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)

Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated by the HIV-1-specific inhibitors bis-heteroarylpiperazine (BHAP), 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)-on e (TIBO) R82913, nevirapine, and the N3-methylthymine derivative of [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide) (TSAO-m3T), as single agents or in combination, at escalating concentrations. When used individually, the compounds led to the emergence of drug-resistant virus strains within two to five subcultivations. The resulting strains were designated HIV-1/BHAP, HIV-1/TIBO, HIV-1/Nev, and HIV-1/TSAO-m3T, respectively. The mutant viruses showed the following amino acid substitutions in their reverse transcriptase (RT): Leu-100-->Ile for HIV-1/BHAP; Lys-103-->Asn for HIV-1/TIBO; Val-106-->Ala for HIV-1/Nev; and Glu-138-->Lys for HIV-1/TSAO-m3T. Both the Tyr-181-->Cys and Val-106-->Ala mutations were found in another mutant emerging following treatment with nevirapine at escalating concentrations. The BHAP-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and TSAO-m3T, whereas the TSAO-m3T-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and BHAP. When different pairs of nonnucleoside RT inhibitors (i.e., BHAP plus TSAO-m3T, nevirapine plus TSAO-m3T, TIBO plus TSAO-m3T, nevirapine plus TIBO, and BHAP plus nevirapine) were used, resistant virus emerged as fast as with single-drug therapy. In all cases the Tyr-181-->Cys mutation appeared; the virus showed markedly reduced sensitivity to all HIV-1-specific inhibitors but retained sensitivity to 2',3'-dideoxynucleoside analogs such as zidovudine, ddC, and ddI. Our findings argue against simultaneous combination of two different nonnucleoside RT inhibitors that are unable to inhibit HIV-1 mutant strains containing the Tyr-181-->Cys mutation when administered as single drugs.
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PMID:Treatment of human immunodeficiency virus type 1 (HIV-1)-infected cells with combinations of HIV-1-specific inhibitors results in a different resistance pattern than does treatment with single-drug therapy. 768 22

Specific mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene that cause zidovudine (3'-azido-2',3'-dideoxythymidine; AZT) and didanosine (2',3'-dideoxyinosine; ddI) resistance were studied. The 50% inhibitory concentrations (IC50s) of nucleosides for cloned viruses containing these mutations were compared with the IC50s of the corresponding triphosphate analogs for mutant recombinant-expressed reverse transcriptases (RTs). Changes in ddATP inhibition of RNA-dependent DNA polymerase activity fully accounted for the ddI resistance of the virus caused by a Leu-74-->Val substitution in RT, including an augmentation by the AZT-selected substitutions Thr-215-->Tyr and Lys-219-->Gln in RT. In contrast, the AZT-selected substitutions studied did not cause as great a change in the IC50 of AZT-triphosphate (AZT-TP) for polymerase as they did in the IC50 of AZT for mutant virus. In addition, the mutation at codon 74 suppressed AZT resistance in the virus caused by the mutations at codons 215 and 219 but did not suppress the AZT-TP resistance of enzyme containing these same mutations in RT. The mutation at codon 74 was found in clinical isolates whether or not the patient had received AZT prior to starting ddI therapy. AZT resistance coexisted with ddI resistance following acquisition of Leu-74-->Val in three clinical isolates, indicating that the suppressive effect of Val-74 on the AZT resistance of the virus does not occur in all genetic contexts. When this suppression of AZT resistance was seen in the virus, Val-74 did not appear to cause mutually exclusive changes in AZT-TP and ddATP binding to RT in vitro. The results of the in vitro experiments and characterization of clinical isolates suggest that there are differences in the functional effects of these AZT and ddI resistance mutations.
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PMID:pol mutations conferring zidovudine and didanosine resistance with different effects in vitro yield multiply resistant human immunodeficiency virus type 1 isolates in vivo. 768 22

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
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PMID:Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity. 769 May 4

