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)

Fluid shear stress can stimulate secretion of tissue plasminogen activator (tPA) by cultured human endothelial cells, while plasminogen activator inhibitor type-1 secretion remains unstimulated. To determine whether hemodynamically induced changes in tPA messenger RNA (mRNA) levels also occur, primary cultures from the same harvest of primary human umbilical vein endothelial cells were either maintained in stationary culture or exposed to arterial levels of shear stress (25 dynes/cm2) for 24 hours. Total cellular RNA was isolated from the shear stressed and stationary cultures and the relative levels of tPA mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were determined using a coupled reverse transcriptase/polymerase chain reaction method. As indicated by the amount of amplification product, tPA mRNA levels were many fold higher (greater than 10) in endothelial cells subjected to shear stress for 24 hours than in stationary controls. In contrast, mRNA levels for GAPDH were similar in control and shear stressed cells. The constancy of the measured GAPDH signal indicated that the tPA response was a selective effect of fluid shear stress. When a similar polymerase chain reaction method was used, the mRNA levels of basic fibroblast growth factor (bFGF) were found not to vary in comparison to GAPDH mRNA after 24 hours of shear stress. These results indicate that enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces could involve transcriptional events.
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PMID:Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. 211 Jan 69

To study mechanisms underlying the modulation of luteinizing hormone-releasing hormone receptor (LHRH-R) during lactation and the estrous cycle, we used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to generate a probe for rat LHRH-R messenger RNA (mRNA). Using primers based on the mouse sequence, we amplified an approximately 300 bp fragment from rat pituitary complementary DNA. This PCR product was shown to be part of LHRH-R cDNA by direct sequencing and by comparing to the rat LHRH-R cDNA reported recently. Then, this PCR fragment was used as a probe for northern blotting analysis. The level of LHRH-R mRNA in the pituitary was significantly decreased during lactation, by approximately 80%, compared to that of ovariectomized and intact (diestrous and metestrous cycling) rats while no statistical difference in glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA level was observed between groups. During the estrous cycle, the level of LHRH-R mRNA in the pituitary was about two-fold higher on diestrous day 2 and the morning of proestrus than that on diestrous day 1 and quickly returned toward control level by noon of proestrus. In addition, we found that GAPDH mRNA levels from a so-called housekeeping gene often thought to be unchanged under different conditions, were significantly higher on proestrus while levels of 18S rRNA were not significantly changed. The large decrease in LHRH-R mRNA during lactation could account for the changes in LHRH binding previously reported.
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PMID:Luteinizing hormone-releasing hormone receptor messenger ribonucleic acid expression in the rat pituitary during lactation and the estrous cycle. 752 39

Erythropoietin (Epo) is a glycoprotein secreted by kidney cells which plays an important role in the regulation of erythropoiesis. Localization of the Epo production by immunohistochemical studies and in situ hybridization has not been definitively established and is still a matter of controversy. Epo and glyceraldehyde 3-dehydrogenase (GAPDH) mRNA levels were determined in total RNA isolated from control and CoCl2-treated rats using a coupled reverse transcriptase/polymerase chain reaction method (RT/PCR). As indicated by the amount of amplification product, Epo mRNA levels were several-fold higher in CoCl2-treated rat kidney. In contrast, GAPDH mRNA levels were similar in control and CoCl2-treated rats. This RT/PCR method was also used to assess the level of Epo and GAPDH mRNA in microdissected nephron segments. All nephron segments tested lacked any detectable levels of Epo mRNA in either control or CoCl2-treated rats. On the other hand, peritubular cells (capillary fraction: afferent/efferent arteriole, vasa recta) were the only cells where the Epo mRNA was detected. Using a specific primer for GAPDH, the RT/PCR method could identify GAPDH mRNA in all microdissected nephron segments where the Epo mRNA was not expressed. Thus, a combination of microdissected nephron segments and RT/PCR enabled us to detect GAPDH mRNA populations in all nephron segments, whereas the failure to detect Epo mRNA in all segments but the capillary fraction, is due to the specific and localized expression of the Epo gene to this fraction.
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PMID:Localization of erythropoietin mRNA in the rat kidney by polymerase chain reaction. 817 98

