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)

Novel beta-L-2',3'-dideoxy-3'-fluoro nucleosides were synthesized and further converted to their 5'-triphosphates. Their inhibitory activities against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) DNA polymerases, human immunodeficiency virus (HIV) reverse transcriptase (RT), and the cellular DNA polymerases alpha, beta, gamma, delta, and epsilon were investigated and compared with those of the corresponding 3'-fluoro-modified beta-d-analogues. The 5'-triphosphates of 3'-deoxy-3'-fluoro-beta-L-thymidine (beta-L-FTTP), 2',3'-dideoxy-3'-fluoro-beta-L-cytidine (beta-L-FdCTP), and 2',3'-dideoxy-3'-fluoro-beta-l-5-methylcytidine (beta-L-FMetdCTP) emerged as effective inhibitors of HBV/DHBV DNA polymerases (IC50 = 0.25-10.4 microM). They were either equally (FTTP) or less (FMetdCTP, FdCTP) effective than their beta-d-counterparts. Also the 5'-triphosphate of beta-L-thymidine (beta-L-TTP) was shown to be a strong inhibitor of these two viral enzymes (IC50 = 0.46/1.0 microM). However, all beta-L-FdNTPs (also beta-L-TTP) were inactive against HIV-RT, a result which contrasts sharply with the high efficiency of the beta-D- FdNTPs against this polymerase. Between the cellular DNA polymerases only the beta and gamma enzymes displayed a critical susceptibility to beta-D-FdNTPs which is largely abolished by the beta-L-enantiomers. These results recommend beta-L-FTdR, beta-L-FCdR, and beta-L-FMetCdR for further evaluation as selective inhibitors of HBV replication at the cellular level.
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PMID:Newly synthesized L-enantiomers of 3'-fluoro-modified beta-2'-deoxyribonucleoside 5'-triphosphates inhibit hepatitis B DNA polymerases but not the five cellular DNA polymerases alpha, beta, gamma, delta, and epsilon nor HIV-1 reverse transcriptase. 962 45

4'-Acylated thymidines represent a new class of DNA chain terminators, since they have been shown to act as post-incorporation chain-terminating nucleotides despite the presence of a free 3'-hydroxyl group. Here, we describe the action of the 4'-acetyl- (MeTTP) and 4'-propanoylthymidine 5'-triphosphate (EtTTP) on HIV-1 reverse transcriptase in RNA- and DNA-dependent DNA synthesis and on DNA synthesis catalyzed by the cellular DNA polymerases alpha, beta, delta and epsilon. MeTTP exhibits a high selectivity towards HIV-1 reverse transcriptase. By the use of the bulkier propanoyl group as the 4'-substituent of the nucleoside 5'-triphosphate, selectivity towards HIV-1 reverse transcriptase could be increased without affecting substrate efficiency. Thus, 4'-modifications may serve as a tool to increase selectivity towards HIV-1 reverse transcriptase.
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PMID:4'-Acylated thymidine 5'-triphosphates: a tool to increase selectivity towards HIV-1 reverse transcriptase. 970 20

To elucidate the role of retinoic acid (RA) in anti-CD40 + interleukin-4 (IL-4)-mediated B-cell activation, the effect of 10(-12) to 10(-6) mol/L RA was studied in anti-CD40 (1 microgram/mL) + IL-4 (5 ng/mL)-mediated proliferation and Ig synthesis by human peripheral blood mononuclear cells (PBMC) and B cells in healthy donors. Anti-CD40 + IL-4-mediated proliferation of PBMC and B cells was inhibited by RA in a dose-dependent manner, with maximal inhibition of 62% +/- 5% in PBMC and 55% +/- 4.4% in B cells by all-trans RA, and 58% +/- 6.7% and 51% +/- 4.7%, respectively by 13-cis RA. IgE synthesis was even more markedly inhibited by RA starting at concentrations of >10(-14) mol/L for B cells and >10(-10) mol/L for PBMC. Maximal inhibition of IgE production for B cells was at 10(-8) mol/L for all-trans RA (94% +/- 1.8%) and 96% +/- 3.2% for 13-cis RA. Low concentrations of RA inhibiting IgE synthesis (10(-10) mol/L) affected neither B-cell proliferation nor the production of IgA, IgG, and IgM. Elucidation of the mechanism involved in this inhibition of IgE production shows that epsilon germline transcription is decreased by RA, whereas production of interferon-gamma (IFN-gamma) was not enhanced in the presence of RA. To differentiate whether the RA effect was mediated by RA receptors alpha, beta, and gamma, the expression of the retinoic acid receptors (RAR) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The data show that unstimulated human peripheral B cells express mRNA of the RA receptor alpha, beta, and gamma. Using retinoids with different receptor binding specificity (CD336, CD437, CD2019, CD367), dose-dependent inhibition of IgE synthesis was shown by all four derivates, but was most marked by an RA binding the alpha receptor with high specificity. Taken together, this study shows that RA inhibits IgE production of anti-CD40 + IL-4-stimulated B cells in vitro.
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PMID:Retinoic acid inhibits CD40 + interleukin-4-mediated IgE production in vitro. 971

