 3, the cells were harvested, the lymphocytes were lysed, and the obtained extracts were assayed for thymidylate synthetase. In 16 other experiments the same procedure was performed, and the incorporation of radioactive thymidine or deoxyuridine by the intact cells was measured. In additional experiments, a deoxyuridine suppression test of treated and untreated stimulated lymphocytes was also performed. The results indicate that nitrous oxide significantly reduces the activity of thymidylate synthetase and that this reduction is significantly corrected by MeCbl, suggesting a causative relation between the vitamin and the enzyme. However, there was no statistically significant effect of nitrous oxide demonstrated on the nucleoside incorporation nor on the deoxyuridine suppression test.
Mol Cell Biochem 1985 Jan
PMID:Methylcobalamin corrects the deleterious in vitro effect of nitrous oxide on thymidylate synthetase. 398 97

Leishmania tropica promastigotes have been selected which are highly resistant to the thymidylate synthetase (TS) inhibitor, 10-propargyl-5,8-dideazafolate (CB3717). As reported for L. tropica resistant to methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), CB3717-resistant organisms have high levels of the bifunctional TS-DHFR and amplified DNA sequences. TS-DHFR represents up to 2% of the protein in cell extracts and does not appear to have a structural alteration that contributes to drug resistance. The amplified unit of DNA has a uniform restriction-site map throughout the selection and is nearly identical to the 30 kb amplified unit of R-region DNA found in MTX-resistant cells, except for a small increase in size of the fragment that contains a junction believed to be the site of DNA rearrangements generated during amplification. CB3717-resistant cells do not possess the amplified H-region DNA found in MTX-resistant cells. The amplified DNA in cells resistant to low levels of CB3717 appears as a 30-kb extrachromosomal circle, similar to the amplified DNA of MTX-resistant organisms. In cells resistant to higher levels of drug, the amplified DNA appeared as higher molecular weight forms. When resistant cells were grown in the absence of drug, the amplified DNA and levels of TS-DHFR gradually fell to approximately 10% of the resistant levels. These findings support the proposal that the R-region DNA possesses the sequences that encode the bifunctional protein.
Mol Biochem Parasitol 1985 Oct
PMID:Selection and properties of Leishmania tropica resistant to 10-propargyl-5,8-dideazafolate, an inhibitor of thymidylate synthetase. 405 89

Thymidine auxotrophic mutants of mouse FM3A cells due to thymidylate synthase deficiency can be transformed into prototrophs by DNA-mediated gene transfer using total human DNA (Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., and Seno, T. (1983) J. Biol. Chem. 258, 48-53). From one such transformed cell clone, cloned recombinant lambda phages containing DNA fragments were obtained recently that were concluded by circumstantial genetic evidence to have been derived from the human thymidylate synthase gene (Takeishi, K., Ayusawa, D., Kaneda, S., Shimizu, K., and Seno, T. (1984) J. Biochem. (Tokyo) 95, 1477-1483). Using a DNA segment derived from the cloned genomic DNA fragment and free of repetitive sequences as a probe, functional cDNA corresponding to thymidylate synthase mRNA could be cloned from a cDNA library of SV40 transformed human fibroblasts constructed by Okayama and Berg (Okayama, H. and Berg, P. (1983) Mol. Cell. Biol. 3, 280-289). The cloned cDNA plasmid containing an insert of approximately 1.7-kilobase transformed mouse thymidine auxotrophic mutant cells to thymidine prototrophic cells at a frequency of 2-3 transformants/micrograms of DNA/10(5) cells, a value almost comparable to the highest so far reported. The resultant transformants retained the introduced cDNA and expressed human thymidylate synthase protein sufficient for supporting normal growth of otherwise auxotrophic mouse cells.
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PMID:Isolation of functional cDNA clones for human thymidylate synthase. 609 54

