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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To combat infection and inhibit viral replication of HIV in the brain, antiretroviral agents must cross the blood-brain barrier (BBB). An in vitro BBB model consisting of bovine brain microvessel endothelial cells grown on porous filters was used to study and compare the transport of nevirapine, a potent and selective nonnucleoside reverse transcriptase inhibitor, with other HIV antiretroviral agents currently in use for the treatment of HIV infection. These included nucleoside reverse transcriptase inhibitors (didanosine, stavudine, zalcitabine, zidovudine), a nonnucleoside reverse transcriptase (delaviridine), and protease inhibitors (indinavir, saquinavir, VX-478). Nevirapine was the most permeable antiretroviral agent studied in the BBB model. The order of in vitro BBB permeability was nevirapine >> VX-478 > didanosine, stavudine, zalcitabine, zidovudine > indinavir > saquinavir. There was an apparent bell-shaped relationship between in vitro BBB permeability and octanol/phosphate-buffered saline distribution coefficient (D) where all lipophilic (log D > 2.5) as well as hydrophilic (log D < -0.5) antiretrovirals were less permeable than nevirapine (log D = 1.8). There were no significant effects on the in vitro BBB permeability of nevirapine in combination with other antiretroviral agents. Saquinavir was the only drug shown to have an affinity for the P-glycoprotein efflux pump, which may have contributed to its very low permeability. The apparent ability of nevirapine to readily permeate the BBB and enter the brain, where it may inhibit replication of HIV, potentially increases its therapeutic value.
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PMID:In vitro blood-brain barrier permeability of nevirapine compared to other HIV antiretroviral agents. 952 83

Mutations in PEX, a phosphate-regulating gene with homology to endopeptidase on the X chromosome, were recently identified in patients with X-linked hypophosphatemia (XLH), an inherited disorder of phosphate homeostasis characterized by growth retardation and rachitic and osteomalacic bone disease. To understand the mechanism by which loss of PEX function elicits the mutant phenotype, a study of its mRNA localization and ontogenesis was undertaken. Using the reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) with polyA+ RNA purified from mouse testis, a 337-bp Pex cDNA fragment was generated and cloned in the pCRII plasmid. The cDNA was used to generate sense and anti-sense Pex riboprobes for in situ hybridization (ISH) and Northern analysis. To survey a large number of different tissues, sagittal sections of embryos and newborn mice were examined. ISH showed the presence of Pex mRNA in osteoblasts and odontoblasts. Pex gene expression was detectable on Day 15 of embryonic development, which coincides with the beginning of intercellular matrix deposition in bones. Finally, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the 7-kb Pex transcript is decreased in adult bones and in nongrowing teeth. The present study demonstrates that Pex mRNA is expressed in bones and teeth and suggests that this putative endopeptidase plays an important role in the development of these tissues.
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PMID:Pex mRNA is localized in developing mouse osteoblasts and odontoblasts. 952 91

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.
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PMID:Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage. 952 44

Quantitation of mRNA expression by endothelial cells in vivo has been limited to larger animals from which sufficient amounts of RNA could be isolated for Northern blot analysis. In the present study, we established a technique to isolate endothelial RNA from rat aortas using en face preparations. This RNA was not contaminated with RNA from smooth muscle cells as demonstrated by the absence of smooth muscle alpha-actin RNA. Following lipopolysaccharide (LPS) administration to rats, quantitation of platelet-derived growth factor (PDGF) ligand and receptor mRNA expression was carried out by competitive reverse transcriptase-polymerase chain reaction and normalized to glyceraldehyde-3 phosphate dehydrogenase. The results of the competitive reverse transcriptase-polymerase chain reaction were compared with those obtained by en face in situ hybridization. Aortic endothelium showed a 140-fold increase in PDGF-A mRNA expression 4 hours after LPS injection. Expression levels of this growth factor declined to near base line levels within 36 hours of the LPS injection. A 52-fold increase in PDGF-B mRNA was seen at 12 hours after LPS injection but expression levels were approximately 300-fold lower than for PDGF-A. These data indicate that changes in PDGF expression by endothelium in vivo can greatly exceed those observed in cultured cells. This method should permit study of endothelial gene regulation in a variety of pathological conditions in vivo.
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PMID:A quantitative method for determination of endothelial mRNA expression in vivo: induction of platelet-derived growth factor by endotoxin. 954 51

