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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycolipid transfer protein (GLTP) is a small (23-24 kDa), basic protein (pI congruent with 9.0) that accelerates the intermembrane transfer of various glycolipids. Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs. The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR. The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR. The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation. The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine. Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein. Regulation of growth and induction conditions led to approximately 50% of expressed fusion protein being soluble and active. Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP. Northern blot analyses of bovine tissues showed a single transcript of approximately 2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen congruent with lung congruent with cerebellum > liver > heart muscle. Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.
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PMID:Cloning and expression of glycolipid transfer protein from bovine and porcine brain. 1067 54

In bacteria, the known pathways for diaminopimelate (DAP) and lysine biosynthesis share two key enzymes, dihydrodipicolinate synthase and dihydrodipicolinate reductase, encoded by the dapA and dapB genes, respectively. In rhizobia, these genes have not yet been genetically characterized. In this work, by sequence analysis, we identified two divergent open reading frames on the 140-MDa plasmid pRmeGR4b of Sinorhizobium meliloti strain GR4. Termed dapA and dapB, these encode products which show significant sequence similarities to DapA and DapB proteins, respectively. Escherichia coli DAP auxotrophs (dapA and dapB mutants) could be complemented with the pRmeGR4b dapA and dapB genes, indicating that these genes code for functional dihydrodipicolinate synthase and dihydrodipicolinate reductase, respectively. Reverse-transcriptase PCR analyses and beta-galactosidase assays using transcriptional dapA-lacZ and dapB-lacZ fusions suggest that these genes are constitutively expressed in S. meliloti. The dapA and dapB genes are not widely distributed in S. meliloti and appear to be specific for strains carrying pRmeGR4b-type plasmids.
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PMID:Characterization of the Sinorhizobium meliloti genes encoding a functional dihydrodipicolinate synthase (dapA) and dihydrodipicolinate reductase (dapB). 1089 25

Gene therapy strategies for the prevention of restenosis postangioplasty are promising. Nonviral gene transfer to the arterial wall in vivo has so far been limited by poor efficiency. This study aimed to optimize transfection of primary vascular smooth muscle cells using cationic nonviral formulations based on cholesterol derivates (DC-, DAC-, DCQ-, and Sp-Chol), double-chained amphiphils (LipofectAMINE, DOTMA, DOSGA, DOSPER, and DOCSPER), or heterogeneous reagents (Superfect, Effectene, and Tfx-50). Estimation of transfection efficiencies was performed using galactosidase assays at different ratios of transfection reagent to plasmid DNA with reporter gene. Toxicity was monitored by analyzing cell metabolism. Transfer efficiency and safety were determined in a porcine restenosis model for local gene therapy using morphometry, histology, galactosidase assays, and reverse-transcriptase polymerase chain reaction. The highest in vitro transfection efficiency was achieved using the recently developed DOCSPER liposomes, with transfer rates of at least 20% in vascular smooth muscle cells. Transfer efficiency was further enhanced up to 20% by complexing with poly-L-lysine. Transfection efficiency in vivo in a porcine restenosis model was up to 15% of adventitial cells using DOCSPER versus 0.1% using LipofectAMINE. Toxicity in vivo and in vitro was lowest using DOCSPER. Increased biological effects were demonstrated following optimization of transfer conditions.
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PMID:Optimization of nonviral gene transfer of vascular smooth muscle cells in vitro and in vivo. 1093 55

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

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).
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PMID:Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture. 1208 28

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNA-dependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 A contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the Lambda 1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive pre-initiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template.
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PMID:Modulation of hepatitis C virus RNA-dependent RNA polymerase activity by structure-based site-directed mutagenesis. 1214 89

Eukaryotic heterochromatin is characterized by a high density of repeats and transposons, as well as by modified histones, and influences both gene expression and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, we deleted the argonaute, dicer, and RNA-dependent RNA polymerase gene homologs, which encode part of the machinery responsible for RNA interference (RNAi). Deletion results in the aberrant accumulation of complementary transcripts from centromeric heterochromatic repeats. This is accompanied by transcriptional de-repression of transgenes integrated at the centromere, loss of histone H3 lysine-9 methylation, and impairment of centromere function. We propose that double-stranded RNA arising from centromeric repeats targets formation and maintenance of heterochromatin through RNAi.
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PMID:Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. 1219 43

