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 neu/erb B2 ligand growth factor (NEL-GF) was purified to homogeneity from bovine kidney by a procedure involving ammonium sulfate fractionation (35-70% saturation) followed by sequential column chromatography on DEAE-cellulose (DE52), Sulfadex (sulfated Sephadex G-50), heparin-Sepharose 4B, and Superdex 75 (fast protein liquid chromatography). NEL-GF was found to be a 25-kDa polypeptide according to the analysis by gel filtration on Superdex 75 and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NEL-GF stimulated the tyrosine-specific autophosphorylation of the neu/erb B2 gene product purified by immunoabsorbent and tyrosine-specific phosphorylation of the neu/erb B2 gene product in intact dihydrofolate reductase (DHFR/G-8 cells (NIH 3T3 cells transfected with rat c-neu). NEL-GF also down-regulated the cell surface neu/erb B2 gene product in DHFR/G-8 cells. NEL-GF was mitogenic toward NIH 3T3 cells, DHFR/G-8 cells, A431 cells (human epidermoid carcinoma cells), and SK-BR-3 cells (human breast carcinoma cells) but inactive toward bovine aorta endothelial cells. NEL-GF was sensitive to 0.1% trifluoroacetic acid but resistant to 5% beta-mercaptoethanol and appeared to be distinct from a neu protein-specific activating factor (Davis, J. G., Hamuro, J., Shim, C. Y., Samanta, A., Greene, M. I., and Dobashi, K. (1991) Biochem. Biophys. Res. Commun. 179, 1536-1542) and a 30-kDa glycoprotein which competed with a monoclonal antibody for binding to the neu/erb B2 gene product (Lupu, R., Colomer, R., Zugmaier, G., Sarup, J., Shepard, M., Slamon, D., and Lippman, M. E. (1990) Science 249, 1552-1555).
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PMID:Purification and characterization of the neu/erb B2 ligand-growth factor from bovine kidney. 135 Jul 85

Resistance to pyrimethamine and proguanil is due to a single point mutation in the gene that codes for dihydrofolate reductase. A single mutation gives rise to resistance to only one of the drugs. Resistance to both drugs results from several mutations. Chloroquine resistance phenotype is due to a rapid efflux of the drug from the parasite's digestive vacuole. This efflux is associated with a transmembrane permeability glycoprotein, or P-gp, which is similar to the protein implicated in the multidrug resistant phenotype of some cancer cells. However, one or several other poorly understood major gene(s) may be involved. Drugs which can inhibit the supposed affinity of P-gp for chloroquine are under study.
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PMID:[Contribution of molecular genetics to the understanding of chemoresistance of Plasmodium falciparum]. 135 39

A major problem in cytostatic treatment of malignant tumors is the development of chemoresistant cell clones. An increased understanding of chemoresistance related mechanisms, improved methods for the detection and localization of resistant cell populations including predictive conclusions on the effectiveness of cytostatic drugs would contribute to the advancement of anti-tumor strategies. This paper reviews current concepts suggested for the development of cellular resistance to natural product drugs (anthracyclines, Vinca alkaloids, epipodophyllotoxines, antibiotics; so-called multidrug resistance substances), alkylating agents (nitrosureas, busulfan and mitomycin C), heavy metal compounds (cisplatin) and antifolates (5-fluorouracil, methotrexate) and describes the role of drug transporting and binding proteins (P-170-glycoprotein), detoxifying enzymes (glutathion-S-transferase, dihydrofolate reductase), DNA repair enzymes (topoisomerase I and II, polymerase alpha and beta), and genomic alterations (amplification, double minutes and homogeneous staining regions) due to resistance. It is focussed on the employment of morphological methods (light microscopy, immunocytochemistry, electron microscopy, fluorescence analysis, in situ hybridization, computer aided morphometric analysis) which will help to detect resistant cell clones in tumor biopsies. First correlations between histological data and clinical course will be reported. In the future, the morphological determination of chemoresistance may play an important role in applied functional tumor pathology.
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PMID:What's new in cytostatic drug resistance and pathology. 175 14

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.
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PMID:Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector. 215 29