The folylpolyglutamate hydrolase activities of mouse liver, kidney, muscle and brain were examined by incorporation of methylenetetrahydrofolate polyglutamate reaction products into a stable ternary complex with tritiated fluorodeoxyuridylate and L. casei thymidylate synthetase. Complexes were separated electrophoretically on the basis of charge associated with the polyglutamyl moieties to determine distribution of chain lengths throughout the time course of the reaction. Tissue folylpolyglutamate hydrolase activities were allowed to utilize endogenous folylpolyglutamate as substrates by incubating crude tissue extracts at pH 7.4 ang pH 4.5. Kidney and muscle contained relatively reactive hydrolases which were capable of generating intermediates of essentially all chain lengths from folylpentaglutamate, the predominant endogenous species. The relatively low activity in brain also gave rise to all possible intermediates. Liver contained a high concentration of methylenetetrahydrofolate but little hydrolase activity. The activity present in liver gave rise to essentially no intermediates but yielded only the monoglutamate form of the cofactor. When purified lysosomal preparations from liver and kidney were allowed to react with synthetic folylpolyglutamates, the same specificity with regard to reaction products was observed as with endogenous substrates.
Mol Cell Biochem 1982 Mar 19
PMID:Comparison of folylpolyglutamate hydrolases of mouse liver, kidney, muscle and brain. 617 12

Trichomonas vaginalis is incapable of de novo pyrimidine biosynthesis because it cannot incorporate bicarbonate, aspartate or orotate into its pyrimidine nucleotides or nucleic acids. The organism can salvage exogenous cytidine greater than uridine greater than uracil and thymidine, and incorporate them into the nucleotide pool. A portion of cytidine is converted to CMP, CDP and CTP by cytidine phosphotransferase and nucleotide kinases. Some cytidine and most of uracil are, however, converted first to uridine by cytidine deaminase and uridine phosphorylase respectively; uridine is then incorporated into UMP, UDP and UTP by uridine phosphotransferase and nucleotide kinases. The two phosphotransferases, found mainly in the non-sedimentable fraction of T. vaginalis, provide the main avenue of pyrimidine salvage. No significant levels of pyrimidine phosphoribosyl transferase or nucleoside kinases can be detected in the extract. T. vaginalis has no appreciable dihydrofolate reductase or thymidylate synthetase; it grows normally in millimolar concentrations of methotrexate, pyrimethamine, or trimethoprim, and cannot incorporate labels from exogenous uracil or uridine into DNA. It has an enzyme thymidine phosphotransferase in the sedimentable fraction which converts thymidine to TMP. Thymidine salvage in T. vaginalis is thus totally isolated from the rest of the pyrimidine salvage.
Mol Biochem Parasitol 1984 Feb
PMID:Salvage of pyrimidine nucleosides by Trichomonas vaginalis. 619 66

Thymidylate synthetase levels in five human gastrointestinal tumor cell lines (two colon, two colorectal, one stomach) were determined. Titration of the enzyme in cell cytosol using the active-site titrant, 5-fluoro-2'-deoxyuridine-5'-monophosphate, demonstrated a 20-fold variation in the level of this enzyme among the tumor lines. Titrations performed in the presence or absence of added methylenetetrahydrofolate gave the same values for enzyme content. The cytotoxicity of 5-fluorodeoxyuridine to these cell lines (expressed as EC50 values) varied from 0.44 nM for SW 403 cells to 16 nM for HuTu 80 cells, and in all cases was reversed by the addition of thymidine. The concentration of 5-fluorodeoxyuridine required for cytotoxicity correlated directly (r = 0.98) with the level of thymidylate synthetase in the particular cell line. An inverse correlation (r = -0.95) was observed between the concentration of methotrexate producing cytotoxicity in these cell lines and their thymidylate synthetase levels. The cells were found to contain similar levels of dihydrofolate reductase and to possess normal transport capability for methotrexate.
Mol Pharmacol 1982 May
PMID:Thymidylate synthetase levels as a factor in 5-fluorodeoxyuridine and methotrexate cytotoxicity in gastrointestinal tumor cells. 621 45