Combinatorial libraries related to spliceosomal U2 and U6 snRNAs were tested for catalytic reactions typical of the splicing of nuclear pre-mRNAs. Ribozymes with four different activities were selected based on covalent bond formation to a substrate RNA. The first activity was reversible self-cleavage; ribozymes self-cleaved then ligated the 5'-hydroxyl group of the substrate oligonucleotide to their 2',3'-cyclic phosphate intermediate. The second activity was 2',5'-branch formation by the attack of a substrate 2'-hydroxyl group on the 5'-terminal triphosphate of the ribozyme transcript, releasing pyrophosphate. The third ribozyme activity was similar to reversible self-cleavage but was a three-step reaction. This ribozyme self-cleaved, then cleaved the substrate in trans, and then ligated the substrate 3' cleavage product to its cyclic phosphate intermediate. This three-step pathway shares similarities with the pathway of tRNA splicing. The fourth activity was 2',3'-branch formation; to form this unusual branch, a 2'-hydroxyl of the substrate attacked an internal phosphate of the ribozyme, releasing an oligonucleotide leaving group. The isolation of branching activities by the in vitro selection protocol was unanticipated and was due to surprising properties of reverse transcriptase, which can read through 2',5'- or 2',3'-branches and efficiently perform non-templated intramolecular jumps.
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PMID:Selection in vitro of novel ribozymes from a partially randomized U2 and U6 snRNA library. 956 46

Biochemical and genetic data on retroviral nucleocapsid (NC) proteins have shown that this viral protein exhibits nucleic acid annealing and strand transfer activities and is required for the formation of infectious viral particles. However, the DNA binding properties of the NC protein of the human T-cell leukemia virus type-I (HTLV-I) has not been extensively studied. In this work we characterize the DNA binding ability of the zinc-bound and zinc-free forms of the p15 NC of HTLV-I. We found that only the zinc-bound form of the p15 NC binds single-stranded and double-stranded DNA fragments, but both forms of the p15 NC protein bind and unwind supercoiled DNA. The unwinding activity of the zinc-bound form was 3-fold higher than that observed with the zinc-free form of the protein. Interestingly, eukaryotic DNA topoisomerase antagonists inhibited this unwinding activity. In addition, we showed the formation of NC protein-DNA cleavable complex, which is the result of a presumably covalent bond formed between the protein and the phosphate moiety of the DNA backbone. Moreover, the presence of the p15 NC in the reverse transcription assay significantly increased the activity of the HTLV-I reverse transcriptase. These results demonstrate new DNA binding properties of the p15 NC protein and shed light on the possibility of a novel physiological function for the HTLV-I NC protein in the viral life cycle.
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PMID:DNA binding properties of the nucleocapsid protein from human T-cell leukemia virus type-I. 973 5

The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3' terminus of the primer strand. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the "primer grip" (the beta12-beta13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the template-primer (including the primer grip, template grip, and helices alphaH and alphaI of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement of nucleic acid and protein during polymerization.
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PMID:Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 A resolution. 983 29