The gene that encodes the membrane-bound Mr 100,000 human melanoma-associated antigen (MAA) defined by mouse mAb 376.96, a leukocyte and fibroblast interferon-modulated glycoprotein having preferential distribution on melanoma and carcinoma cells, has been transfected into the mouse melanoma cell line B78H1 as a step toward molecular cloning and characterization of the MAA. Primary, secondary, and tertiary B78H1 transfectants expressing the Mr 100,000 MAA gene were generated by treatment with coprecipitated DNA from Mr 100,000 MAA+ human or transfectant mouse cells and they were detected by an indirect RBC rosetting assay. The Mr 100,000 MAA gene was also transferred into K-1735 mouse melanoma cells and into nonmalignant and malignant mouse fibroblast lines. The species immunoprecipitated by mAb 376.96 from human melanoma cells (Mr 100,000) and from mouse melanoma transfectant cells (Mr 97,000-100,000) were both converted to molecule(s) having an Mr of approximately 70,000 by enzymatic removal of asparagine-linked carbohydrate residues. Two independent secondary transformant clones of B78H1 cells express Mr 100,000 MAA antigenicity at levels significantly higher than those observed when one or two copies of the gene are present. Clone Mr 100,000 secondary-A spontaneously overexpresses Mr 100,000 MAA at least 5-fold and has greater than or equal to 10 times elevated levels of putatively Mr 100,000 MAA gene-associated human alu family repeat element (h-alu)-positive restriction fragments relative to "single" copy secondary transfectant cells. Clone Mr 100,000 secondary-B has increased copy number and expression of Mr 100,000 MAA as a consequence of a selective co-amplification procedure which is targeted to a mouse wild type dihydrofolate reductase (dhfr) gene expression vector. This vector was co-introduced into B78H1 cells in addition to the DNA of Mr 100,000 MAA+ primary transfectant cells and the initially selected aminoglycoside phosphotransferase (neo) gene vector. Stepwise selections of a secondary Mr 100,000 MAA+ transfectant clone with increasing concentrations of the dihydrofolate reductase-inhibitory antimetabolite methotrexate led to progressive increases in copy numbers of the introduced dhfr gene and to parallel increases in h-alu sequences, in cellular levels of dihydrofolate reductase protein, and in cellular mAb 376.96 reactivity. Levels of these entities ultimately reached 50-fold, relative to levels expressed prior to amplification. The array of h-alu+ restriction fragments amplified in Mr 100,000 secondary-B cell DNA is very similar to that observed in Mr 100,000 secondary-A cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interspecific DNA-mediated transfer and amplification of a gene specifying a Mr 100,000 human melanoma-associated cell surface glycoprotein. 230 16

Methotrexate-induced gene amplification increased the expression of biologically active bovine luteinizing hormone (bLH) approximately 11-fold after stable transfection of a line of Chinese hamster ovary cells with genes encoding dihydrofolate reductase and the alpha and beta subunits of bLH. Subsequent analysis of the bovine genes revealed that while the alpha gene was amplified in response to methotrexate selection, the LH beta subunit gene remained unaffected. This effect was probably due to the linkage of the alpha subunit gene with the dihydrofolate reductase gene, the selectable and methotrexate-sensitive marker in the plasmid construct. Prior to methotrexate selection, the concentration of LH beta mRNA and the rate of LH beta synthesis exceeded that of alpha subunit mRNA and protein. Stepwise selection with methotrexate led to a progressive increase in the synthesis and secretion of biologically active bLH. Enhanced production of bLH correlated directly with similar increases in both the steady-state level of alpha subunit mRNA and the relative synthesis rate of alpha subunit protein. Despite progressive changes in alpha subunit concentration, formation of the alpha/beta heterodimer was always incomplete, even when the concentration of alpha subunit exceeded that of LH beta. Cumulatively, these results are consistent with a model in which the extent of steady-state combination of the subunits is determined by the mutual affinity and concentration of both subunits within the lumen of the secretory pathway. This stands in contrast to the long held view that the extent of glycoprotein hormone assembly is limited by the concentration of the beta subunit.
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PMID:Methotrexate-induced amplification of the bovine lutropin genes in Chinese hamster ovary cells. Relative concentration of the alpha and beta subunits determines the extent of heterodimer assembly. 245 62

We have developed an ELISA which detects, with high specificity, antibodies against a major surface protein of P. falciparum merozoites which is a processing product of the precursor glycoprotein gp190. This assay can be used in the diagnosis of acute malaria in individuals with primary infection. Two partial sequences of gp190 were expressed in E. coli as beta-galactosidase (beta-Gal) fusion proteins. The same sequences fused to chloramphenicol acetyltransferase (CAT) or mouse dihydrofolate reductase (DHFR) react with high frequency when sera of acute malaria patients are analyzed in immunoblots. Antibodies from such sera crosslink, via their antigen binding sites, the beta-Gal fusions to the corresponding CAT or DHFR fusions adsorbed to a solid phase as demonstrated by the captured beta-Gal activity. The assay is highly specific, shows extremely low cut off values and should therefore be widely applicable.
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PMID:A new tool for the serodiagnosis of acute Plasmodium falciparum malaria in individuals with primary infection. 266 17

Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV-1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA- mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild-type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody.
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PMID:Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line. 300 54

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.
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PMID:Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis. 302 1

The gene for glycoprotein gB1 of herpes simplex virus type 1 strain Patton was expressed in stable Chinese hamster ovary cell lines. Expression vectors containing the dihydrofolate reductase (dhfr) cDNA plus the complete gB1 gene or a truncated gene lacking the 194 carboxyl-terminal amino acids of gB1 were transfected into CHO DHFR-deficient cells. Radioimmunoprecipitation demonstrated that the complete gB1 protein expressed in CHO cell lines was cell associated, whereas the truncated protein was secreted from the cells due to deletion of the transmembrane and C-terminal domains of gB1. Cells expressing the truncated gB1 protein were subjected to stepwise methotrexate selection, and a cell line was isolated in which the gB1 gene copy number had been amplified 10-fold and the level of expression of gB1 had increased over 60-fold. The truncated gB1 protein was purified from medium conditioned by the amplified cell line. N-terminal amino acid sequence analysis of this purified protein identified the signal peptide cleavage site and predicted the cleavage of a 30-amino-acid signal sequence from the primary protein. The immunogenicity of the truncated gB1 protein was also tested in mice, and high levels of antibody and protection from virus challenge were observed.
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PMID:Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells. 302 63


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