We describe the isolation and characterization of a series of 5-fluorodeoxyuridine (FdUrd)-resistant mouse 3T6 cell lines that overproduce thymidylate synthetase (TS) by up to 50-fold compared with the parental cells. The resistant cells were selected by growing 3T6 cells or a methotrexate-resistant 3T6 cell line (M50L3, isolated previously in our laboratory) in gradually increasing concentrations of FdUrd. Uridine and cytidine were included in the culture medium to reduce toxicity from metabolic products of FdUrd. Cells that were resistant to the drug by virtue of loss of thymidine kinase activity were eliminated by selection in medium containing hypoxanthine, methotrexate, and thymidine. M50L3 cells were found to adapt to FdUrd more readily than 3T6 cells. A number of clones were isolated that were able to grow in the presence of 3 microM (M50L3 derived) or 0.3 microM (3T6 derived) FdUrd. Several were found to overproduce TS by 10 to 50-fold compared with normal 3T6 cells. All were found to have thymidine kinase activity, although the enzyme level was significantly reduced in some clones. The overproduced TS was inactivated by 5-fluorodeoxyuridylic acid at the same concentration as the enzyme from 3T6 cells. TS was purified from the LU3-7 clone (50-fold overproducer) by affinity chromatography on methotrexate-polyacrylamide. The monomer molecular weight was about 38,000, which was the same as the molecular weight of the monomer in 3T6 cells. The overproduction trait was gradually lost (half-life, 3 weeks) when LU3-7 cells were grown in the absence of FdUrd. The overproducing cells will provide an abundant supply of TS and (very likely) its mRNA and may serve as a convenient model system for detailed studies of the regulation of TS gene expression during the cell cycle.
Mol Cell Biol 1982 Sep
PMID:Thymidylate synthetase overproduction in 5-fluorodeoxyuridine-resistant mouse fibroblasts. 621 15

5-Fluoro-5'-O-nitro-2'-deoxyuridine (FdUMN), a neutral isostere of 5-fluoro-2'-deoxyuridine 5'-monophosphate, inhibited the growth of L1210 cultures. The inhibition of L1210 cultures by FdUMN was prevented by thymidine, but not by 2'-deoxyuridine. Like 5-fluoro-2'-deoxyuridine (FdUrd), FdUMN inhibited the incorporation of 2'-deoxyuridine into DNA, but the onset of this inhibition was not immediate, as was seen with FdUrd. FdUMN did not inhibit the activity of purified thymidylate synthetase from Lactobacillus casei and was a poor inhibitor of thymidylate synthetase activity in homogenates of L1210 ascites cells. However, after incubation with homogenates of these cells and subsequent addition of ATP, FdUMN inhibited this enzyme effectively. These results indicate that intracellular activation of FdUMN is required for its inhibition of thymidylate synthetase.
Mol Pharmacol 1982 Nov
PMID:Cytotoxicity of 5-fluoro-5'-O-nitro-2'-deoxyuridine, a new fluorinated pyrimidine derivative, in L1210 cultures. 621 91

Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.
Mol Cell Biochem 1984
PMID:Effects of Mg2+ and adenine nucleotides on thymidylate synthetase from different mouse tumors. 623 70

The inhibitors of DNA synthesis, 5-fluoro-2'-deoxyuridine and hydroxyurea, caused an inhibition of thymidine kinase, replicative DNA polymerase and CDP reductase activities in stimulated lymphocytes when they were exposed to the inhibitors during the early transformation period (0-17 hr). However, the enzyme activities were unaffected when the inhibitors were added to cells stimulated for more than 17 hr. As opposed to these enzymes the deoxycytidylate deaminase activity was unaffected by the inhibitors during the entire transformation period (0-28 hr). This indicates a close regulatory mechanism in lymphocytes between DNA synthesis and induction of enzymes involved in DNA replication. The inhibitory mechanism exerted by the inhibitors is for the moment unknown. It might be independent of the well-known inhibition of the target enzymes, thymidylate synthetase and ribonucleoside diphosphate reductase, since there was no immediate apparent correlation in time between depletion of the pool sizes and the inhibition of the enzyme activities.
Mol Cell Biochem 1984 Jun
PMID:Effect of 5-fluoro-2'-deoxyuridine and hydroxyurea on the phytohemagglutinin-induced increase of thymidine kinase, replicative DNA polymerase, deoxycytidylate deaminase and CDP reductase activities in human lymphocytes. 623 43


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