Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells treated with methotrexate and labeled with [(3)H]dUrd contained dUMP in DNA in readily detectable amounts ( approximately 0.8 pmol of dUMP per mumol of total DNA nucleotide), and this was increased approximately 3-fold if the cells were also treated with Ura at the same time. No dUMP (<1 fmol/mumol of DNA) could be detected by these methods in DNA from cells not treated with methotrexate, regardless of whether Ura was present or absent. The presence of dUMP in DNA from cells treated with methotrexate is a result of the great increase in intracellular concentration of dUTP and the fall in dTTP that accompany inhibition of thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) by the drug. These changes are apparently sufficient to overcome the normal mechanisms that exclude dUMP from DNA, and the enhancement by Ura reflects suppression of one of the mechanisms, Ura removal from DNA by the enzyme Ura-DNA glycosylase. The results suggest an active lesion of DNA in cells in which thymidylate synthetase is inhibited. Under these conditions there appears to be a cyclic incorporation and removal of dUMP resulting from reinsertion of dUMP during gap repair at sites of Ura removal. This consequence of the normal excision-repair process, which occurs when intracellular levels of dUTP approach those of dTTP, may have effects related to the cytotoxicity of drug inhibitors of thymidylate synthetase, clinical deficiencies of folate and vitamin B-12, and thymineless death, in general.
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PMID:Methotrexate-induced misincorporation of uracil into DNA. 692 29

The lipophilic diaminopyridopyrimidine BW 301U (2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine) is as active as methotrexate as an inhibitor of dihydrofolate reductase and mammalian cell growth. This compound was selected from among related pyridopyrimidines and other lipid-soluble diaminoheterocyclic compounds as having the most favorable combination of properties as a potent inhibitor of dihydrofolate reductase with minimal effects on histamine metabolism. In contrast to methotrexate, entry of BW 301U into cells is rapid and is not temperature dependent, indicating passage across cell membranes by diffusion. There is no competition between BW 301U and leucovorin (folinic acid) for uptake into Sarcoma 180 cells in culture. When BW 301U is added to culture medium, deoxyuridine incorporation ceases within the first few min, and this inhibition persists when cells are transferred to drug-free medium. Both leucovorin and thymidine are required to protect cells in culture from the cytotoxicity of BW 301U. The effect on thymidine biosynthesis appears to be indirect since BW 301U is inactive as an inhibitor of thymidylate synthetase. Hypoxanthine and thymidine restore growth by only 50% in cultures containing BW 301U, and complete restoration of growth requires the further addition of adenosine and either uridine or cytidine to the medium. In vivo, BW 301U is active against Walker 256, L1210, P388, Sarcoma 180, and Ehrlich ascites tumors.
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PMID:Biochemical and chemotherapeutic studies on 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine (BW 301U), a novel lipid-soluble inhibitor of dihydrofolate reductase. 695 6

DNA of a mutant of the bacteriophage T4, which contains cytosine instead of glucosylated hydroxymethylcytosine, was shown to direct the synthesis of enzymatically active dihydrofolate reductase in a coupled in vitro transcription-translation system. The DNA-directed synthesis of the enzyme was used to localize the dihydrofolate-reductase gene frd on a 2300 bp long restriction-nuclease-generated DNA fragment. Fine structure mapping showed that the gene is encoded on a segment of less than 1850 bp but more than 700 bp length. The enzyme, which is synthesized in vitro from the DNA fragment, has a molecular weight of 18 500 to 19 500. A restriction map was constructed which extends about 10 kb to both sides of the reductase gene and which covers the T4 genome between the genes 55 and 63. The two genes which flank the frd gene, genes 32 and td (thymidylate synthetase), were mapped in detail. A correlation between the physical and genetic maps was established.
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PMID:Isolation and characterization of DNA fragments containing the dihydrofolate-reductase gene of coliphage T4. 699 88

The Saccharomyces cerevisiae tmp3 mutant is deficient in the mitochondrial enzyme complex that participates in the formation of one-carbon-group-tetrahydrofolate coenzymes, serine transhydroxymethylase, dihydrofolate reductase, and thymidylate synthetase, thus leading to multiple nutritional requirements of dTMP, adenine, histidine, and methionine. The tmp3 mutant quickly loses its mitochondrial genome even when grown on fully supplemented medium or on a high concentration of 5-formyl tetrahydrofolate, which replaces all the four requirements. A study of the loss of the mitochondrial genome by following several mitochondrial genetic markers showed that there was a preferential specific loss of a large region of the mitochondrial genome, covering mit ts983, Er, Cr, and mit ts982 up to OrI, and retention of the region of Pr and mit tscs1297. A kinetic study showed that there was a preferentially rapid loss of the region covering the mit+ alleles ts983 to tscs902 at the rate of 10% per generation.
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PMID:Influence of the nuclear gene tmp3 on the loss of mitochondrial genes in Saccharomyces cerevisiae. 705 Jun 73

N-Substituted indan-1.3-diones have proven to be potent cytotoxic agents effective against the growth of single cell leukemia tumors and cell lines derived from solid tumors. A number of the derivatives were active against growth of solid tumors e.g. colon, lung bronchogenic and osteosarcoma for which few effective agents are available to inhibit their growth. These agents inhibited DNA and RNA synthesis of L1210 cells. The de novo purine synthetic pathway was inhibited at PRPP amido transferase and IMP dehydrogenase. The pyrimidine synthetic pathway was inhibited at aspartate transcarbamylase. Other sites which demonstrate minor inhibition were DNA polymerase alpha, r- and t-RNA polymerase, ribonucleoside reductase, dihydrofolate reductase, nucleoside kinases and thymidylate synthetase. In addition d(NTP) pool levels were reduced by the drugs. L1210 DNA strand scission was evident after exposure to drugs for 24 hr. at 100 microM.
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PMID:Cytotoxicity and mode of action of substituted indan-1, 3-diones in murine and human tissue cultured cells. 784 49

