Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A macromolecular binder of folic acid and folic acid derivatives has been identified in the particulate fraction of homogenates of rabbit choroid plexus. Within the choroid plexus, there are 2.3 nmol of folate-binding activity (binder) per g of tissue. The molecular weight of the folate binder complex, separated from the particulate fraction after solubilization with Triton X-100, was 340,000 to 400,000 by Sephadex gel filtration. The partially purified binder, when freed of endogenous folates, bound equivalent amounts of both [3H]folic acid and [methyl-14C]methyltetrahydrofolic acid per mg of protein. Folic acid, homofolic acid, 5-methyltetrahydrofolic acid, and to a lesser degree, methotrexate, inhibited the binding of both [3H]folic acid and [14C]methyltetrahydrofolic acid. Binding activity, which decreased below pH = 7.0, was unaffected by pretreatment with ribonuclease but was eliminated completely by papain and a protease (Streptomyces griseus). Although dihydrofolate reductase was present in choroid plexus, the binder was distinct from dihydrofolate reductase as judged by gel filtration and methotrexate sensitivity. This high affinity binder of folates may be responsible, in part, for the rapid, saturable uptake of folic acid and methyltetrahydrofolic acid by rabbit choroid plexus in vitro.
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PMID:Identification of folate binding macromolecule in rabbit choroid plexus. 1 98

Antisense oligonucleotides containing either anionic diester or neutral methylphosphonate internucleoside linkages were prepared by automated synthesis, and were compared for their ability to arrest translation of human dihydrofolate reductase (DHFR) mRNA in a nuclease treated rabbit reticulocyte lysate. In the case of oligodeoxyribonucleotides, tandem targeting of three 14-mers resulted in synergistic and complete selective inhibition of DHFR synthesis at a total oligomer concentration of 25 microM. Hybrid arrest by three or six tandem oligodeoxyribonucleoside methylphosphonates was dramatically less effective. This difference does not result from preferential recognition of hybrids involving oligodeoxyribonucleotides by endogenous RNaseH activity. A ribonuclease protection assay demonstrated that antisense oligodeoxyribonucleoside methylphosphonates bind selectively to target RNA sequences, but with 275 fold lower affinity than the corresponding oligodeoxyribonucleotides. This low binding affinity results in poor arrest of translation, and may be related to the stereochemistry of the methylphosphonate linkage.
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PMID:Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system. 283 93

Methotrexate (MTX) dose-escalation studies were conducted in two inbred lines of FVB/N transgenic mice expressing distinct drug-resistant dihydrofolate reductases (DHFRs) and in animals transplanted with transgenic marrow. Survival of animals expressing a tryptophan-31 variant DHFR transgene was only slightly improved over that of normal animals, and survival of tryptophan-31 variant DHFR marrow transplant recipients was indistinguishable from that of normal animals (at a MTX dose of 4 mg/kg i.p. daily). In contrast, extended survival was observed for animals expressing an arginine-22 variant (Arg22) DHFR transgene, with the last three of eight animals in this group succumbing at a final MTX dose of 14 mg/kg i.p. daily. Survival was slightly reduced for normal animals transplanted with Arg22 marrow. Interestingly, demise of animals in both Arg22 groups was not associated with the profound drop in hematocrit levels usually observed in MTX-treated animals. These animals were instead characterized by severe atrophy of the gastrointestinal tract, whereas hematocrit levels and marrow histology were relatively normal. Kidney pathology (mesangiocapillary glomerulopathy) was also observed in Arg22 marrow recipients but not in Arg22 transgenics, consistent with expression of the drug-resistance gene in kidney tissues of the transgenics, as demonstrated by ribonuclease protection analysis. Immediate dose-response studies in Arg22 marrow transplant recipients defined a maximum tolerated dose of 4 mg/kg/day MTX, 2 to 3 times that of animals transplanted with normal marrow or of normal untransplanted animals. These results define the extent of chemoprotection afforded by drug-resistant DHFR expression and serve to identify alternate sites of toxicity in animals administered the higher levels of MTX afforded by drug-resistant DHFR expression in the marrow.
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PMID:Methotrexate dose-escalation studies in transgenic mice and marrow transplant recipients expressing drug-resistant dihydrofolate reductase activity. 881 32

Expression of the arg22, drug-resistant variant of dihydrofolate reductase (DHFR) in hematopoietic cells has been demonstrated to confer resistance to methotrexate (MTX) in mice, even though this variant suffers from low catalytic activity. The recently reported tyr22 variant has the advantage of higher catalytic activity combined with significant resistance to MTX. To evaluate the resistance conferred by tyr22-DHFR in vivo, we generated several transgenic mouse lines carrying a tyr22-DHFR minigene regulated by its natural promoter. The transgene copy number in 11 lines ranged from 6 to 68 copies and ribonuclease protection analysis demonstrated that 4 of these lines expressed significant transgenic DHFR mRNA at 20 to 68% of the endogenous DHFR mRNA level. Marrow from 4 of the 11 lines conferred significant increases in MTX-resistance in comparison with normal marrow when transplanted into lethally irradiated recipients. The ability of the tyr22-DHFR transgenic marrow to confer MTX-resistance to bone marrow transplant (BMT) recipients did not correlate with the level of mRNA expression or the number of transgene copies. However, two lines (lines 11 and 15) that were most effective in maintaining normal hematocrit levels in BMT recipients receiving 1 mg/kg/day MTX exhibited the greatest ability to form MTX-resistant hematopoietic progenitor colonies in vitro. Furthermore, MTX dose escalation studies demonstrated that line 11 marrow conferred resistance in BMT recipients receiving up to 6 mg/kg/day MTX. Southern blot analysis of the BMT recipients 7 months posttransplantation showed a preponderance of transgenic donor-derived cells in bone marrow and spleen, as well as a surprisingly high level in the small intestine. These results indicate that tyr22-DHFR is likely to be superior to arg22-DHFR in conferring MTX-resistance in BMT recipients, illustrating its usefulness for chemoprotection during MTX chemotherapy and also potentially for in vivo selection of transduced cells in gene therapy trials.
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PMID:Transgenic mice expressing the tyr22 variant of murine DHFR: protection of transgenic marrow transplant recipients from lethal doses of methotrexate. 935 73

This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, nuclear magnetic resonance, and single-molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme-substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conformations, and cooperative conformational changes are important features of this model.
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PMID:Flexibility, diversity, and cooperativity: pillars of enzyme catalysis. 2202 78