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)

We studied the components of the hepatic phenylalanine hydroxylating system in a child with phenylketonuria who showed substantial neurologic impairment despite early dietary control of elevated blood phenylalanine levels. Phenylalanine hydroxylase, dihydropteridine reductase and dihydrofolate reductase activities were normal. In contrast the level of hydroxylation cofactor, tetrahydrobiopterin, in liver was only 10 per cent of normal. In addition to this hepatic deficiency, serum and urinary levels of biopterin-like compounds were low, and the serum biopterin did not increase in response to a phenylalanine load as it does in normal and phenylketonuric subjects. The phenylalanine hydroxylase activity in this child, as determined by an in vivo tritium-release assay, was 2.3 per cent of the normal value. These results indicate that the child suffers from a variant form of phenylketonuria--a deficiency of a functional phenylalanine hydroxylating system secondary to a defect in biosynthesis of biopterin.
N Engl J Med 1978 Sep 28
PMID:Hyperphenylalaninemia due to a deficiency of biopterin. A variant form of phenylketonuria. 68 51

A competitive protein binding assay has been developed for methotrexate based on the tight binding of this drug to Lactobacillus casei dihydrofolate reductase (= tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3). Free drug may be separated from that bound to reductase by adsorption with dextran--albumin coated charcoal. Scatchard plot analysis of the enzyme--drug interaction confirmed the presence of a single homogeneous class of binding sites with an association constant Ka of 2.1 X 10(8) M-1. This high affinity binding permits detection of methotrexate in the range of 0.3--30 pmol with a coefficient of variation of 15% or less. The predominant circulating folate, 5-methyl tetrahydrofolate, and the clinically useful rescue agent leucovorin (5-formyl tetrahydropteroyl-glutamic acid) do not interfere with the assay, nor does the methotrexate metabolite 4-amino-4-deoxy-10-methylpteroic acid. Assay of clinical samples, including plasma and cerebrospinal fluid, showed close agreement between the previously described enzyme inhibition assay and the more rapid competitive binding method.
Proc Natl Acad Sci U S A 1975 Sep
PMID:Competitive protein binding assay for methotrexate. 81 Aug 4

Methotrexate (MTX) inhibits the enzyme dihydrofolate reductase, which in turn limits the body's ability to perform transmethylation reactions. This study examined the hypothesis that the consequent deficiency of an important methylated compound, choline, may have contributed to the MTX-induced fatty change in the liver of W rats. Groups of rats were given MTX alone or MTX plus choline in varying dose combinations. All groups but one receiving the combined treatment showed a significantly lower triglyceride concentration in their livers and much less visible hepatocytic fat on histologic examination than did those given MTX alone. The protective effect of choline on the liver was dose related, the unaffected group having received a very small amount. Growth rate, survival, and hematopoietic depression due to MTX were unaltered by choline administration.
J Natl Cancer Inst 1977 Sep
PMID:Chronic toxicity of methotrexate in rats: partial to complete projection of the liver by choline: Brief communication. 89 41

Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td(+), whereas particles produced after infection with td(-) had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from td(ts) to td(+) simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both td(ts) and frd(ts) genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frd(ts) mutants to frd(+) not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles.
J Virol 1977 Sep
PMID:Bacteriophage T4 virion baseplate thymidylate synthetase and dihydrofolate reductase. 89 93

Using the technology of metaphase chromosome transfer, evidence has been obtained in CHO cells that genes controlling enzymes in a common pathway in folate metabolism are closely linked. MtxRI and MtxRIII are co-dominant mutations which affect the structure and level of dihydrofolate reductase. Gat- is a glycine-, adenosine- and thymidine-requiring auxotrophic mutant with a lesion in folylpolyglutamate synthetase, an enzyme responsible for addition of glutamates to folate residues. GlyB- is an auxotrophic glycine-requiring mutant whose phenotype may be reversed by folinic acid. Using purified metaphase chromosomes, the MtxR genes were co-transferred into recipient cells with the auxotrophic markers, as demonstrated by the isolation of transferents when two of the phenotypes, either Mtx and Gat, or Mtx and GlyB, were selected at the same time. When recipient cells were selected for Gat+ or GlyB+ alone, the transferents carried the MtxR markers. The GlyA mutation, another glycine-requiring auxotrophic change, is not co-transferred with methotrexate resistance. Because of previous evidence that only a small fragment is involved in chromosomal transfer experiments, these results seem to provide the first indication that some genes which control enzymes on a common metabolic pathway in eucaryotes are closely linked or are at least syntenic.
Cell 1977 Sep
PMID:Linkage of markers controlling consecutive biochemical steps in CHO cells as demonstrated by chromosome transfer. 90 14

Data are presented on the causal prophylactic action of about 100 compounds of various types against Plasmodium yoelii nigeriensis N67 in mice. Examples are given to show how action against pre-erythrocytic schizonts may be differentiated from action on emerging erythrocytic stages. In a series of 35 8-aminoquinolines, all but 10 showed definite causal prophylactic activity at tolerated doses. The data permit the compounds to be ranked in order of activity, and many are shown to be more active in this test system than primaquine. Marked causal prophylactic activity is displayed by a variety of quinone structures, several of which show a significant residual action on blood stages. A high level of activity is found in dihydrofolate reductase inhibitors within several chemical classes. Rorguanil is more effective as a causal prophylactic than a blood schizontocide in the mouse as in man. Sulphonamides and sulphones are also effective in this system. The active levels are influenced by the content of PABA in the diet of the hosts. Causal prophylactic action has been detected in a number of experimental compounds including some antibiotics (such as tetracycline and clindamycin). The pyrocatechol RC 12 shows only slight activity at the maximum tolerated dose. Chloroquine, mepacrine, quinine, quinolinemethanols and phenanthrenemethanols are inactive as causal prophylactics. It is concluded that a rodent malaria-mouse model does provide a relatively simple model for the screening of drugs for causal prophylaxis, and the data so obtained are of relevance to the detection of causal prophylactics against human malaria.
Ann Trop Med Parasitol 1975 Sep
PMID:The chemotherapy of rodent malaria, XXIII Causal prophylaxis, part II: Practical experience with Plasmodium yoelii nigeriensis in drug screening. 109 90

