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)

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.
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PMID:Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. 1115 51

The potential roles of an amino acid deletion at codon 67 (Delta67) and a Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase of human immunodeficiency virus (HIV) type 1 in drug sensitivity and relative replication fitness were studied. Our results suggest that the Delta67 and T69G changes can be categorized as mutations associated with multidrug resistance. The combination of both mutations with an L74I change (Delta67+T69G/L74I) leads to a novel 3'-azido-3'-deoxythymidine resistance motif and compensates for impaired HIV replication.
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PMID:Amino acid deletion at codon 67 and Thr-to-Gly change at codon 69 of human immunodeficiency virus type 1 reverse transcriptase confer novel drug resistance profiles. 1126 89

Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by "semiquantitative" reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower.
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PMID:Differential expression of 1-aminocyclopropane-1-carboxylate synthase genes during orchid flower senescence induced by the protein phosphatase inhibitor okadaic acid. 1135 Oct 88

An amino acid mutation at residue 284 (Ala to Thr) in the VP2 protein of infectious bursal disease viruses (IBDVs) has been correlated with the ability to replicate in cell culture. In this study, we designed a molecular test for this mutation. The reverse transcriptase/polymerase chain reaction (RT/PCR) was used to amplify a 743-bp region of the VP2 gene that contained the codon for amino acid 284. The restriction endonuclease NgoMIV was selected for this study because the first three nucleotides of its six-base recognition sequence are the codon responsible for the amino acid alanine at residue 284. The RT/PCR products from 10 known pathogenic and 16 vaccine strains of IBDV were examined for the presence or absence of the NgoMIV site. We also examined 189 field strains of IBDV for the NgoMIV site. All 10 known pathogenic IBDV strains contained the NgoMIV site, indicating they contained alanine at residue 284. None of the vaccine strains had the NgoMIV site, suggesting they had threonine or another amino acid at residue 284. The results suggest that the presence of this NgoMIV site can be used as a marker for the identification of wild-type (nonvaccine) IBDV strains. The RT/PCR products from 152 (80.4%) of the field strains had the NgoMIV site and thus have the potential to be wild-type pathogenic viruses. The RT/PCR products from 37 (19.6%) of the field strains were not cleaved by NgoMIV and thus are potentially attenuated vaccine strains. Molecular diagnostic assays have been used to place IBDV strains into genetically related groups. The identification of this genetic marker now makes it possible to identify viruses that are wild-type strains that have the potential to be pathogenic viruses.
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PMID:Use of a genetic marker for wild-type potentially pathogenic infectious bursal disease viruses. 1156 47

Human endogenous retroviruses (HERVs) have been implicated as causative agents in diseases characterized by inflammation and macrophage activation, such as multiple sclerosis. Because monocyte activation and differentiation influence retroviral transcription and replication, we investigated the contribution of these processes to the expression of four HERV families (HERV-W, HERV-K, HERV-E, and HERV-H) in human monocytes, and autopsied brain tissue from patients with brain diseases associated with increased macrophage activity. Reverse transcriptase-polymerase chain reaction analysis of primary macrophages and U937 monocytoid cells stimulated with phorbol-12-myristate-13-acetate or lipopolysaccharide revealed three- to ninefold increases in HERV-W, HERV-K, and HERV-H RNA levels. In addition, elevated reverse transcriptase activity and HERV RNA were detectable in supernatants from PMA-stimulated U937 cultures, properties that could be attenuated with the inhibitor of monocyte differentiation threonine-lysine-proline. In contrast, stimulation of monocytes decreased or had no effect on HERV-E expression. Compared with controls, HERV-W and HERV-K expression was increased in brain tissue from patients with multiple sclerosis or human immunodeficiency virus infection or AIDS, with concomitant elevated tumor necrosis factor-alpha levels. Similarly, elevated HERV-W levels were detected in patients with Alzheimer's dementia only when tumor necrosis factor-alpha expression was also evident (2 of 6 cases). The detection of several HERVs in inflammatory brain diseases and the capacity to augment HERV expression in monocytes with compounds that influence cellular activity suggest that increased expression of these viruses is a consequence of increased immune activity rather than causative of distinct diseases.
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PMID:Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases. 1160 92

