Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this work was to investigate the effect of c-fos antisense oligonucleotide (c-fos-AS-ON) on proliferative vitreoretinopathy (PVR). Cultures of human retinal pigment epithelial (hRPE) cells were established from adult human corneal donors. These cells were positively stained for cytokeratins. C-fos-AS-ON effect on serum-stimulated cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine (BrdU) into cellular DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were respectively performed to quantify the serum-stimulated c-fos gene mRNA and protein expression in hRPE cells. Eight rabbits (16 eyes) were divided into c-fos-AS-ON treatment group and control group. 2.5 x 10(5) cultured hRPE cells were injected into the vitreous cavity of eyes to establish a PVR model. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection and by pathological study on days 28 post-injection. The results showed that blocking the expression of c-fos by the addition of c-fos-AS-ON to the culture medium significantly inhibited the hRPE cells proliferation. This effect of c-fos-AS-ON was found to be sequence specific (the use of a sense or a mismatch sense oligonucleotide had no such an effect) and dose-dependent (0.375 microM was the lowest effective dose tested). Growth inhibition by c-fos-AS-ON remained for at least 72 h. By using RT-PCR and Western blotting, we found that the c-fos-AS-ON could specifically inhibit c-fos mRNA and protein synthesis in cultured hRPE cells. Though the eyes injected with c-fos-AS-ON also developed features of PVR, the severities of days 14, 21 and 28 post-injection were significantly lower than those in the control eyes (P<0.05). We conclude that c-fos-AS-ON can inhibit cultured hRPE cell proliferation, which mechanism may relate to blocking the expression of c-fos and can reduce the prevalence of experimental PVR. These findings establish a rationale for investigating the potential use of a c-fos-AS-ON as a novel therapeutical tool in the treatment of PVR.
 ...
PMID:Antisense oligonucleotide targeting c-fos mRNA limits retinal pigment epithelial cell proliferation: a key step in the progression of proliferative vitreoretinopathy. 1697 60

Discovery and development of effective antiviral agents to combat hepatitis C virus (HCV) is the focus of intensive research both in academia and in pharmaceutical companies. One of the HCV nonstructural proteins, NS5B (an RNA-dependent RNA polymerase), represents an attractive target in light of the clinical success of human immunodeficiency virus reverse transcriptase inhibitors. To identify and evaluate NS5B inhibitors, we developed a homogeneous, solid-phase, high-throughput biochemical assay for detecting NS5B enzymatic activity. In this assay, a biotinylated oligo(dT(12)) primer was immobilized onto streptavidin-coated scintillation proximity assay (SPA) beads, and after addition of homopolymeric A template, NS5B enzyme, and radiolabeled uridine 5'-triphosphate, the primer-dependent RNA synthesis occurred on beads. Optimization of the on-bead reaction resulted in the use of significantly less RNA template and NS5B enzyme while producing a faster steady state reaction rate compared to the solution-phase or off-bead SPA. The newly developed solid-phase assay is functionally comparable to the solution-phase assay as similar potencies of HCV NS5B inhibitors tested were obtained with the two assays. Furthermore, the solid-phase assay offers the advantage of delaying initiation, mimicking a physical preincubation step required for evaluating time-dependent inhibitors.
 ...
PMID:A homogeneous, solid-phase assay for hepatitis C virus RNA-dependent RNA polymerase. 1705 98

RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain 'natural' pyrimidine bases, but possess a beta-methyl substituent at the 2'-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA+), single-stranded negative (ssRNA-) or double-stranded (dsRNA), revealed potent activities for D-2'-C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5'-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2'-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development.
 ...
PMID:2'-C-Methyl branched pyrimidine ribonucleoside analogues: potent inhibitors of RNA virus replication. 1790 80

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5'-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5'-triphosphate of the uridine congener, beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5'-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.
 ...
PMID:The mechanism of action of beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine involves a second metabolic pathway leading to beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine 5'-triphosphate, a potent inhibitor of the hepatitis C virus RNA-dependent RNA polymerase. 1799 67