Myelin basic proteins (MBPs) are a family of alternatively spliced isoforms present in myelin sheaths of most vertebrates. A reverse transcriptase-polymerase chain reaction (RT-PCR) approach was used to clone MBP isoforms in species representing two superorders of elasmobranchs: Squalus acanthias, representing Squalomorph sharks, and Raja erinacia, representing Batoidea rays. Two products were generated from each species. The larger product encoded a 155 amino acid protein, the same size as MBPs from two Galeomorph sharks, Heterodontus francisci and Carcharhinus obscurus, which, based upon alignment with other vertebrate MBPs, contained six of the seven MBP exons; only exon II was absent. The smaller product encoded a 141 amino acid protein that lacked exon II and exon V. There were 26 and 30 nucleotide differences between Squalus and Heterodontus, and Raja and Heterodontus, respectively. Sequences from Squalus and Raja were far more similar, having only five nucleotide differences. Both isoforms of elasmobranch MBP contain 18.5% basic (lysine plus arginine) amino acids, compared with 17.5% in mammalian MBPs comprised of the corresponding exons. Northern blot analysis of whole brain total RNA revealed a single band of 2.5 kb in Squalus, and three bands of 1.2, 1.4, and 2.3 kb in Raja. The finding that MBPs of a Squalomorph shark and a Batoidea ray are closer to one another than either is to the Galeomorph sharks suggests that MBP sequence information may prove useful in classifying modern day Chondrichthytes.
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PMID:Molecular cloning of the myelin basic proteins in the shark, Squalus acanthias, and the ray, Raja erinacia. 769 75

We recently characterized (Moebius, F. F., Burrows, G. G., Striessnig, J., and Glossmann H. (1993) Mol. Pharmacol. 43, 139-144) and purified (Moebius, F. F., Hanner, M., Knaus, H. G., Weber, F., Striessnig, J., and Glossmann, H. (1994) J. Biol. Chem. 269, 29314-29320) a binding protein for the phenylalkylamine Ca2+ antagonist emopamil. The emopamil-binding protein (EBP) acts as a high affinity acceptor for several antiischemic drugs and thus represents a potential common molecular target for antiischemic drug action. Degenerate oligonucleotides were synthesized according to the N-terminal amino acid sequence of purified EBP and used to amplify a guinea pig cDNA with reverse transcriptase-polymerase chain reaction and to clone full-length cDNAs from guinea pig and human liver cDNA libraries. The cDNAs coded for 229 (guinea pig) and 230 (human) amino acid 27-kDa polypeptides without significant sequence homology with any known protein. However, EBP shared structural features with pro- and eukaryotic drug transport proteins. The amino acid identity between human and guinea pig EBP was 73%. Hydrophobicity plots predicted four transmembrane segments. The C terminus contained a lysine-rich consensus sequence for the retrieval of type I integral membrane proteins to the endoplasmic reticulum. The heterologous expression of human and guinea pig EBP in Saccharomyces cerevisiae demonstrated that the expression of EBP alone is sufficient to form high affinity drug- and cation-binding domains identical to the [3H]-emopamil-binding site of guinea pig liver. Northern and Western blot analysis revealed high abundance of EBP in guinea pig epithelial tissues as liver, bowel, adrenal gland, testis, ovary, and uterus and low densities in brain, cerebellum, skeletal muscle, and heart. EBP is suggested to be the first structurally characterized member of a family of high affinity microsomal drug acceptor proteins carrying so called sigma-binding sites.
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PMID:Phenylalkylamine Ca2+ antagonist binding protein. Molecular cloning, tissue distribution, and heterologous expression. 770 2

We have reported previously mitogenic effects of gastrin on several immortalized and neoplastic cell lines, including Swiss 3T3 fibroblasts. Receptor subtypes, cholecystokinin (CCK)-A and CCK-B, for a closely related peptide, cholecystokinin, were recently cloned. These studies were undertaken to investigate if CCK-A- and CCK-B receptors were perhaps mediating the mitogenic effects of gastrin on Swiss 3T3 cells. Receptor antagonists that inhibit the biological effects and binding of peptides to the CCK-A (L-364,718 (L18)) and CCK-B (L-365,260 (L60)) receptors were ineffective toward inhibiting the binding and proliferative effects of gastrin on Swiss 3T3 cells. Radiolabeled L18 and L60 demonstrated no binding to the cells, indicating that CCK-A and CCK-B receptors may be absent on Swiss 3T3 cells. Radiolabeled CCK-8, gastrin, L18, and L60, on the other hand, demonstrated specific binding to a pancreatic cancer cell line (AR42J cells) (used as a positive control). In cross-linking studies the molecular mass of the major band of gastrin receptors (GR) on Swiss 3T3 cells was determined to be approximately 45 kDa. The mitogenic potency of 0.1-1.0 nM gastrin-like peptides on Swiss 3T3 cells was in the order of G1-17 > or = G1-17-Gly > G5-17 > or = G5-17-Gly > G2-17 > CCK-8-Gly > or = G1-17-Lys > or = CCK-8. The relative binding affinity of the peptides (based on the dose-dependent inhibition of binding of 125I-G1-17 to Swiss 3T3 cells) was similar to the relative mitogenic potency of the peptides as given above. Furthermore, G1-17-Gly was equally effective as G1-17 in displacing the binding of 125I-G1-17 to the 45-kDa GR from the Swiss 3T3 cells. Based on these studies it became evident that the novel gastrin preferring GR, expressed by Swiss 3T3 cells, binds and mediates the mitogenic effects of not only the mature (amidated) forms of gastrin-like peptides but also binds and mediates the mitogenic effects of glycine-extended forms of gastrin-like peptides. Possible mRNA expression of CCK-A and CCK-B receptor subtypes by gastrin-responsive rodent intestinal and fibroblast cell lines (Swiss 3T3, IEC-6, CA) was measured by the methods of Northern blot analysis and reverse transcriptase-polymerase chain reaction. mRNA from rat pancreas, AR42J cells, and rat antrum served as positive controls.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Novel gastrin receptors mediate mitogenic effects of gastrin and processing intermediates of gastrin on Swiss 3T3 fibroblasts. Absence of detectable cholecystokinin (CCK)-A and CCK-B receptors. 772 37