Phosphorylation/dephosphorylation of proteins is a general mechanism of hormonal signal transduction, including ABA, and serine/threonine protein phosphatases 2C (PP2C, EC 3.1.3.16) have been suggested to play an important role in this process. By means of differential reverse transcriptase-polymerase chain reaction (RT-PCR) and further screening of a cDNA library made from mRNA of ABA-treated Fagus sylvatica L. seeds, a full-length cDNA clone (FsPP2C2) encoding a putative PP2C was obtained. Comparison to the databases revealed high homology to plant PP2C and most features of these enzymes, but unusual characteristics were found within the catalytic domain and the N-terminal region of the amino acid sequence. The coding region of FsPP2C2 was expressed in Escherichia coli as histidine tag fusion protein and shows Mg2+-dependent in vitro phosphatase activity. Transcription of the FsPP2C2 gene is low during seeds stratification at 4 degrees C or under gibberellic acid (GA3) treatment and clearly increases when seeds are treated with ABA and calcium (Ca2+) together, while the addition of calcium chelators (EGTA or TMB-8) decreases its expression. Furthermore, FsPP2C2 is only expressed in ABA-treated tissues, preferentially in seeds, which suggests that this PP2C is specifically induced by ABA in dormant seeds, in a Ca2+-dependent manner, and also in other ABA-treated tissues.
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PMID:Molecular cloning of a functional protein phosphatase 2C (FsPP2C2) with unusual features and synergistically up-regulated by ABA and calcium in dormant seeds of Fagus sylvatica. 1206 Feb 71

Two different enzyme assays, both based on the interaction of native reverse transcriptase (RT) and 3'-azido-2',3'-deoxythymidine triphosphate (AZT-TP), were used to characterize the enzymes from 18 HIV-1 isolates with decreased sensitivity to AZT in cell culture. The first assay, which measures the balance between incorporation and excision of AZT monophosphate in the presence of dNTP substrate (in terms of IC(50)), gave an approx. 9-fold variation in sensitivity to AZT-TP. There was a correlation between the IC(50) values and the sensitivity of the corresponding virus to AZT in cell culture (r=0.60, P<0.01). The second assay, which was designed specifically for measurement of chain termination in the absence of dNTP substrate (as the concentration of AZT-TP giving 50% residual primer function, or CT(50)), revealed a more than 600-fold difference between the different isolate RTs. For the majority of enzymes there was a strict correlation between the results from the two assays; however, four isolates exhibited significantly higher CT(50)/IC(50) ratios than the other isolates. These differences were not related to sensitivity of the corresponding viruses to AZT but to the occurrence of certain mutations in their pol gene. The four deviating isolates contained either a minimum of four AZT-specific substitutions, including Thr-215-->Tyr (isolates 134 and 143), or some of the known specific substitutions combined with Thr-39-->Ala (isolates 80 and 157). The Thr-39-->Ala substitution has previously been recorded in connection with AZT/Foscarnet combination therapy.
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PMID:Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3'-azido-2',3'-deoxythymidine-resistant HIV isolates. 1207 93