d4T-5'-[p-Bromophenyl methoxyalaninyl phosphate] (d4T-pBPMAP), a novel phenyl phosphate derivative of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) that has an enhanced ability to undergo hydrolysis due to the electron withdrawing properties of its single bromo substituent at the para-position of the phenyl moiety, was found to yield substantially more of the key metabolite alaninyl d4T monophosphate (A-d4T-MP) than the unsubstituted d4T-5'-phenyl methoxyalaninyl phosphate or para-methoxy substituted d4T-5'-phenyl methoxyalaninyl phosphate. d4T-pBPMAP was tested for its anti-HIV-1 activity in peripheral blood mononuclear cells (PBMNC) and thymidine kinase (TK)-deficient CEM T-cells. d4T-pBPMAP was 12.6-fold more potent than the parent compound d4T in inhibiting p24 production (IC50 values: 44 nM vs 556 nM) and 41.3-fold more potent than d4T in inhibiting the reverse transcriptase (RT) activity (IC50 values: 57 nM vs 2355 nM) in HIV-1-infected TK-deficient CEM cells. Similarly, d4T-pBPMAP was more potent than the unsubstituted or para-methoxy substituted phenyl methoxyalaninyl phosphate derivatives of d4T. d4T-pBPMAP did not exhibit any detectable cytotoxicity to PBMNC or CEM cells at concentrations as high as 10,000 nM. Notably, d4T-pBPMAP was capable of inhibiting the replication of a zidovudine (ZDV/AZT)-resistant HIV-1 strain as well as HIV-2 in PBMNC at nanomolar concentrations. To our knowledge, this is the first demonstration that the potency of the d4T-aryl-phosphate derivatives can be substantially enhanced by introducing a single para-bromo substituent in the aryl moiety.
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PMID:Enhancing effects of a mono-bromo substitution at the para position of the phenyl moiety on the metabolism and anti-HIV activity of d4T-phenyl methoxyalaninyl phosphate derivatives. 987 88

Three aryl phosphate derivatives of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) were tested for their anti-human immunodeficiency virus (HIV) activity in peripheral blood mononuclear cells (PBMC) and thymidine kinase (TK)-deficient CEM T cells. Compared to the parent compound d4T, the lead compound d4T-5'-[p-bromophenyl methoxyalaninylphosphate] with a para-bromo substituent in the aryl moiety was 12.6-fold more potent in inhibiting p24 production (IC50 values: 44 nM versus 556 nM) and 41.3-fold more potent in inhibiting the reverse transcriptase (RT) activity (IC50 values: 57 nM versus 2355 nM) in HIV-infected TK-deficient CEM cells. None of the compounds exhibited any detectable cytotoxicity to PBMC or CEM cells at concentrations as high as 10,000 nM. To our knowledge, this is the first demonstration that the potency as well as selectivity index of the d4T aryl phosphate derivatives in TK-deficient cells can be substantially enhanced by introducing a single para-bromo substituent in the phenyl moiety.
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PMID:D4T-5'-[p-bromophenyl methoxyalaninyl phosphate] as a potent and non-toxic anti-human immunodeficiency virus agent. 987 98

By means of successive Mono Q and glycyrrhizin (GL)-affinity column chromatography (HPLC), recombinant HIV-1 RT (rRT) was purified to apparent homogeneity from the Superdex 200 pg fraction of the crude protein extract of E. coli BL21 transfected with pET 21a(+)/HIV-1 PR-RT. It was found that (i) rRT functioned as an effective phosphate acceptor for recombinant human casein kinase II (rhCK-II) in vitro; (ii) this phosphorylation was inhibited by anti-HIV-1 substances [a glycyrrhetinic acid derivative (oGA) and quercetin] and a high dose (100 microM) of GL; (iii) RNA-dependent DNA polymerase (RDDP) activity was stimulated about 2.5-fold after full phosphorylation of rRT by rhCK-II; and (iv) oGA as well as NCS-chromophore effectively prevented the CK-II-mediated stimulation of RDDP activity. These results suggest that the anti-HIV-1 effect of oGA may be involved in the selective inhibition of the CK-II-mediated stimulation of HIV-1 RT at the cellular level.
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PMID:Biochemical characterization of recombinant HIV-1 reverse transcriptase (rRT) as a glycyrrhizin-binding protein and the CK-II-mediated stimulation of rRT activity potently inhibited by glycyrrhetinic acid derivative. 988 39


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