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 have examined the metabolism of the folate analog methotrexate (MTX) in the human parasite Leishmania major. These cells readily hydrolyzed MTX to N-10-methyl-4-deoxy-4-aminopteroate (MAPA), such that following a 24-h incubation in 1 microM [3H]MTX approximately 30% of the cell-associated radioactivity was MAPA. MAPA also accumulated in the culture medium, exceeding the concentration of MTX after 24 h. Neither 7-hydroxy-methotrexate nor MTX polyglutamates were observed in cells or medium, even after a 72-h incubation with MTX. In contrast to MTX, folate is extensively polyglutamylated in L. major (Santi, D. V., Nolan, P., and Shane, B. (1987) Biochem. Biophys. Res. Commun. 146, 1089-1092). MAPA was found to be 190-fold less potent than MTX as an inhibitor of leishmanial growth and to bind less tightly than MTX to leishmanial dihydrofolate reductase-thymidylate synthase. We therefore examined the possibility that MTX resistance is mediated by increased MTX hydrolysis to MAPA in drug-resistant Leishmania. However, enzymatic assays show that the rate of MTX hydrolysis was unaltered in the MTX-resistant R1000-3 line and the primaquine-resistant PQ-R30 line (which is 24-fold cross-resistant to MTX). In addition to MAPA, several minor unidentified metabolites were observed in the LT252, R1000-5B, and PQR30 cells but no consistent differences in the amounts of these metabolites were evident among these lines. These data indicate that alterations in the rate of MTX hydrolysis in vitro, or in the characteristics of MTX metabolites formed in vivo, do not underly the MTX resistance displayed by the H region-amplified R1000-5B and PQ-R30 lines.
J Biol Chem 1989 Sep 25
PMID:Wild-type and drug-resistant Leishmania major hydrolyze methotrexate to N-10-methyl-4-deoxy-4-aminopteroate without accumulation of methotrexate polyglutamates. 247 35

We have examined the transcriptional organization of the R region of the protozoan parasite Leishmania major. This region encodes the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) and is frequently amplified as a 30-kilobase (kb) extrachromosomal circular DNA in methotrexate-resistant lines. Northern (RNA) blot analysis shows that the R region encodes at least 10 stable cytoplasmic polysomal poly(A)+ RNAs, ranging in size from 1.7 to 13 kb and including the 3.2-kb DHFR-TS mRNA. Transcriptional mapping reveals that these RNAs are closely spaced and collectively cover more than 95% of the 30-kb amplified R region. The organization is complex, including several overlapping RNAs 3' of DHFR-TS and two examples of antisense RNAs 5' of DHFR-TS. The R region RNAs can be grouped into two empirical domains, with eight contiguous RNAs transcribed in the same direction as that of DHFR-TS and two contiguous RNAs transcribed in the orientation opposite to that of DHFR-TS. The two 5'-most RNAs of the DHFR-TS-containing domain overlap the RNAs transcribed from the opposite strand. These data are relevant to models of transcription, including recent studies suggesting polycistronic transcription in trypanosomatids. The abundance of R region RNAs increases uniformly 10- to 15-fold in the amplified R1000-3 line relative to the wild type, and no new RNAs were observed. This suggests that all elements required in cis for DHFR-TS expression are contained within the 30-kb circular DNA. Quantitative analysis reveals that the steady-state DHFR-TS mRNA and protein levels are not growth phase regulated, unlike the monofunctional mouse DHFR. DHFR-TS is developmentally regulated, however, declining about fivefold in lesion amastigotes relative to promastigotes.
Mol Cell Biol 1989 Sep
PMID:Transcriptional mapping of the amplified region encoding the dihydrofolate reductase-thymidylate synthase of Leishmania major reveals a high density of transcripts, including overlapping and antisense RNAs. 247 67

We have constructed multicistronic vectors containing the cDNAs for murine dihydrofolate reductase (DHFR), hygromycin phosphotransferase (HyPR), and human protein C (HPC), an antithrombotic factor. Using a sequential selection protocol with hygromycin (Hy) and methotrexate (MTX), we demonstrate the selective amplification of the murine dhfr cDNA in the adenovirus-transformed human kidney cell line 293, and the coamplification of the cDNA for HPC. Such recombinant 293 cell lines secreted HPC at levels as high as 25 micrograms/10(6) cells/day. In addition, we found that the complex vitamin K-dependent posttranslational modification of gamma-carboxylation of glutamate was not limiting at these high secretion levels, although the proteolytic processing of the protein was slightly reduced. Further, the HPC secreted from the gene-amplified cell lines had full anticoagulant activity when compared to plasma-derived HPC.
Gene 1989 Sep 01
PMID:Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C. 247 19

Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vgB/+ flies are reared on these substances (vgB is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies. Our data suggest that the vg+ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.
Mol Gen Genet 1989 Sep
PMID:Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant. 253 Dec 71

A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with [125l]methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found. published HPLC method was found.
J Pharm Sci 1989 Sep
PMID:Competitive protein binding assay for piritrexim. 258 69