The rate of DNA synthesis in cultures of human lymphoblasts decreased more than 80% within 30 min after the cells were exposed to methotrexate, a potent inhibitor of dihydrofolate reductase. Despite this rapid initial inhibition, DNA continued to be synthesized for at least an additional 6 h. The mode of this subsequent replication appeared to be semiconservative, as indicated by the buoyant density of 5-bromodeoxyuridine-substituted DNA in alkaline CsCl gradients. The growth rates of DNA chains in cells exposed to methotrexate were determined by sedimentation rate analysis in alkaline sucrose gradients. DNA synthesized during 2-min or 10-min pulses with labeled deoxycitidine in the presence of methotrexate had about the same sedimentation coefficient, 35 S, as controls. When methotrexate-treated cultures were pulse-labeled for 10 min and then chased for various times, DNA fragments of about 80 S accumulated. DNA synthesized in the presence of methotrexate was stable and elongated to bulk-size DNA after methotrexate inhibition of growth was removed by addition of thymidine and deoxycytidine. The data suggest that methotrexate reduces the rate of DNA replication by inhibiting chain initiation independently of chain elongation.
Eur J Biochem 1975 Sep 15
PMID:DNA replication in methotrexate-treated human lymphoblasts. 117 48

Gene amplification is characteristic of tumors and continuous cell lines but not of primary, normal, diploid, senescing cells. However, the rat cell line REF52, which resembles primary cells in requiring expression of cooperating oncogenes for transformation, is unusual among cell lines as it is not permissive for amplification. REF52 cells did not form colonies in N-(phosphonacetyl)-L-aspartate (PALA), a drug for which the only known mechanism of resistance is amplification of the carbamoylphosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD) gene. Colonies did form in a low concentration of methotrexate but did not contain amplified dihydrofolate reductase genes. Expression of two cooperating oncogenes in REF52 cells converted them to a state permissive for amplification. Cells expressing only the 12S E1A mRNA of adenovirus 5 did not give rise to PALA-resistant colonies, but expression of an activated ras gene together with E1A readily allowed the cells to form resistant colonies in which the CAD gene was amplified. Cells expressing E1A plus ras were fully transformed, but expression of simian virus 40 large tumor antigen alone converted REF52 cells to a state permissive for amplification without transforming them fully. The ability to manipulate gene amplification in REF52 cells by expression of oncogenes should contribute to an understanding of the nature of the permissive state.
Proc Natl Acad Sci U S A 1992 Sep 01
PMID:Simian virus 40 large tumor antigen alone or two cooperating oncogenes convert REF52 cells to a state permissive for gene amplification. 132 47

We previously reported the expression of human beta-interferon (beta-IFN) (Miyaji et al., 1989) and human lymphotoxin (Miyaji et al., 1990) in Namalwa KJM-1 cells adapted to serum-free medium. To establish an efficient gene expression system, a dihydrofolate reductase (dhfr) gene coamplification method was applied to this cell line. A beta-IFN expression plasmid was introduced with a dhfr expression plasmid into KJM-1 and methotrexate (MTX)-resistant derivatives were selected by a stepwise increase of MTX concentration. Among them, derivatives which showed higher expression levels of beta-IFN than that achieved by the parental transformants were obtained, suggesting that a dhfr gene coamplification method can be used for efficient expression of foreign genes in KJM-1 which contains endogenous dhfr genes. Then, an improved beta-IFN expression vector was constructed, which contains a dhfr transcription unit. This plasmid was introduced into KJM-1 and then, MTX-resistant derivatives were selected. Among them, the highest producer, clone 40-10-24, secreted beta-IFN at a level as high as 5 micrograms/ml, which is about 100-fold higher than that obtained by the G418-resistant parental transformants. In addition, beta-IFN produced by recombinant KJM-1 cells had the same molecular weight of that produced by fibroblasts.
Cytotechnology 1990 Sep
PMID:Efficient expression of human beta-interferon in Namalwa KJM-1 cells adapted to serum-free medium by a dhfr gene coamplification method. 136 43

We have examined the characteristics of protein synthesis in an improved continuous flow cell-free translation system prepared from wheat germ extract with dihydrofolate reductase (dhfr) mRNA as the translated message. Continuous buffer flow and separation of product from the reaction mixture were accomplished by the use of a modified Amicon ultrafiltration chamber as reaction vessel. The system produced protein for more than 20 h, and the product had an activity of dhfr comparable to that of authentic enzyme from E. coli. Analysis of RNA recovered from the filtrate supports the notion that a functionally active protein-synthesizing machinery is superorganized in a dynamic complex.
J Biotechnol 1992 Sep
PMID:Production of an enzymatic active protein using a continuous flow cell-free translation system. 136 1


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