Platelet-derived growth factor (PDGF) is a potent mitogen and chemotactic agent which may be involved in the formation of proliferative lesions of the arterial system, such as intimal hyperplasia and atherosclerosis. To examine the regional variation in vessel wall production of this mitogen, PDGF production and PDGF A chain mRNA expression by normal arterial wall was studied as a function of vessel location. PDGF production by canine aortic segments was measured after 72 h in organ culture, revealing significantly more PDGF produced by the distal compared to proximal aorta at 77 +/- 10 versus 14 +/- 6 pg/cm2/72 h (p<0.05). Endothelial cells (EC) and smooth muscle cells (SMC), isolated from analogous aortic sites, were grown in tissue culture and the conditioned medium was assayed for PDGF. EC in vitro demonstrated a similar geographic trend in PDGF production (distal=1,501 +/- 389 pg/microgram DNA/72 h, proximal=759 +/- 230 pg/microgram DNA/72 h; p=0.17). PDGF production by SMC in cell culture had a similar pattern with cells from the distal aorta producing 58 +/- 28 pg PDGF/microgram DNA/72 h, compared to cells from the proximal aorta producing 37 +/- 15 pg PDGF/microgram DNA/72 h (p=0.13). Freshly harvested EC and SMC, isolated from the same aortic sites, were subjected to quantitation of PDGF mRNA levels using a coupled reverse transcriptase and polymerase chain reaction amplification method, with glyceraldehyde-phosphate dehydrogenase (GAPDH) as a control. The ratio of PDGF A chain:GAPDH mRNA was significantly greater in distal aortic SMC, 2.30 +/- 0.99, compared to proximal aortic SMC, 1.27 +/- 0.46 (p=0.05), but was not significantly different between proximal and distal aortic EC (p=0.86). These findings demonstrate significant regional differences in PDGF production in the normal canine aorta. Additionally, SMC are implicated as a significant contributor to the regional variation in PDGF production.
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PMID:Regional differences in platelet-derived growth factor production by the canine aorta. 860 28

Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein. The size of the protein was confirmed by in vitro coupled transcription/translation. Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring. The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase. Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis. Its expression was detected in cultures of M. bovis BCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.
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PMID:A serine/threonine protein kinase from Mycobacterium tuberculosis. 911 30

The family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae.
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PMID:The genome of Melanoplus sanguinipes entomopoxvirus. 984 59

There are 3 groups of drugs available for the treatment of patients with HIV disease. These are the nucleoside reverse transcriptase inhibitors ('nucleoside analogues') [zidovudine, didanosine, zalcitabine, lamivudine and abacavir]; the non-nucleoside reverse transcriptase inhibitors (nevirapine, delavirdine and efavirenz); and the protease inhibitors (saquinavir, ritonavir, indinavir, nelfinavir and amprenavir). The preferred initial regimen should reduce and maintain plasma HIV RNA below the level of detection. Presently, the regimen of choice consists of 2 nucleoside analogues plus a protease inhibitor with high in vivo efficacy. An alternative combination consists of 2 nucleoside analogues plus a non-nucleoside reverse transcriptase inhibitor. Drug interactions are one of the major problems associated with these multidrug regimens. Changes in plasma concentrations of the nucleoside analogues are unlikely to be of clinical relevance as drug effect is mainly dependent on the rate and extent of intracellular phosphorylation. Combinations of zidovudine plus stavudine, and probably zalcitabine plus lamivudine, should be avoided as competition for phosphorylating enzymes may occur. The antiviral efficacy of some nucleoside analogues, e.g. stavudine, may be compromised by prior treatment with other nucleosides (e.g. zidovudine). However, these data need to be clarified in further studies. It is unlikely that administration of other antiretrovirals will influence the activity of nucleoside analogues. Protease inhibitors are metabolised by hepatic cytochrome P450 (CYP) 3A4. Combination protease inhibitor therapy can result in drug interactions mediated by enzyme inhibition. Ritonavir is the most potent inhibitor, saquinavir the least. The protease inhibitors also interact with the non-nucleoside reverse transcriptase inhibitors. Nevirapine and efavirenz induce drug metabolising enzymes and may reduce plasma concentrations of protease inhibitors. A study in healthy volunteers showed that nelfinavir concentrations are increased by combination with efavirenz. Delavirdine inhibits drug metabolising enzymes and increases the plasma concentration of coadministered protease inhibitors. The nucleoside analogues would not be expected to interact with the protease inhibitors. Apart from the ability of didanosine to reduce the area under the concentration-time curve of delavirdine, there are no reports of clinically significant interactions of other antiretrovirals with the non-nucleoside reverse transcriptase inhibitors. Triple therapy is the current standard of care for patients with HIV disease. However, studies of quadruple therapy are already under way. Drug interactions are likely to remain one of the major considerations when selecting a therapeutic regimen for patients with HIV.
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PMID:Pharmacokinetics and potential interactions amongst antiretroviral agents used to treat patients with HIV infection. 1032 Sep 51