Pseudouridine, the so-called fifth nucleoside due to its ubiquitous presence in ribonucleic acids (RNAs), remains among the most challenging modified nucleosides to characterize. As an isomer of the major nucleoside uridine, pseudouridine cannot be detected by standard reverse-transcriptase-based DNA sequencing or RNase mapping approaches. Thus, over the past 15 years, investigators have focused on the unique structural properties of pseudouridine to develop selective derivatization or fragmentation strategies for its determination. While the N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate (CMCT)-reverse transcriptase assay remains both a popular and powerful approach to screen for pseudouridine in larger RNAs, mass-spectrometry-based approaches are poised to play an increasingly important role in either confirming the findings of the CMCT-reverse transcriptase assay or in characterizing pseudouridine sequence placement and abundance in smaller RNAs. This review includes a brief discussion of pseudouridine including a summary of its biosynthesis and known importance within various RNAs. The review then focuses on chemical derivatization approaches that can be used to selectively modify pseudouridine to improve its detection, and the development of mass-spectrometry-based assays for the identification and sequencing of pseudouridine in various RNAs.
 ...
PMID:Mass spectrometry of the fifth nucleoside: a review of the identification of pseudouridine in nucleic acids. 1862 Sep 15

To elucidate the pharmacological profile of P2X receptors and the probable expression of P2Y receptors in otic ganglion neurons from 17-day-old rats, single neurons were enzymatically isolated and maintained in tissue culture for up to 30 h. Whole-cell voltage-clamp recording was carried out at a holding potential of -60 mV. Most otic ganglion neurons responded to adenosine 5'-triphosphate (ATP), 2-methylthio ATP (2-MeSATP) and alpha,beta-methylene ATP (alphabeta-meATP) with sustained currents and EC(50) values of 19 microM, 47 microM and 94 microM, respectively. 2',3'-O-trinitrophenyl-ATP (TNP-ATP) inhibited the response to alphabeta-meATP and ATP with an IC(50) values of 3.9 nM and 18.3 nM, respectively, which was closed to that observed in nodose neurons. The response to ATP was antagonized by suramin and cibacron blue. The dose-response curve of suramin against ATP response at a pH of 6.5 was shifted to the left compared to that at a pH of 7.4. Diinosine pentaphosphate (Ip(5)I), which blocks P2X(3), but not P2X(2/3)-mediated responses, had no effect on the currents evoked by ATP or alphabeta-meATP. In some neurons, uridine 5'-triphosphate (UTP) induced a tiny, but long-lasting current with a mean amplitude of 0.034+/-0.011 nA. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the expression of mRNAs for P2X(2), P2X(3), P2X(4), P2X(6) and P2X(7), but not for P2X(1) and P2X(5) receptors in otic ganglion. In conclusion, in rat otic ganglion neurons, P2X(2/3) heteromultimer receptors dominate, but P2X(7) and P2Y(2) or P2Y(4) receptors also play roles.
 ...
PMID:Pharmacological properties of P2 receptors on rat otic parasympathetic ganglion neurons. 1862 50

The 2 M LiCl-soluble RNA fraction extracted from tobacco mosaic virus (TMV)-infected tobacco plants contains, in addition to the viral replicative form of 4 x 10(6) MW, three smaller double-stranded (ds) RNA species with apparent molecular weights (estimated by polyacrylamide gel electrophoresis, using ds RNAs as markers) of 2.25, 1.1, and 0.23 x 10(6). The synthesis of all four ds RNAs is insensitive to actinomycin D. They are completely RNase insensitive at high salt concentrations and are found both in directly inoculated and in apical tissues. In tissues incubated in the presence of 3H-uridine and actinomycin D, the three small ds RNAs accounted for 6 to 11.5% of the total radioactivity incorporated into viral ds RNA. On a molar basis, however, in apical leaves the smallest ds RNA was synthesized to almost the same level as the replicative form. By molecular hybridization, the three small ds RNAs have been shown to be of viral origin, and each contains sequences represented in the 5' end of complementary (negative strand) TMV RNA. Based on molecular weight data, none of the ds RNAs can be considered to be a ds form of the subgenomic TMV coat protein mRNA (the LMC), suggesting that it is not replicated independently. None of the small ds RNAs was found to be an endogenous product of the bound TMV RNA replicase.
 ...
PMID:The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants. 1863 83

