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)

Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.
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PMID:Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity. 138 19

A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was used quantitatively to measure accumulated levels of RNA transcripts in total mouse RNAs derived from male germ cells at various spermatogenic stages. RNA levels for two X-linked enzymes, phosphoglycerate kinase (PGK-1) and hypoxanthine phosphoribosyl transferase (HPRT), both decrease during spermatogenesis, although the transcript levels decrease much more rapidly for PGK-1. RNA for the Y-linked ZFY (zinc finger protein) is elevated in all spermatogenic cell fractions tested, being particularly high in leptotene/zygotene spermatocytes and round spermatids. RNA for adenine phosphoribosyltransferase (APRT) increases 5-fold to a peak during late pachynema. RNA for PGK-2, undetectable in spermatogonial cells, increases at least 50-fold by the round spermatid stage. DNA (cytosine-5-)-methyltransferase (MTase) transcript levels are over an order of magnitude higher throughout spermatogenesis than in non-dividing liver cells.
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PMID:Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis. 169 Aug 74

The intronless autosomal phosphoglycerate kinase gene (Pgk-2) is a functional retroposon expressed in a tissue-specific manner in the meiotic and postmeiotic stages of mammalian spermatogenesis. The nucleotide sequence of the promoter region of this gene and its transcription start point are compared with those of Pgk-1, an intron-containing, X-linked, housekeeping gene expressed constitutively in all somatic cells and premeiotic germ cells. The location of flanking direct repeats and apparent conservation of specific regulatory sequences suggest the Pgk-2 retroposon arose from reverse transcriptase-mediated processing of an aberrant Pgk-1 transcript that included the endogenous Pgk-1 promoter elements. Specific sequences that may be involved in mediating differences observed in both the level and cell-type specificity of expression of these genes in spermatogenesis are identified.
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PMID:Nucleotide sequence of the promoter region of a tissue-specific human retroposon: comparison with its housekeeping progenitor. 344 75

The ability to accurately measure mRNA levels in samples of total RNA is essential for studies on control of gene expression. The mRNAs from the housekeeping gene for phosphoglycerate kinase (PGK-1) can serve as a quality control for RNA samples. We describe an enzyme-linked immunosorbent assay (ELISA) method for mRNA determination by Q-RT-PCR, a quantitative reverse transcriptase-mediated PCR assay with competitive internal standards. After PCR, two biotinylated capture primers, one specific for PGK-1 cDNA and another one for internal standard, are annealed in separate assays so that each can attach DNA to a streptavidin-coated microplate. The captured DNA is either internally labeled with digoxigenin (DIG) or is "developed" after annealing with DIG-labeled primers. Bound DNA is then quantitated by adding DIG-specific antibody with attached alkaline phosphatase and measuring phosphatase activity with a chromogenic substrate and a plate reader. We compared different capturing methods and various primers labeled with DIG at their 3' ends. We determined that amplified PGK-1 DNA specifically captured with biotinylated primers was efficiently assayed with random p(dN)6-DIG.
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PMID:Random primer p(dN)6-digoxigenin labeling for quantitation of mRNA by Q-RT-PCR and ELISA. 770 58

