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)

Although anti-human immunodeficiency virus type 1 (HIV-1) therapy has prolonged the lives of patients, drug resistance is a significant problem. Of particular concern are mutations that cause cross-resistance to a particular class of drugs. Among the mutations that cause resistance to several nucleoside analogs are the insertion of amino acids in the fingers subdomain of HIV-1 reverse transcriptase (RT) at positions 69 and 70. These insertions are usually associated with changes in the flanking amino acids and with a change to F or Y at position 215. We have proposed that the T215F/Y mutation makes the binding of ATP to HIV-1 RT more effective, which increases the excision of 3-azido-3'-deoxythymidine-5'-monophosphate (AZTMP) in vitro and increases zidovudine (AZT) resistance in vivo. Although the mechanism of AZT resistance involves enhanced excision, resistance to 3TC involves a block to incorporation of the analog. We measured the effects of fingers insertion mutations on the misincorporation and excision of several nucleoside analogs. RT variants with the amino acid insertions in the fingers and T215Y have a decreased level of misincorporation of ddATP and 3TCTP. These mutants also have the ability to excise AZTMP by ATP-dependent pyrophosphorylysis. However, unlike the classic AZT resistance mutations (M41L/D67N/K70R/T215Y or F/K219E or Q), the combination of the amino acid insertions in the fingers and the T215Y mutation allows efficient excision of ddTMP and d4TMP, even when relatively high levels of deoxynucleoside triphosphates are present in the reaction. Although the dideoxynucleoside analogs of other nucleosides were excised more slowly than AZTMP, ddTMP, and d4TMP, the mutants with the fingers insertion and T215Y excised all of the nucleoside analogs that were tested more efficiently than wild-type RT or a mutant RT carrying the classical AZT resistance mutations. In the ternary complex (RT/template-primer/dNTP), the presence of the bound dNTP prevents the end of the primer from gaining access to the nucleotide binding site (N site) where excision occurs. Gel shift analysis showed that the amino acid insertions in the fingers destabilized the ternary complex compared to wild-type HIV-1 RT. If the ternary complex is unstable, the end of the primer can gain access to the N site and excision can occur. This could explain the enhanced excision of the nucleoside analogs.
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PMID:Nucleoside analog resistance caused by insertions in the fingers of human immunodeficiency virus type 1 reverse transcriptase involves ATP-mediated excision. 1218 98

Melanin pigments often co-purify during preparation of nucleic acids from cells or tissues of melanocytic origin. Contaminating melanin can severely impede subsequent analyses of RNA. We attempted to eliminate melanin in RNA preparations using selected gel matrices. We show here that co-purified melanin pigments can be largely eliminated from RNA samples after passing through polyacrylamide-based beads (Bio-Gel P-60). After isolation from the pigment-containing cells or tissues, RNA was subsequently processed through batch or column purification under acidic pH conditions. The resulting RNA was devoid of contaminating melanin pigments and amenable to molecular reactions such as polymerase chain reaction and cDNA synthesis by reverse transcriptase. Although the process results in some loss of input RNA, this purification procedure is simple, robust and can easily be adopted in any laboratory for the molecular analysis of RNA that requires removal of melanin contamination.
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PMID:A versatile method for the removal of melanin from ribonucleic acids in melanocytic cells. 1239 86

Purified water is a reagent used in a variety of molecular biology experiments, for sample and media preparation, in mobile phases of liquid chromatography techniques, and in rinsing steps. The combination of several technologies in water purification systems allows delivering high-purity water adapted to each application and technique. Through a series of examples, the importance of water quality on biotechnology experiments, such as single nucleotide polymorphism (SNP) analysis by denaturating HPLC, RNA preparation and PCR, is presented. Results obtained on DNA mutation and single nucleotide polymorphism analysis using the denaturating HPLC (DHPLC) technique highlight the benefits of organic removal by UV photooxidation process. Comparative gel electrophoresis data show that ultrafiltration is as efficient as diethylpyrocarbonate (DEPC) treatment for suppressing RNase activity in water. Gel electrophoresis and densitometry measurement also point out the benefits of ultrafiltration to carry out reverse transcriptase-polymerase chain reaction quantitatively.
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PMID:Impact of purified water quality on molecular biology experiments. 1274 91

ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis. Two full-length cDNA clones, pVR-EIL1 and pVR-EIL2, encoding EIN3-LIKE proteins were isolated by reverse transcriptase-polymerase chain reaction and by screening the cDNA library of mung bean (Vigna radiata) hypocotyls. VR-EIL1 and VR-EIL2 share 70% identity and display varying degrees of sequence conservation (39%-65%) with previously isolated EIN3 homologs from Arabidopsis, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) plants. Gel retardation assay revealed that both VR-EILs were able to interact specifically with optimal binding sequence-1, the recently identified optimal binding sequence for tobacco TEIL, with the binding of VR-EIL2 being more efficient than that of VR-EIL1. Transient expression analysis using a VR-EIL::smGFP fusion gene in onion (Allium cepa) epidermal cells indicated that the VR-EIL proteins were effectively targeted to the nucleus. The fusion protein of VR-EIL2 with GAL4 DNA-binding domain strongly activated transcription of a reporter gene in yeast cells, and an essential domain for transcription-stimulating activity was localized to the amino-terminal acidic region that consists of 50 amino acid residues. In contrast with what has been previously found in EIN3- and TEIL-overexpressing Arabidopsis plants, transgenic tobacco seedlings expressing the VR-EIL genes under the control of cauliflower mosaic virus 35S promoter did not exhibit a constitutive triple response. Instead, they displayed a markedly enhanced proliferation of root hairs, one of the typical ethylene response phenotypes, and increased sensitivity to exogenous ethylene. In addition, the pathogenesis-related (PR) genes encoding beta-1,3-glucanase, osmotin, and PR1 were constitutively expressed in 35S::VR-EIL lines without added ethylene, and were hyperinduced in response to ethylene treatment. These results indicate that VR-EILs are functional in tobacco cells, thereby effectively transactivating the GCC-box-containing PR genes and enhancing sensitivity to ethylene. The possible physiological role of VR-EILs is discussed in the light of the suggestion that they are active components of the ethylene-signaling pathway and their heterologous expressions constitutively turn on a subset of ethylene responses in tobacco plants.
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PMID:Molecular and biochemical characterization of VR-EILs encoding mung bean ETHYLENE INSENSITIVE3-LIKE proteins. 1285 28

Before birth, the compliance of the heart is limited predominantly by extracardiac constraint. Reduction of this constraint at birth requires that myocardial compliance be determined mainly by the heart's own constituents. Because titin is a principal contributor to ventricular passive tension (PT), we studied the expression and mechanics of cardiac-titin isoforms during perinatal rat heart development. Gel electrophoresis and immunoblotting revealed a single, 3.7-MDa, N2BA isoform present 6 days before birth and an additional, also previously unknown, N2BA isoform of 3.5 to 3.6 MDa expressed in the near-term fetus. These large isoforms rapidly disappear after birth and are replaced by a small N2B isoform (3.0 MDa) predominating in 1-week-old and adult rats. In addition, neonatal pig hearts showed large N2BA-titin isoforms distinct from those present in the adult porcine myocardium. By quantitative reverse transcriptase-polymerase chain reaction, developmentally expressed titin-mRNA species were detected in rat heart. Titin-based PT was much lower (approximately 15 times) in fetal than adult rat cardiomyocytes, and measured PT levels were readily predictable with a model of worm-like chain titin elasticity. Immunofluorescence microscopy showed the extensibility of the differentially spliced molecular spring regions of fetal/neonatal titin isoforms in isolated rat cardiomyofibrils. Whereas the titin-isoform shift by 700 kDa ensures high passive stiffness of the postnatal cardiac myofibrils, the expression of specific fetal/neonatal cardiac-titin isoforms may also have important functions for contractile properties, myofibril assembly or turnover, and myocardial signaling during perinatal heart development.
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PMID:Developmentally regulated switching of titin size alters myofibrillar stiffness in the perinatal heart. 1508 25