In plants, animals and fungi, active centromeres are associated with arrays of repetitive DNA sequences. The outer repeats at fission yeast (Schizosaccharomyces pombe) centromeres are heterochromatic and are required for the assembly of an active centromere. Components of the RNA interference (RNAi) machinery process transcripts derived from these repeats and mediate the formation of silent chromatin. A subfragment of the repeat (dg) is known to induce silencing of marker genes at euchromatic sites and is required for centromere formation. We show that the RNAi components, Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1), are required to maintain silencing, lysine 9 methylation of histone H3 and association of Swi6 via this dg ectopic silencer. Deletion of Ago1, Dcr1 or Rdp1 disrupts chromosome segregation leading to a high incidence of lagging chromosomes on late anaphase spindles and sensitivity to a microtubule poison. Analysis of dg transcription indicates that csp mutants, previously shown to abrogate centromere silencing and chromosome segregation, are also defective in the regulation of non-coding centromeric RNAs. In addition, histone H3 lysine 9 methylation at, and recruitment of Swi6 and cohesin to, centromeric repeats is disrupted in these mutants. Thus the formation of silent chromatin on dg repeats and the development of a fully functional centromere is dependent on RNAi.
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PMID:RNA interference is required for normal centromere function in fission yeast. 1273 40

Although randomized trials have shown enhancement of efficacy for combination therapy with interferon (IFN) alpha-2b and ribavirin compared with IFN monotherapy as first-line treatment for chronic hepatitis C, infection with genotype 1b and high viremia are still associated with significantly low response rates compared with non-1 genotypes and low viremia. We analysed amino acid sequences of the viral RNA-dependent RNA polymerase (RdRP) or nonstructural protein 5B (NS5B), responsible for ribavirin misincorporation into RNA products in patients with genotype 1b-related chronic hepatitis C and high viremia, and examined the relationship between such RdRp polymorphisms, and the initial decline in viral load induced by combination therapy with IFN-alpha and ribavirin. Substitution of glutamic acid to lysine at the 124th position (E124K) and of isoleucine to valine at the 85th position (I85V) were found to be closely associated with a potent decline of viral load and viral clearance at 8 weeks of treatment (five of five patients, coincidence rate 100%). In conclusion, our results suggest that the polymorphisms of E124K and I85V identified in NS5B protein are crucial for early viral clearance in patients with genotype 1b and high viremia by combination therapy with IFN and ribavirin, and that detection of amino acid sequence motifs might enable prediction of clinical efficacy.
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PMID:Polymorphisms of NS5B protein relates to early clearance of hepatitis C virus by interferon plus ribavirin: a pilot study. 1511 24

Retinoids have shown significant activities in cancer prevention and therapy. Many of their effects are mediated by nuclear retinoid receptors including retinoic acid receptors (RARs alpha, beta and gamma) and retinoid X receptors (RXRs alpha, beta and gamma). Human retinoic acid receptor beta (RARbeta) has three different isoforms: beta1, beta2 and beta4. The tumor suppressive characteristics of RARbeta2, its silencing by promoter hypermethylation, and its reexpression following demethylation have been reported. In contrast, RARbeta1, an embryonic isoform with restricted expression in adult tissues has been linked to carcinogenesis. However, factors regulating RARbeta1 expression have not yet been clarified. During studies on the head and neck squamous cell carcinoma cells, we found that the expression of RARbeta increased in cells grown to high density. Real-time reverse-transcriptase polymerase chain reaction revealed that the isoform increased in these cells was RARbeta1. Epigenetic modifications of this isoform were tested using combined bisulfite restriction analysis and chromatin immunoprecipitation assays. The UMSCC38 cell line showed significant RARbeta1 expression (p < 0.001), which was dependent on cell density and culture duration. The increased expression of RARbeta1 was not due to demethylation of its promoter. However, higher cell densities were associated with increased acetylation of histone 3 at lysine 9 in RARbeta1 but not in RARbeta2. These findings reveal that the expression of RARbeta1 is regulated by cell density through changes in histone acetylation.
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PMID:Regulation of RARbeta1 expression in head and neck cancer cells by cell density-dependent chromatin remodeling. 1546 35


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