The leukemia-specific AML1/ETO fusion gene has been shown to be detected by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. In the present study, the AML1/ETO mRNA could be detected by RT-PCR in bone marrow (BM) and/or peripheral blood (PB) samples from all 18 patients who had been maintaining complete remission for 12 to 150 months (median, 45 months) following chemotherapy or PB stem cell transplantation (PBSCT), whereas it could not be detected in four patients who had been maintaining remission for more than 30 months following allogeneic BM transplantation (BMT). We surveyed the expression of AML1/ETO mRNA in clonogenic progenitors from BM in these cases. Notably, 51 of 2,469 colonies from clonogenic progenitors (2.1%) expressed the AML1/ETO mRNA in 18 cases who were RT-PCR+ in BM and/or PB samples. Expression was observed in various clonogenic progenitors, including granulocyte-macrophage colonies, mixed colonies, erythroid colonies, and megakaryocyte colonies. Furthermore, we analyzed the clonality of these progenitors by X-chromosome inactivation patterns of the phosphoglycerate kinase (PGK) gene in four female patients. The AML1/ETO mRNA+ progenitors showed the PGK allele identical to that detected in the leukemic blasts from the time of initial diagnosis. Normal constitutive hematopoiesis was sustained by polyclonal BM reconstitution in these patients. Accordingly, these committed progenitor cells that express AML1/ETO mRNA during remission likely have arisen from common t(8;21)+ pluripotent progenitor cells with at least trilineage differentiation potential. These data strongly suggest that the origin of the clonogenic leukemic progenitors of t(8;21) AML may be multipotent hematopoietic progenitors that acquired the t(8;21) chromosomal abnormality.
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PMID:Persistence of multipotent progenitors expressing AML1/ETO transcripts in long-term remission patients with t(8;21) acute myelogenous leukemia. 863 50

Quinapril, an angiotensin-converting enzyme (ACE) inhibitor with high affinity for cardiac ACE, prevents increases in both plasma and cardiac angiotensin II (ANG II) and development of cardiac hypertrophy after aortocaval shunt in rats. In contrast, enalapril, an ACE inhibitor with low affinity for cardiac ACE, only prevents the increase in plasma ANG II. In the present study, we assessed whether these differences between enalapril and quinapril reflect different inhibition of cardiac tissue ACE and local ANG II by measuring their effects on cardiac ACE mRNA. Treatment with enalapril (250 mg/l) and quinapril (200 mg/l in drinking water) was started 3 days before the shunt and sham surgery. After 1 wk of aortocaval shunt, the hearts were excised and the left ventricle and right ventricle were weighed and used for reverse transcriptase-polymerase chain reaction (RT-PCR) assays for ACE and phosphoglycerate kinase-1 (internal standard). Quinapril, but not enalapril, inhibited the development of cardiac hypertrophy by aortocaval shunt. The shunt increased ACE mRNA in both left and right ventricles about twofold. In animals with aortocaval shunt, quinapril markedly further upregulated ACE mRNA in both ventricles, whereas enalapril did not cause significant changes. In sham rats, both ACE inhibitors increased ACE mRNA, but the increase was more pronounced by treatment with quinapril. These studies show that in vivo ACE inhibitors with low (enalapril) vs. high (quinapril) affinity for cardiac ACE differ in their effects on cardiac ACE mRNA. This difference is more pronounced in volume overload-induced cardiac hypertrophy, presumably reflecting their different effects on cardiac ANG II.
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PMID:ACE inhibitors and cardiac ACE mRNA in volume overload-induced cardiac hypertrophy. 927 79

Chlamydia trachomatis is an obligate intracellular eubacteria that is dependent on a eukaryotic host cell for a variety of metabolites. For years, it has been speculated that chlamydiae are energy parasites, totally dependent on their host cell for ATP and other high-energy intermediates. To determine whether C. trachomatis contains functional enzymes that produce energy or reducing power, four enzymes involved in glycolysis or the pentose phosphate pathway, specifically pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase, were cloned, sequenced and expressed as recombinant proteins in Escherichia coli. The deduced amino acid sequences obtained show high homology to other pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase enzymes. In contrast to numerous other bacterial species, chlamydial glycolytic genes are not arranged in an operon, but are dispersed throughout the genome. Results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicate that all four genes are maximally expressed in the middle of the chlamydial developmental cycle. The chlamydial genes are capable of complementing mutant E. coli strains lacking the respective enzyme activities. In vitro enzyme analysis indicates that recombinant chlamydial enzymes expressed in E. coli are active and, interestingly, recombinant chlamydial pyruvate kinase is not regulated allosterically by fructose 1,6 bisphosphate or AMP, as found with other bacterial pyruvate kinases. In summary, identification and characterization of these glucose-catabolizing enzymes indicate that chlamydia contains the functional capacity to produce its own ATP and reducing power.
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PMID:Glucose metabolism in Chlamydia trachomatis: the 'energy parasite' hypothesis revisited. 1041 34