Oxidative stress is one of the characteristics of diabetes and is thought to be responsible for many of the pathophysiological changes caused by the disease. We previously identified an insulin response element in the promoter of plasminogen activator inhibitor 1 (PAI-1) that was activated by an unidentified member of the forkhead/winged helix (Fox) family of transcription factors. This element mediated a 5-7-fold increase in PAI-1 transcription because of insulin. Here we report that oxidative stress also caused a 3-fold increase in PAI-1 transcription and that the effect was additive with that of insulin. Antioxidants prevent this response. Mutational analysis of the PAI-1 promoter revealed that oxidative stress acted at an AP-1 site at -60/52 of the promoter. Gel mobility shift analysis demonstrated that binding to an AP-1 oligonucleotide was increased 4-fold by oxidative stress. Jun levels were increased by oxidants as assessed by reverse transcriptase-PCR. Western blotting demonstrated that a rapid and prolonged nuclear accumulation of phospho-c-Jun followed oxidant stimulation. The nuclear c-Jun phosphorylation was not observed in cells treated with reduced glutathione. Finally, JNK/SAPK activity was found to increase in response to oxidants, and inhibition of JNK/SAP blocked TBHQ-increased PAI-1-luciferase expression. Thus, oxidative stress stimulated AP-1 and activated the PAI-1 promoter.
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PMID:Oxidative stress activates the plasminogen activator inhibitor type 1 (PAI-1) promoter through an AP-1 response element and cooperates with insulin for additive effects on PAI-1 transcription. 1506 77

We have previously shown that transforming growth factor (TGF)-beta1, a crucial molecule in metastatic bone cancer, stimulates collagenase-3 expression in the human breast cancer cell line, MDA-MB231. To understand the molecular mechanisms responsible for TGF-beta1 response on collagenase-3 promoter activity, a functional analysis of the promoter region of the collagenase-3 gene was carried out, and we identified the distal runt domain (RD) and proximal RD/activator protein-1 (AP-1) sites as necessary for full TGF-beta1-stimulated collagenase-3 promoter activity. Gel shift, real time reverse transcriptase-PCR, and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-beta1 treatment in MDA-MB231 cells. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2, whereas Smad3 interacted with both. Chromatin immunoprecipitation experiments confirmed interaction of Smad3 with JunB and Cbfa1/Runx2. Under basal conditions, Cbfa1/Runx2 bound to both the proximal RD/AP-1 and distal RD sites. In response to TGF-beta1, Cbfa1/Runx2 was seen only at the distal RD site, whereas JunB occupied the proximal RD/AP-1 site. An assemblage of Smad3, JunB, and Cbfa1/Runx2 at the distal RD site of the collagenase-3 promoter occurred in response to TGF-beta1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-beta type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-beta1. Taken together, our results suggest that TGF-beta1 stimulated JunB and Cbfa1/Runx2 to bind to their respective DNA consensus sites and that Smad3 is likely to stabilize their interaction to confer functional TGF-beta1-stimulation of collagenase-3 expression in MDA-MB231 cells.
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PMID:Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-beta1-stimulated collagenase-3 expression in human breast cancer cells. 1508 95

A female controlled drug delivery system (FcDDS) containing sodium dodecyl sulfate as a microbicide, ethylenediaminetetraacetic acid (EDTA) as a synergistic microbicide, and lactic acid as a pH modulator was developed as an intravaginal barrier device against sexually transmitted diseases. The host response of the vagina to the FcDDS was evaluated through biocompatibility tests including cell viability, estrogenicity, and cytotoxicity assays on HeLa cervical cells and NIH:Ovcar-3 ovarian cells. Gel electrophoresis and reverse transcriptase polymerase chain reaction assays on HeLa cervical cell lines were also performed to elucidate the effects of EDTA on the expression of particular proteins of interest. The results of the cell viability test showed no significant difference in viability of cells upon exposure to EDTA at concentrations less than 0.035% that was reported to exert spermicidal activity. EDTA at concentrations less than 0.035% did not cause any cytotoxicity. The results of reverse transcriptase polymerase chain reaction analysis revealed that EDTA induced the expression of a 67-kDa protein in HeLa cells, which was identified as elastin binding protein (a part of the elastin receptor complex). This work has demonstrated that FcDDS containing EDTA is biocompatible and safe to be used as an intravaginal barrier device.
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PMID:Biocompatibility of components of a female controlled drug delivery system. 1537 89

Previous work demonstrated both acid and neutral, bile salt-independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification, and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, phenyl-Sepharose hydrophobic interaction, concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular mass of approximately 62 kDa, as measured by mass spectrometry. Gel filtration chromatography of the purified protein revealed a native molecular mass of approximately 176 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross-reaction with an anti-ES-10 antibody. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a K(m) of 86 microm. Substrate specificity studies showed that ES-10 is also able to catalyze hydrolysis of triolein. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time reverse transcriptase-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis, and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4, and ES-3 were expressed at significantly higher levels than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.
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PMID:Isolation and characterization of a microsomal acid retinyl ester hydrolase. 1576 60

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi <or= 0.005.
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PMID:Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase. 1604 16


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