We have expressed the dihydrofolate reductase (DHFR) part of the DHFR-thymidylate synthetase complex of P. falciparum in Escherichia coli, by constructing a gene with synthetic oligonucleotides that changed the gene's codon usages. The induced expression in an E. coli cell of the synthetic gene yielded a product that constituted about 30% of the total bacterial protein. The product was precipitated in an inclusion body in a cell. Its enzymatic activity was restored after denaturation and renaturation procedures with guanidine-HCl. Recombinant DHFRs with Ser or Thr at position 108 were prepared. Kinetic characterization showed that the DHFRSer108 has less of an affinity for NADPH and dihydrofolate than the DHFRThr108.
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PMID:Purification and characterization of dihydrofolate reductase of Plasmodium falciparum expressed by a synthetic gene in Escherichia coli. 800 23

Rat Sertoli cell-conditioned medium (SCCM) or low molecular weight filtrate of SCCM (< 1,000 Da molecular weight) inhibited the uptake of [3H]thymidine into cells in culture; [3H]thymidine is incorporated into DNA by means of a salvage pathway. The incorporation of radioactivity into DNA from [14C]N5,N10-methylene-tetrahydrofolate, required for the thymidylate synthetase reaction, was not inhibited by SCCM and reflected the increase in cell number. SCCM specifically inhibited the incorporation of pyrimidines ([3H]uracil, [3H]thymidine) with no effect on the transport or incorporation of [3H]adenosine. Inhibition of thymidine uptake by SCCM could have occurred by a direct competition, i.e., the secretion of thymidine into the medium by the Sertoli cells, or by an indirect mechanism that would result in an inhibition of transport. The activity was partially purified by membrane ultrafiltration, ion exchange chromatography, and sequential extraction. Addition of SCCM filtrate (< 1,000 Da molecular weight) to growth-arrested cells cultured in the presence of inhibitors of dihydrofolate reductase resulted in cell proliferation, suggesting that the factor is involved in thymidine biosynthesis. This activity may play a role in the regulation of nucleoside biosynthesis and/or utilization in the testis.
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PMID:Sertoli cell-conditioned medium affects nucleoside utilization in vitro. 805 34

Antifolates have demonstrated effective antineoplastic activity in the treatment of disorders of cell proliferation, e.g., acute lymphocytic leukemia, breast cancer, and mycosis fungoides. The enzymatic pathways involved in DNA biosynthesis, specifically dihydrofolate reductase and thymidylate synthetase, are the biochemical targets of antifolates. Methotrexate (MTX) and its analogs, 10-ethyl-10-deazaaminopterin (edatrexate), and trimetrexate (TMT) are paradigms for cytotoxicity at the biochemical level. Understanding the cellular pharmacology of MTX and other antifolates has provided a strong rationale for the use of high-dose MTX with leucovorin (LV) rescue. The combination of MTX and LV prevents severe toxicity without diminishing the antitumor activity of the drugs. The efficacy of antifolate drugs is related to the extent of intracellular polyglutamation in normal and cancer cells. Since toxicity in patients is difficult to predict, monitoring drug concentrations is critical. Antifolates, specifically MTX and edatrexate, are among a growing class of chemotherapeutic agents that require assiduous and rapid monitoring to help prevent severe systemic toxicity. Chemical and physical properties, mechanism of chemotherapeutic activity, and analytical methodology for measurement of serum concentrations of antifolates will be discussed.
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PMID:Antifolate analogs: mechanism of action, analytical methodology, and clinical efficacy. 812 87

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg83b27/vgBG heterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.
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PMID:The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis. 823 10

The effects of different sulphonamides, dihydrofolate reductase inhibitors and other inhibitors of folate metabolism on growth of Acanthamoeba culbertsoni in a chemically defined medium are reported. Among the sulphonamides, sulphamethoxazole and sulphadiazine were most effective followed by sulphanilamide and sulphaguanidine. Inhibition by each sulphonamide was reversed by p-aminobenzoic acid as well as folic acid. 7-Methylguanosine, a pteridine synthesis-inhibitor, did not inhibit multiplication of A. culbertsoni. Among the dihydrofolate reductase inhibitors, pyrimethamine blocked the amoebic growth at 100 micrograms/ml, while trimethoprim and cycloguanil palmoate failed to cause significant inhibition of growth even at 250 micrograms/ml. Metoprine inhibited amoebic growth completely at 50 micrograms/ml. Methotrexate and a thymidylate synthetase inhibitor 5-fluorouracil inhibited growth strongly, with IC50 values (the concentration of the drug which causes 50% inhibition of the growth at 72 h) of 1.97 and 2.45 micrograms/ml, respectively. Inhibition by methotrexate, metoprine or 5-fluorouracil could not be reversed by folic acid, folinic acid, thymidine, or folinic acid plus thymidine. The results indicate unusual features in A. culbertsoni folate metabolism.
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PMID:In vitro susceptibility of Acanthamoeba culbertsoni to inhibitors of folate biosynthesis. 845 98


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