Gap junction coupling between neurons is important for the temporal and spatial co-ordination of neocortical development and can be visualised by dye-coupling. Neuronal dye-coupling in the rat neocortex is extensive during the first 2 postnatal weeks and diminishes rapidly thereafter. We used RT (reverse transcriptase)-PCR to investigate the time-related changes in mRNA expression for the connexins (Cx) Cx 26, Cx 30, Cx 32, Cx 36, Cx 37, Cx 40, Cx 43, Cx 45 and Cx 46 as well as for beta-actin and GAPDH in rat neocortex during the first 6 postnatal weeks. The time courses for mRNA expression for GAPDH, Cx 30, Cx 36 and Cx 43 were also investigated by northern blotting. Cx 30 and Cx 45 mRNA abundance showed no time-dependent changes during the early postnatal period. The relative abundance of Cx 32, Cx 43 and Cx 46 mRNA increased significantly during the first 2-3 weeks and then remained relatively constant during weeks 3-6. The relative abundance of Cx 26, Cx 36, Cx 37 and Cx 40 mRNA also increased significantly during the first 10-15 postnatal days but then declined significantly from their peak values during weeks 3-6. beta-actin mRNA expression showed no time-related changes but GAPDH mRNA expression increased significantly during the first postnatal week, then remained constant. The time-dependent changes in mRNA relative abundance for GAPDH, Cx 36 and Cx 43 determined by northern blotting corroborate the results from the RT-PCR study. None of the Cx exhibited time-dependent changes in mRNA expression in homogenates of rat neocortex which parallel the changes in neuronal dye-coupling during postnatal development.
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PMID:Time-related changes in connexin mRNA abundance in the rat neocortex during postnatal development. 1064 78

Natural D-nucleosides are no longer the sole basis for designing effective antiviral analogues. Many antivirals with an opposite (L) configuration were reported, with lamivudine being the most notable example. In contrast, carbocyclic nucleoside analogues are significantly more enantioselective, and enantiomers with a configuration corresponding to D-nucleosides are usually favored. In the series of acyclic nucleoside analogues, the antiviral potency resides in a single enantiomer. Allenic analogues with an axial dissymmetry are R-enantioselective, in contrast to structurally similar methylenecyclopropanes, where the enantioselectivity strongly depends on the type of virus. Enantioselectivity of acyclic nucleotide analogues exhibits a more complex pattern. The overall enantioselectivity of the antiviral effects is determined by responses of activating (phosphorylating) enzymes, as well as target DNA polymerases (reverse transcriptase), toward enantiomers of active analogues.
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PMID:Enantioselectivity of the antiviral effects of nucleoside analogues. 1073 80

This review is primarily intended for synthetic bio-organic chemists and enzymologists who are interested in new strategies in the design of virus inhibitors. It is an attempt to assess the importance of the enzymatic properties of L-nucleosides and their analogues, particularly those that are active against viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpes simplex virus (HSV), etc. Only data obtained with purified enzymes have been considered and discussed. The examined enzymes include nucleoside- or nucleotide-phosphorylating enzymes, catabolic enzymes, viral target enzymes and cellular polymerases. The enantioselectivities of these enzymes were determined from existing data and are significant only when a sufficient number of enantiomeric pairs of substrates could be examined. The reported data emphasize the weak enantioselectivities of cellular or viral nucleoside kinases and some viral DNA polymerases. Thus, cellular deoxycytidine kinase has a considerably relaxed enantioselectivity with respect to a large number of nucleosides or their analogues, and it occupies a strategic position in the intracellular activation of the compounds. Similarly, HIV-1 reverse transcriptase often has a relatively weak enantioselectivity and can be inhibited by the 5-triphosphates of a large series of L-nucleosides and analogues. In contrast, degradation enzymes, such as adenosine or cytidine deaminases, generally demonstrate strict enantioselectivities favouring D-enantiomers and are used by chemists in asymmetric syntheses. The weak enantioselectivities of some enzymes involved in nucleoside metabolism are more or less pronounced, and one enantiomer or the other is favoured depending on the substrate. This suggests that the low enantioselectivity is fortuitous and does not result from evolutionary pressure, since these enzymes do not create or modify asymmetric centres in substrates. The combined enantioselectivities of the enzymes examined in this review strongly suggest that the field of L-nucleosides and their analogues should be systematically explored in the search for new virus inhibitors.
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PMID:The enantioselectivity of enzymes involved in current antiviral therapy using nucleoside analogues: a new strategy? 1090 Dec 89


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