Alterations in gene expression represent key events in carcinogenesis. We have studied HPV-induced cervical carcinogenesis, using an HPV-33-positive cell line (UT-DEC-1) established from a low-grade vaginal dysplasia (VAIN-I). Early-passage cells contained HPV-33 in episomal form, but these were superseded at later passages by cells carrying only integrated virus. To gain insight into the biologic significance of HPV integration, we compared the level of gene expression in normal vaginal keratinocytes, early-passage and late-passage UT-DEC-1 cells, using cDNA microarrays. Total RNA was isolated from cells by CsCl-gradient centrifugation, reverse-transcribed with MMLV reverse transcriptase and labeled with alpha-(32)P ATP. A cDNA microarray expression profile analysis was performed with Clontech's Human Cancer 1.2 cDNA expression array kit. The 16 upregulated genes (cut-off 2-fold), identified by comparing both cell types to control keratinocytes, appeared to support cell-cycle progression or to be functional in mitosis. These included, e.g., MCM4 DNA replication licensing factor, cdc2p34 and chromatin assembly factor 1 p48 subunit. Downregulated genes (44 altogether) interfered with apoptosis and cell adhesion, including the apoptosis-inducing genes FRAP, Bik and caspase-9 precursor. The most significant differences between the late and early passages (29 and 46 constantly up- and downregulated genes without any fluctuation) were overexpression of the transcription factors E2F5 with its dimerization partner DP1, NF-kappa B and serine/threonine kinases and underexpression of enzymes of the MAPK pathway. Acquisition of a selective growth advantage after viral integration might be explained by a major shift from a MAPK pathway to cell-cycle dysregulation (G(2)/M).
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PMID:Transcriptional profiling of a human papillomavirus 33-positive squamous epithelial cell line which acquired a selective growth advantage after viral integration. 1211 47

The synthesis of a protected form of the hypermodified nucleoside, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, (ms2t6A) is reported. The hypermodified nucleoside was subsequently elaborated to the protected nucleoside phosphophoramidite using a protecting group strategy compatible with standard RNA oligonucleotide chemistry. The phosphoramidite reagent was then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodon stem-loop (ASL) domain of human tRNA(Lys,3), the primer for HIV-1 reverse transcriptase. Introduction of the modification at position 37 of the tRNA ASL modestly decreases the thermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t6A nucleoside lacking the 2-methylthio substituent. 2D NOESY NMR spectra of the ms2t6A containing tRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in the solution structure of the analogous t6A containing E. coli tRNA(Lys), despite the presence of the bulky methylthio group. This synthetic approach allows for site-specific incorporation of the hypermodified nucleoside and should facilitate future studies directed at understanding the roles of nucleoside modification in modulating the stability and specificity of biologically important RNA-RNA interactions. Our synthesis of the ms2t6A containing RNAs demonstrates that this methodology is suitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.
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PMID:Synthesis of the tRNA(Lys,3) anticodon stem-loop domain containing the hypermodified ms2t6A nucleoside. 1212 27

Cystathionine-beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously described essential transactivating roles for specificity protein 1 (Sp1), Sp3, nuclear factor Y (NF-Y), and USF-1 in the regulation of the CBS-1b promoter. Differential binding of Sp1/Sp3 to the CBS-1b promoter due to differences in Sp1/Sp3 phosphorylation, and Sp1/Sp3 synergism with NF-Y might, in part, explain cell-specific patterns of CBS expression. In this report, the roles of various NF-YA isoforms in influencing cell-specific differences in CBS gene expression were determined in HT1080 and HepG2 cells. Seven unique NF-YA isoforms were detected in HT1080 by reverse transcriptase-PCR (RT-PCR) and DNA sequencing, characterized by deletions in the glutamine-rich and/or serine/threonine-rich domains. Only four of the seven NF-YA isoforms were found in HepG2 cells. The six alternatively spliced NF-YA isoforms all showed significantly less synergistic transactivation of the CBS-1b promoter with Sp1 than wild-type NF-YA, as determined by cotransfections in Drosophila SL2 cells with NF-YB and NF-YC. Further, all six alternatively spliced NF-YA isoforms inhibited the synergistic transactivation of the CBS-1b promoter by wild-type NF-Y and Sp1. Thus, the cellular distributions of these alternatively spliced NF-YA isoforms could impart an important cell-specific component to CBS transcriptional regulation, by virtue of their abilities to directly synergize with Sp1/Sp3 and interfere with transactivation of the CBS-1b promoter by wild-type NF-Y. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down's syndrome (DS).
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PMID:Synergistic regulation of human cystathionine-beta-synthase-1b promoter by transcription factors NF-YA isoforms and Sp1. 1242 42


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