To analyze messenger RNA (mRNA) levels for the alpha, beta, and gamma subunits of the human amiloride-sensitive epithelial Na+ channel (hENaC) in respiratory epithelia, we developed a competitive quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) assay specific for each subunit, using two human respiratory epithelial-cell lines. We next determined the relation between hENaC mRNA levels and the biologic activity of the hENaC in the respiratory epithelium of eight normal men. The electrical potential difference (PD) between the epithelium of the inferior nasal turbinate and the subcutaneous space was measured, using control and amiloride (100 microM) solutions. QRT-PCR measurement of hENaC-subunit mRNAs and epithelial-specific cytokeratin 18 mRNA allowed us to normalize hENaC expression to epithelial-cell RNA. Respective values for alpha, beta, and gamma hENaC mRNA levels in epithelium obtained at the site of maximal PD were 39 +/- 4.0, 7.5 +/- 0.92, and 1.8 +/- 0.25 attomol/fmol cytokeratin mRNA, respectively. Respiratory epithelial PD exhibited a significant negative correlation with gamma hENaC (r2 = 0.72, p < 0.01), tended to increase with increasing alpha hENaC, and was unaffected by beta hENaC mRNA levels. Our results suggest that hENaC activity in vivo is influenced by expression of the gene for gamma hENaC. The assay used in the study provides a useful tool for evaluating Na+-channel expression in clinically relevant patient populations.
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PMID:Relation between alpha, beta, and gamma human amiloride- sensitive epithelial Na+ channel mRNA levels and nasal epithelial potential difference in healthy men. 976 84

Monte Carlo (MC) simulations in combination with a linear response approach were used to estimate the free energies of binding for a series of 12 TIBO nonnucleoside inhibitors of HIV-1 reverse transcriptase. Separate correlations were made for the R6 and S6 absolute conformations of the inhibitors, as well as for the analogous N6-monoprotonated species. Models based upon the neutral unbound inhibitors produced overall better fits to experimental values than did those using the protonated unbound inhibitors, with only slight differences between the neutral R6 and S6 cases. The best results were obtained with a three-parameter linear response equation containing van der Waals (alpha), electrostatic (beta), and solvent accessible surface area (SASA, gamma) terms. The averaged (R6 and S6) rms error was approximately 0.88 kcal/mol for the observed range of 4.06 kcal/mol in inhibitor activities. The averaged values of alpha, beta, and gamma were -0.150, 0.114, and 0. 0286, respectively. Omission of the alpha term gave beta 0.152 and gamma 0.022 with a rms of 0.92. The unweighted van der Waals components were found to be highly attractive but failed to correlate well across the series of inhibitors. Contrastingly, while the electrostatic components are all repulsive, they show a direct correlation with inhibitor activity as measured by DeltaGbinding. The role of gamma is primarily to produce an overall negative binding energy, and it can effectively be replaced with a negative constant. During the MC simulations of the unbound solvated inhibitors, the R6 and S6 absolute conformations do not interconvert due to the formation of a favorable hydrogen bond to solvent. In the complex, however, interconversion of these conformations of the inhibitor is observed during the course of the simulations, a phenomenon which is apparently not observed in the crystalline state of the complex. Hydrogen bonding of the inhibitor to the backbone NH of K101 and the lack of such an interaction with the C=O of K101 or with solvent correlate with enhanced activity, as does the ability to assume a number of different orientations of the inhibitor dimethylallyl moiety with respect to residues Y181 and Y188 while retaining contact with W229. Overall, the use of a combination of MC simulation with a linear response method shows promise as a relatively rapid means of estimating inhibitor activities. This approach should be useful in the preliminary evaluation of potential modifications to known inhibitors to enhance activity.
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PMID:Prediction of binding affinities for TIBO inhibitors of HIV-1 reverse transcriptase using Monte Carlo simulations in a linear response method. 985 95