The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar to TS from other organisms and is most closely related to the enzyme from Saccharomyces cerevisiae with 65% identity. TS is found on a 330-kilobase-pair chromosome in P. carinii. While TS and dihydrofolate reductase reside on a single polypeptide chain in all protozoa studied to date, TS is not linked to dihydrofolate reductase in P. carinii. The TS gene shows the presence of four small intervening sequences, some of which interrupt the coding sequence in highly ordered structural regions of the protein. Heterologous expression of P. carinii TS in E. coli was accomplished by cloning the coding sequence into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein.
Proc Natl Acad Sci U S A 1989 Sep
PMID:Isolation and expression of the Pneumocystis carinii thymidylate synthase gene. 267 92

Starting from 4,4'-diamino-diphenylsulfone (DDS) as a lead structure, new 2-substituted analogues as well as new 2-substituted 4-alkylamino-4'-amino diphenylsulfones have been designed and synthetized in different ways. This has led to compounds the inhibitory activity of which against 7,8-dihydropteroic acid synthase of plasmodia and mycobacteria is clearly superior to that of sulfadoxine and in most cases to that of DDS. Of special interest is 4'-amino-4-n-propylamino-2-methyl-diphenylsulfone. Together with inhibitors of 7,8-dihydrofolate reductase in vitro and in vivo it possesses a marked synergistic inhibitory activity against plasmodia. In contrast to DDS in doses up to 200 mg/kg p.o. (cat) no methemoglobin formation is observed. The compound has been selected for further studies.
Arzneimittelforschung 1989 Sep
PMID:Preparation and biological activity of new substituted antimalarial diaminodiphenylsulfones. 268 56

To test the possibility that long-range interactions might influence the folding and stability of dihydrofolate reductase, a series of single and double mutations at positions 28 and 139 were constructed and their urea-induced unfolding reactions studied by absorbance and circular dichroism spectroscopy. The alpha carbons of the two side chains are separated by 15 A in the native conformation. The replacement of Leu 28 by Arg and of Glu 139 by Gln resulted in additive effects on both kinetic and equilibrium properties of the reversible unfolding transition; no evidence for interaction was obtained. In contrast, the Arg 28/Lys 139 double replacement changed the equilibrium folding model from two state to multistate and showed evidence for interaction in one of the two kinetic phases detected in both unfolding and refolding reactions. The results can be explained in terms of a long-range, repulsive electrostatic interaction between the cationic side chains at these two positions.
Biochemistry 1989 Sep 19
PMID:Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase. 269 6

We developed a system for rapid, manual and automated sequence analysis by utilizing and modifying methods used in conjunction with the polymerase chain reaction (PCR). We are using these techniques to detect single base mutations in the dihydrofolate reductase (DHFR) gene giving rise to methotrexate (MTX) resistance of tumor cells obtained from patients with malignancies. Amplifying in vitro both genomic DNA and transcripts of the human DHFR we are able to reproducibly generate single-stranded templates. Utilizing [alpha-35S]dATP and both the universal and reverse sequencing primers we obtain sequence information from either strand. The methods described have been successfully used for automated sequencing with the Applied Biosystems Model 370A Sequencer using both modified T7 DNA polymerase and Taq I. DNA polymerase for dideoxy-termination sequencing. The use of this methodology to detect a single base change in a human colon carcinoma cell line, HCT-8, is illustrated.
Biotechniques 1989 Sep
PMID:Sequence analysis of a human gene responsible for drug resistance: a rapid method for manual and automated direct sequencing of products generated by the polymerase chain reaction. 269 61

The novel tetrahydrofolate, 5,10-dideazatetrahydrofolic acid (DDATHF), was designed as an inhibitor of folate metabolism at a site other than dihydrofolate reductase. DDATHF has been shown to inhibit glycinamide ribonucleotide transformylase, a folate-requiring enzyme that catalyzes the first of two one-carbon transfer reactions in the de novo purine nucleotide biosynthetic pathway. Incubation of HL-60 promyelocytic leukemia cells with 5 x 10(-8) to 10(-5) M DDATHF resulted in a marked inhibition of growth after 48 h, with a complete cessation of cellular replication by day 4. Cell cycle analyses of DDATHF-treated HL-60 cells demonstrated an initial block in early S phase by day 3 followed by an accumulation of cells in the G1 and G2 + M phases of the cell cycle. Inhibition of growth was accompanied by a concentration-dependent increase in the percentage of mature myeloid cells that expressed nitroblue tetrazolium positivity, and a small increase in nonspecific esterase activity. Induction of differentiation and inhibition of growth by DDATHF were completely prevented by hypoxanthine and 5(4)-amino-4(5)-imidazole carboxamide, suggesting that depletion of intracellular purine nucleotide pools has an important role in the biological effects of this inhibitor. This possibility was confirmed by the finding that DDATHF caused a pronounced reduction in intracellular GTP and ATP levels within 2 h, with maximum decreases being observed by 24 h, a time interval which preceded the inhibition of cellular proliferation by this agent. Pyrimidine nucleoside triphosphate levels were markedly increased under these conditions. The findings indicate the importance of purine nucleotides to both the inhibition of growth and the induction of differentiation of HL-60 leukemia cells by DDATHF.
Cancer Res 1989 Sep 01
PMID:Induction of HL-60 leukemia cell differentiation by the novel antifolate 5,10-dideazatetrahydrofolic acid. 275 15


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