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

Rat glomerular epithelial cells were grown to confluency on semipermeable tissue culture inserts and the plasminogen system of these cells was analyzed using enzyme assays, Western analysis, zymography, and reverse transcriptase-PCR. The glomerular epithelial cells are capable of activating exogenous plasminogen to plasmin by endogenous plasminogen activators. The cells produce both tissue-plasminogen activator and urokinase-plasminogen activator with urokinase being the prominent activator. Both activators are present primarily on the basolateral side of the cells with urokinase found primarily at the cell surface presumably bound to its receptor and tissue-plasminogen activator found primarily in the matrix secreted by the cells on the semipermeable insert. The cells also produce plasminogen activator inhibitor-1 and urokinase-plasminogen activator receptor. Inhibition of plasminogen activation occurred with plasminogen activator inhibitor-1, anti-catalytic anti-tissue-plasminogen activator antibody, epsilon-aminocaproic acid, which inhibits the binding of plasminogen through its lysine binding sites, and amiloride, which specifically inhibits urokinase.
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PMID:Analysis of the plasminogen system on rat glomerular epithelial cells. 779 90

Complementary DNA fragments encoding the prepro-salmon gonadotropin-releasing hormone ([Trp7, Leu8]GnRH, sGnRH) of the red seabream Pagrus major were amplified from mRNA of the olfactory bulbs using a reverse transcriptase-polymerase chain reaction (RT-PCR), and the full-length cDNA was cloned from a cDNA library using the PCR-amplified cDNA as a probe. The cDNA consisted of 442 bp, including an open reading frame of 270 bp which encoded the prepro-sGnRH (90 amino acid residues). The prepro-sGnRH had the same architecture as that reported in other species. It was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP), which was connected to sGnRH by a Gly-Lys-Arg sequence. The prepro-sGnRH of the red seabream had 90% amino acid identity to the prepro-sGnRH from an African cichlid Haplochromis burtoni which belongs to the same suborder as the red seabream; however, identity was lower to the prepro-sGnRH from Atlantic salmon Salmo salar (74%) and masu salmon Oncorhynchus masou (70%). The GnRH peptide itself and the Gly-Lys-Arg sequence in the prepro-GnRH are highly conserved among vertebrates. The red seabream GAP also shows significant amino acid identity to the GAPs of the African cichlid (89%), Atlantic salmon (74%), and masu salmon (67%), but exhibits no significant identity to chicken or mammalian GAP.
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PMID:Molecular cloning of a cDNA encoding the prepro-salmon gonadotropin-releasing hormone of the red seabream. 785 23

We have created a temperature-sensitive (ts) mutant of human immunodeficiency virus type 1, using the technique of charge-cluster-to-alanine scanning mutagenesis to introduce specific changes into the integrase coding region. In the ts mutant virus, the lysine at amino acid 136 and the glutamic acid at amino acid 138 of integrase have been replaced with alanines (K136A/E138A). When K136A/E138A is synthesized at 35 degrees C, it replicates to a similar degree as wild-type virus during infection of CEM cells at 35 degrees C on the basis of syncytium formation, levels of core antigen, and reverse transcriptase activity. However, during infection at the nonpermissive temperature of 39.5 degrees C, K136A/E138A is capable of only one round of integration. Mutant virions formed at 39.5 degrees C do not integrate but are indistinguishable from wild-type virions when scored for activity of reverse transcriptase and correct expression and processing of Gag and Pol proteins. We demonstrate that the defect responsible for the ts phenotype of K136A/E138A is localized to a step after proviral formation and integrase protein synthesis but prior to particle maturation. It is the temperature at which the K136A/E138A virion is synthesized, not the temperature at which infection occurs, which determines the ability of the virus to integrate.
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PMID:Identification and characterization of a temperature-sensitive mutant of human immunodeficiency virus type 1 by alanine scanning mutagenesis of the integrase gene. 798 62


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