The epithelial Na+ channel (ENaC) functions as the rate-limiting factor in aldosterone-regulated transcellular Na+ transport. In the study described here, the effect of aldosterone on ENaC mRNA levels, protein synthesis and benzamil-sensitive Na+ transport was investigated using primary cultures of immunodissected rabbit kidney connecting tubule and cortical collecting duct cells (CNT and CCD, respectively). After a lag time of 3 h, aldosterone caused transepithelial Na+ transport to increase, reaching maximal level of 260+/-44% after 16 h of incubation. The alpha, beta and gamma rabbit ENaC (rbENaC) mRNA levels, measured by semi-quantitative reverse transcriptase-polymerase chain reaction, were not changed by aldosterone during the first 3 h, but a twofold increase was apparent after 6 h; levels remained elevated for up to 16 h of incubation. Immunoprecipitation of [35S]methionine-labeled rbENaC revealed a rise in protein levels of the alpha and beta subunits, but the protein level of the gamma subunit remained constant. In conclusion, our data suggest that in rabbit CNT and CCD the early increase in Na+ transport caused by aldosterone is due to the activation or insertion of existing Na+ channels into the apical membrane, and that the late response is mediated by increased synthesis of the alpha and beta rbENaC subunits.
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PMID:Time-dependent regulation by aldosterone of the amiloride-sensitive Na+ channel in rabbit kidney. 1039 66

The racemic nucleoside analogue 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) is in clinical development for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection. dOTC is structurally related to lamivudine (3TC), but the oxygen and sulfur in the furanosyl ring are transposed. Intracellular metabolism studies showed that dOTC is phosphorylated within cells via the deoxycytidine kinase pathway and that approximately 2 to 5% of dOTC is converted into the racemic triphosphate derivatives, which had measurable half-lives (2 to 3 hours) within cells. Both 5'-triphosphate (TP) derivatives of dOTC were more potent than 3TC-TP at inhibiting HIV-1 reverse transcriptase (RT) in vitro. The K(i) values for dOTC-TP obtained against human DNA polymerases alpha, beta, and gamma were 5,000-, 78-, and 571-fold greater, respectively, than those for HIV RT (28 nM), indicating a good selectivity for the viral enzyme. In culture experiments, dOTC is a potent inhibitor of primary isolates of HIV-1, which were obtained from antiretroviral drug-naive patients as well as from nucleoside therapy-experienced (3TC- and/or zidovudine [AZT]-treated) patients. The mean 50% inhibitory concentration of dOTC for drug-naive isolates was 1.76 microM, rising to only 2.53 and 2.5 microM for viruses resistant to 3TC and viruses resistant to 3TC and AZT, respectively. This minimal change in activity is in contrast to the more dramatic changes observed when 3TC or AZT was evaluated against these same viral isolates. In tissue culture studies, the 50% toxicity levels for dOTC, which were determined by using [(3)H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 microM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 microM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells was observed. When administered orally to rats, dOTC was well absorbed, with a bioavailability of approximately 77%, with a high proportion (approximately 16.5% of the levels in serum) found in the cerebrospinal fluid.
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PMID:Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine. 1042