The brain efflux index (BEI), a measurement of blood-brain barrier (BBB) efflux transport, was estimated at 15 s, 30 s, 1 min, 3 min and 10 min after intracerebral injection of [14C]pyrimidines. An initial steep increase of the BEI values over time was observed for [14]uracil and [14C]thymine, followed by a more moderate increase after 1 min. For the corresponding nucleosides, [14C]uridine and [14C]thymidine, the increase of BEI values over time was less steep and linear between 30 s and 3 min. The apparent BBB efflux clearances for [14C]uridine, [14C]thymidine, [14C]uracil and [14C]thymine were (microl/min/g): 95.2 +/- 12.1, 125.3 +/- 18.4, 290.4 +/- 28 and 358.5 +/- 32.5, respectively, which is at least several folds higher than the predicted BBB influx clearances of uridine, uracil and thymidine. Quick depletion of brain parenchyma from brain microvasculature has revealed that [14C] radioactivity accumulated in brain microvessels after injection of nucleosides [14C]thymidine and [14C]uridine, but that was not observed when nucleobases, [14C]thymine and [14C]uracil, were injected. Reverse transcriptase-PCR revealed that the rat brain and liver (positive control) express dihydropyrimidine dehydrogenase, a key enzyme in pyrimidine nucleobase catabolism. Two bands representing spliced variants have been detected with the relative density of the bands (expressed relative to the density of glyceraldehyde3-phosphate dehydrogenase bands, mean +/- SEM from 3 separate samples) 0.16 +/- 0.06 and 0.04 +/- 0.01 (brain) and 0.49 +/- 0.1 and 0.07 +/- 0.01 (liver). Overall, these results indicate that the net direction of pyrimidine BBB transport is the efflux transport; rapid BBB efflux transport and metabolic breakdown of pyrimidine nucleobases appear to be important for brain homeostasis.
 ...
PMID:Blood-brain barrier efflux transport of pyrimidine nucleosides and nucleobases in the rat. 1875 95

Our understanding of picornavirus RNA replication has improved over the past 10 years, due in large part to the discovery of cis-active RNA elements (CREs) within picornavirus RNA genomes. CREs function as templates for the conversion of VPg, the Viral Protein of the genome, into VPgpUpU(OH). These so called CREs are different from the previously recognized cis-active RNA sequences and structures within the 5' and 3' NTRs of picornavirus genomes. Two adenosine residues in the loop of the CRE RNA structures allow the viral RNA-dependent RNA polymerase 3D(Pol) to add two uridine residues to the tyrosine residue of VPg. Because VPg and/or VPgpUpU(OH) prime the initiation of viral RNA replication, the asymmetric replication of viral RNA could not be explained without an understanding of the viral RNA template involved in the conversion of VPg into VPgpUpU(OH) primers. We review the growing body of knowledge regarding picornavirus CREs and discuss how CRE RNAs work coordinately with viral replication proteins and other cis-active RNAs in the 5' and 3' NTRs during RNA replication.
 ...
PMID:Cis-active RNA elements (CREs) and picornavirus RNA replication. 1877 30

RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNAs) and microRNAs, several types of endogenously produced small RNAs have important roles in gene regulation, germ cell maintenance and transposon silencing. The production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases to make double-stranded RNA. The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a new class of small RNAs in the filamentous fungus Neurospora crassa. This class of small RNAs, known as qiRNAs because of their interaction with QDE-2, are about 20-21 nucleotides long (several nucleotides shorter than Neurospora siRNAs), with a strong preference for uridine at the 5' end, and originate mostly from the ribosomal DNA locus. The production of qiRNAs requires the RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3 and dicers. qiRNA biogenesis also requires DNA-damage-induced aRNAs as precursors, a process that is dependent on both QDE-1 and QDE-3. Notably, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. Furthermore, the Neurospora RNA interference mutants show increased sensitivity to DNA damage, suggesting a role for qiRNAs in the DNA-damage response by inhibiting protein translation.
 ...
PMID:qiRNA is a new type of small interfering RNA induced by DNA damage. 1944 17


<< Previous 1 2 3 4 5 Next >>