BACKGROUND To evaluate the impact of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes on methotrexate (MTX) resistance in osteosarcoma cells in relation to retinoblastoma (RB1) gene status. MATERIALS AND METHODS A series of human osteosarcoma cell lines-either sensitive or resistant to MTX-and 16 osteosarcoma tumour samples were used in this study. RESULTS In U-2OS MTX-...

Many examples of internal translation initiation in eucaryotes have accumulated in recent years. In many cases terminators of upstream reading frames precede the internal initiation site, suggesting that translational reinitiation may be a mechanism for initiation at internal AUGs. To test this idea, a series of recombinants was constructed in the mammalian expression vector pSV2. Each containe...

Dihydrofolate reductase (DHFR) was measured during the development in rats of brain tumors induced following inoculation with avian sarcoma virus. Increasing activity of this enzyme in brain was correlated with the course of primary brain tumor growth. The specific activities of DHFR in primary human brain tumor tissues were comparable to those found in avian sarcoma virus-induced brain tumors ...

BACKGROUND Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to u...

We describe the isolation and the sequence of the gene for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS; EC 1.5.1.3 and EC 2.1.1.45, respectively) from two pyrimethamine-resistant clones of Plasmodium falciparum, HB3 and 7G8. We have also derived the sequence of the DHFR portion of the gene, by amplification using polymerase chain reaction, for the pyrimethamine...

Large quantities of a catalytically active protein have been produced in a cell free system. More than 10(9) copies of protein were produced from each DNA plasmid containing DNAfol, the bacterial gene encoding dihydrofolate reductase (DHFR). The strategy employed, denoted gene amplification with transcription/translation (GATT), involves sequential coupling of (i) DNA amplification by the polym...

Modeling studies were performed on known inhibitors of the quadruple mutant Plasmodium falciparum dihydrofolate reductase (DHFR). GOLD was used to dock 32 pyrimethamine derivatives into the active site of DHFR obtained from the x-ray crystal structure 1J3K.pdb. Several scoring functions were evaluated and the Molegro Protein-Ligand Interaction Score was determined to have one of the best correl...

Crithidia fasciculata cells grown in defined medium are sensitive to methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR). When cells are challenged with 2-5 microM MTX, cell division ceases after 3-4 divisions and the cells become rounded and immotile for approximately 60 h, with a 40% decrease in cell viability occurring during this period. The cells then recover normal morpholo...

OBJECTIVES The aim of the present study was to evaluate the genetic variation of Pneumocystis jirovecii dihydrofolate reductase (DHFR) gene in an immunocompromised Portuguese population and to investigate the possible association between DHFR genotypes and P. jirovecii pneumonia (PcP) prophylaxis with co-trimoxazole. METHODS One hundred and thirty-eight P. jirovecii isolates were submitted to...

Ðb-Carboline-benzoquinolizidine plant alkaloid deoxytubulosine (DTB) was evaluated and assessed for the ®rst time for its biochemical and biological activity employing the biomarker dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADP oxidoreductase, EC 1.5.1.3) as the probe enzyme, a key target in cancer chemotherapy. DHFR, employed in the present investigations was puri®ed from Lact...