Although nematodes like Caenorhabditis elegans are incapable of de novo cholesterol biosynthesis, they can utilize nonfunctional sterols by converting them into cholesterol and other sterols for cellular function. The results reported previously and presented here suggest that blocking of sterol conversion to cholesterol in C. elegans by 25-azacoprostane-HCl (azacoprostane) treatment causes a serious defect in germ cell development, growth, cuticle development, and motility behavior. To establish a biochemical basis for these physiological abnormalities, we performed proteomic analysis of mixed stage worms that had been treated with the drug. Our results from a differential display proteomic analysis revealed significant decreases in the levels of proteins involved in collagen and cytoskeleton organization such as protein disulfide isomerase (6.7-fold), beta-tubulin (5.41-fold), and NEX-1 protein (>30-fold). Also reduced were enzymes involved in energy production such as phosphoglycerate kinase (4.8-fold) and phosphoenolpyruvate carboxykinase (8.5-fold), a target for antifilarial drugs such as azacoprostane. In particular, reductions in the expression of lipoprotein families such as vitellogenin-2 (7.7-fold) and vitellogenin-6 (5.4-fold) were prominent in the drug-treated worms, indicating that sterol metabolism disturbance caused by azacoprostane treatment is tightly coupled with suppression of the lipid transfer-related proteins at the protein level. However, competitive quantitative reverse transcriptase polymerase chain reaction showed that the transcriptional levels of vit-2, vit-6, and their receptors (e.g. rme-2 and lrp-1) in drug-treated worms were 3- to 5-fold higher than those in the untreated group, suggesting a presence of a sterol regulatory element-binding protein (SREBP)-like pathway in these genes. In fact, multiple predicted sterol regulatory elements or related regulatory sequences responding to sterols were found to be located at the 5'-flanking regions in vit-2 and lrp-1 genes, and their transcriptional activities fluctuated highly in response to changes in sterol concentration. Thus, many physiological abnormalities caused by azacoprostane-mediated sterol metabolism disturbance appear to be exerted at least in part through SREBP pathway in C. elegans.
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PMID:Proteomic changes during disturbance of cholesterol metabolism by azacoprostane treatment in Caenorhabditis elegans. 1290 48

Cardiovascular diseases are associated with multiple changes in gene expression. In general, cardiac tissue is not accessible to expression analysis. This study was designed to investigate expression of cardiac significant genes in white blood cells of heart failure patients and to identify differentially expressed genes. The quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method was used for quantification of messenger RNA (mRNA) transcripts in blood samples of 20 patients (NYHA III-IV) with severe heart failure and of 20 healthy controls (NYHA I). Total RNA was extracted from leukocytes, reverse transcribed into cDNA, amplified and quantitated by SYBR Green detection. Relative mRNA expression was calculated using phosphoglycerate kinase-1 ( PGK-1) gene expression as an endogenous reference. Identified were 14 genes relevant to cardiomyocyte excitability or contractility. Most of them had not been previously reported to be expressed in blood cells. Data was based on 0.5 micro g total RNA applied to RT-PCR and on leukocyte number. In both, an increased transcription level of the Na/Ca exchanger ( NCX) was found in blood of heart failure patients as compared to controls (p < 0.02), in line with an upregulated NCX expression known from myocardial tissue of heart failure patients. This pilot study demonstrates that NCX transcription increased in potential relation to heart failure disease.
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PMID:Real-time RT-PCR for gene expression profiling in blood of heart failure patients-a pilot study: gene expression in blood of heart failure patients. 1508 8

Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and/or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G1/S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.
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PMID:L1 retrotransposition in nondividing and primary human somatic cells. 1669 26


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