Proopiomelanocortin (POMC) is a 31 kDa prohormone that is processed to various bioactive peptides, including adrenocorticotropin (ACTH), melanotropins (alpha, beta, gamma-MSH), lipotropins, and endorphins. POMC is expressed not only in the pituitary gland but also in a variety of nonpituitary organs and tumors, including melanomas. We previously showed that normal human melanocytes produce and secrete alpha-MSH and ACTH, and furthermore, that advanced melanoma cells generally produce higher amounts of POMC peptides that correlate with tumor progression. To elucidate the mechanism of this upregulation, the expression of genes encoding corticotropin-releasing hormone (CRH) and its receptor, CRH-R, as well as POMC and the MSH receptor (MC1-R), was evaluated by reverse transcriptase-polymerase chain reaction using cultured human melanoma cells, nevus cells, and normal melanocytes. Our results show that all melanocytic cells express CRH, CRH-R, POMC, and MC1-R, with highest intensities in melanoma cells. Furthermore, immunohistochemistry shows that CRH as well as POMC is strongly expressed in advanced melanomas, such as vertically growing lesions of acral lentiginous, nodular and metastatic melanomas, in contrast to negative expression in nevus cells. These results indicate that tumor progression accentuates CRH, CRH-R, and POMC expression by melanoma cells.
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PMID:Expression of proopiomelanocortin, corticotropin-releasing hormone (CRH), and CRH receptor in melanoma cells, nevus cells, and normal human melanocytes. 1053 83

DNA polymerases may be differentially expressed by cells during periods of quiescence and proliferation. Murine B cells are an ideal population to study because their division time varies widely in vivo, and different subsets can be easily isolated. Consequently, we analyzed RNA from resting cells (B220(+)peanut agglutinin(-)) and activated germinal center cells (B220(+)peanut agglutinin(+)) from spleens by reverse transcriptase-PCR using primers for five nuclear polymerases and their associated subunits. Gel analyses of the amplified products showed that the rapidly-dividing germinal center B cells expressed DNA polymerases alpha, beta, delta, epsilon, and zeta. The resting B cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta, and zeta. Thus, polymerase epsilon, as well as alpha, appears to have a primary role in chromosomal replication of murine B lymphocytes. Further, the lack of expression of polymerase epsilon in resting cells indicates that this enzyme is not used in any DNA repair pathways by these cells. The expression of polymerase zeta by resting cells suggests that it has another role in DNA repair, perhaps recombination, in addition to its function of bypassing damage during chromosomal replication.
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PMID:Differential expression of DNA polymerase epsilon in resting and activated B lymphocytes is consistent with an in vivo role in replication and not repair. 1086 11

Extracellular ATP is a neurotransmitter and mediates a variety of responses. In the endocrine system, there are data suggesting a physiological role for ATP in Ca(2+) signalling and hormone secretion. However, the ATP receptor subtype involved has not been clearly elucidated in GH3 cells, a rat anterior pituitary cell line. BzATP- and ATP-induced [Ca(2+)](i) responses had EC(50) values of 18 and 651 microM, respectively. The maximal response to ATP was only 59+/-8% of that for BzATP. The BzATP-induced [Ca(2+)](i) increase was dependent upon the extracellular Ca(2+) concentration. Preincubation with oxidized ATP (oATP) nearly abolished the ATP- and BzATP-induced [Ca(2+)](i) increases. Both BzATP and ATP induced depolarization in GH3 cells, with EC(50) values of 31 microM and 1 mM, respectively. The maximal depolarization to BzATP and ATP were 152+/-21 and 146+/-16% of that elicited by 30 mM KCl. The rank order of agonist potency for [Ca(2+)](i) and depolarization responses was BzATP > > ATP >2-MeSATP and purine derivatives such as ADP, AMP, adenosine were ineffective. Neither UTP nor alpha, beta-methylene ATP showed any effect. In low-divalent conditions BzATP evoked non-desensitizing inward currents, which were reversed at approximately 0 mV. This nonselective cationic conductance was increased by repeated applications of BzATP and the cells became very permeable to NMDG. Longer applications (30 min) of BzATP stimulated ethidium bromide influx in low divalent conditions, suggesting increased permeability to larger molecules. We also identified the existence of P2X(7) mRNA on GH3 cells by using reverse transcriptase (RT)-polymerase chain reaction (PCR). These results suggest that the GH3 cells have an endogenous P2X(7) receptor and purinergic stimulation may play a potential role in neuroendocrine modulation on these cells.
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PMID:ATP-induced [Ca(2+)](i) changes and depolarization in GH3 cells. 1095 73


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