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 modified enzymatic amplification (PCR) method has been used to detect overexpression of the dihydrofolate reductase gene in human leukemic cells resistant to methotrexate (MTX) from both patient samples and cell lines in culture. Quantitation of DHFR mRNA by the PCR assay has been demonstrated to be more accurate and more sensitive than Northern blotting. This assay be useful in screening patients relapsing with drug resistance.
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PMID:Dihydrofolate reductase gene expression characterized by the PCR assay in human leukemia cells. 193 30

Pyrimethamine resistance in cultivated laboratory isolates of Plasmodium falciparum is linked to the dihydrofolate reductase mutation Asn-108, a mutation that acts by interrupting drug binding within the active site of the enzyme. To determine the prevalence of this mutation in endemic regions harboring pyrimethamine-resistant malaria, we used a mutation-specific polymerase chain reaction assay to survey P. falciparum strains from a wide section of the Brazilian Amazon. Mutations were identified directly from blood samples without intervening steps of in vitro cultivation. Of 42 samples collected from four states in Brazil, 38 (90%) contained the Asn-108 codon AAC that confers pyrimethamine resistance, four samples contained only the wild-type Ser-108 codon AGC, and none contained the Thr-108 codon ACC found in cycloguanil-resistant pyrimethamine-sensitive strains. These findings indicate that a very high incidence of the Asn-108 DHFR mutation is responsible for pyrimethamine resistance in the Amazon, and they are consistent with recent failure rates reported for Fansidar (pyrimethamine-sulfadoxine). We suggest that limited use of proguanil be evaluated as an alternative to pyrimethamine.
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PMID:Prevalence of the dihydrofolate reductase Asn-108 mutation as the basis for pyrimethamine-resistant falciparum malaria in the Brazilian Amazon. 195 58

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate-induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.
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PMID:Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells. 196 17

In nontransformed DHFR/G-8 cells (NIH 3T3 cells transfected with normal rat neu gene), the normal neu gene product was initially synthesized as a 170-kDa protein bearing endoglycosidase H-sensitive oligosaccharide chains and was then processed to a 175-kDa mature form with endoglycosidase H-resistant, endoglycosidase F-sensitive oligosaccharide chains. Most of this 175-kDa mature form appeared on the cell surface 2 h following synthesis and showed a half-life of approximately 3 h. In the presence of a growth factor(s) partially purified from bovine kidney, the half-life of this 175-kDa normal neu gene product was shortened to less than 30 min. In B104-1-1 cells (NIH 3T3 cells transfected with neu gene activated oncogenically by a point mutation that changes a valine residue to a glutamic acid residue in the putative transmembrane region), the oncogenically activated neu gene product was also synthesized as a 170-kDa precursor with endoglycosidase H-sensitive oligosaccharide chains. However, this 170-kDa precursor diminished very fast and was only partially processed to a 185-kDa mature form which exhibited a half-life of less than 30 min. The 185-kDa activated neu gene product possessed an unidentified post-translational modification in addition to N-linked oligosaccharide chains. Both the precursor and mature forms of the mutationally activated neu gene product showed increased tyrosine-specific phosphorylation as compared with those of their normal counterparts in DHFR/G-8 cells. The mutationally activated neu gene product in B104-1-1 cells shared several features which have been reported previously for the ligand-activated platelet-derived growth factor receptor in v-sis- or c-sis-transformed cells. These properties include: 1) accelerated turnover of the precursor and mature forms compared with the rates of turnover of its normal counterparts, 2) insensitivity of this rapid turnover to lysosomotropic amines, and 3) increased in vivo tyrosine-specific phosphorylation of both the precursor and mature forms. These findings suggest that the mutationally activated neu gene product may transform the cells by mimicking ligand-induced activation.
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PMID:Differential processing and turnover of the oncogenically activated neu/erb B2 gene product and its normal cellular counterpart. 196 62

The neu proto-oncogene product has been found to exist in two interconvertible forms in G8/DHFR mouse fibroblasts. The 185-kilodalton form (p185) present in growing cells is replaced by a 175-kilodalton form (p175) under conditions of serum starvation. This low molecular weight form accounts almost exclusively for the phosphotyrosine content of the receptor and is associated with increased tyrosine kinase activity. Addition of serum, platelet-derived growth factor or tumor promoter induces conversion of p175 to p185 within minutes, and this increase in molecular weight is associated with phosphorylation of serine and threonine; removal of serum growth factors is followed by replacement of p185 with p175 over several hours. Unlike G8/DHFR cells, the human breast cancer cell line SK-Br-3 expresses a high molecular weight neu/HER2 receptor with unchanged phosphotyrosine content in both serum-starved and serum-stimulated cultures. These findings indicate that activation of the neu proto-oncogene product in G8/DHFR cells may be regulated in part by protein kinase C-mediated receptor transmodulation rather than by ligand availability alone.
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PMID:Modulation of a Mr 175,000 c-neu receptor isoform in G8/DHFR cells by serum starvation. 197 80

A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics. This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements. The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100). The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E. coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme. The role of Glu-30 (Asp-27 in E. coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences. While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes. The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases. 197 47

Two collections of trimethoprim R plasmids, isolated from strains of Escherichia coli during 1978-83 and 1987-8 respectively, were retrospectively screened with specific biotinylated DNA probes for the presence of genes encoding particular DHFR enzymes. The results confirmed that the type I DHFR gene was the predominant plasmid-encoded gene conferring trimethoprim resistance in strains of E. coli from the Nottingham area of the UK, but indicated that genes encoding the more recently recognized types of DHFR enzymes had appeared in the bacterial gene pool and could be recognized with increased frequency in the latter plasmid collection. This was particularly true of the type IIIa and type VII enzymes which together accounted for 27% of the trimethoprim R plasmids examined in 1987-8.
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PMID:Detection of novel trimethoprim resistance determinants in the United Kingdom using biotin-labelled DNA probes. 199 53

A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning greater than 50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.
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PMID:A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration. 199 14

A conformationally restricted analogue of trimethoprim (1a) has been prepared by connecting the ortho position of the benzene ring to the methylene linkage with two methylene groups, thus forming a dihydroindene derivative (2b). The chemistry involved the condensation of barbituric acid with an indanone derivative, followed by a three-step conversion to a 2,4-diaminopyrimidine. The S isomer of 2b was found to have a minimum-energy conformation very similar to that of 1a when bound to Escherichia coli dihydrofolate reductase, in contrast to that of 1a in vertebrate DHFR. Theoretically such a derivative might have increased specificity and activity against the bacterial enzyme. Molecular modeling experiments suggested that the actual decreased activity was due to crowding in the enzyme, caused by the extra atoms needed to restrict the conformation.
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PMID:2,4-Diamino-5-benzylpyrimidines as antibacterial agents. 14. 2,3-Dihydro-1-(2,4-diamino-5-pyrimidyl)-1H-indenes as conformationally restricted analogues of trimethoprim. 199 76

Two-dimensional 1H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate. The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant. These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures. During the initial assignment process, it became evident that many of the resonances in this complex, unlike those of the folate complex, are severely broadened or doubled. The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers. At 303 K, NOESY spectra with mixing times of 100 ms did not show interconversion between these isomers. However, exchange cross-peaks were observed in a 700-ms NOESY spectrum at 323 K which demonstrated that these isomers are interconverting slowly on the NMR time scale. Many of the side chains with clearly doubled resonances were located in the beta-sheet and the active site. Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding. In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form. These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.
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PMID:Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli. 199 78


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