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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of cytosolic phospholipase A2 (cPLA2) by TNF has been shown to be an important component of the signaling pathway leading to cell death. The role of cPLA2 in the cytotoxic action of TNF was investigated in a panel of human leukemic cell lines. TNF could activate cPLA2 only in U937 and HL60 TNF-sensitive leukemic cells, but not in KG1a, CEM, and CEM/VLB100 cells that are relatively resistant to TNF. Pretreatment with 4-bromophenacyl bromide, a cPLA2 inhibitor, rendered U937 and HL60 cell lines resistant to the cytotoxic effect of TNF. Immunoblot and reverse-transcriptase PCR demonstrated that cPLA2 expression was detectable at both transcriptional and translational levels in all leukemic cell lines studied, although CEM and CEM/VLB100 cells expressed cPLA2 mRNA and protein at lower levels. The protein synthesis inhibitor, cycloheximide, increased TNF-induced cPLA2 activity and cytotoxicity in both CEM and CEM/VLB100 cell lines. Low levels of cPLA2 activity in the KG1a cell line could be activated by the cPLA2 activator mellitin, or the calcium ionophore A23187. The data suggest that cPLA2 activity is involved in TNF-induced cytotoxicity in leukemic cells. Resistance to TNF-induced cytotoxicity may involve either protein inhibitors that act upstream of cPLA2 in the TNF-signaling pathway or constitutive defects of cPLA2 itself, possibly involving calcium utilization.
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PMID:Failure to activate cytosolic phospholipase A2 causes TNF resistance in human leukemic cells. 963 6

Oligodendrocytes in multiple sclerosis brain may be under a direct attack by proinflammatory cytokines, particularly tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma). In this study, we have examined the in vitro cytotoxic effects of the two cytokines, individually and in combination, on oligodendrocyte lineage cells using morphological criteria, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay (MTT), terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL), and agarose-gel electrophoretic analysis of fragmented DNA. IFNgamma exerted a dose-dependent cytotoxic effect on cultured CG4 cells, an oligodendrocyte progenitor cell line, and in primary cultures of purified oligodendrocyte progenitors. TNFalpha, while by itself being only mildly toxic, greatly potentiated the cytotoxicity of IFNgamma. The cytokine effects were developmentally modified in that their cytotoxic and cooperative effects became less evident in more differentiated cells. A cell-permeable peptide inhibitor (i.e., z-VAD.fmk) of caspases partially suppressed apoptotic changes elicited by the cytokine combination in CG4 cells but not in primary oligodendrocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of mRNA prepared from cytokine-treated cultures revealed an increased expression of the death receptor, Fas. The results suggest particular vulnerability of oligodendrocyte progenitors to a combination of TNFalpha and IFNgamma involving an activation of the cell death program.
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PMID:TNFalpha potentiates IFNgamma-induced cell death in oligodendrocyte progenitors. 984 48

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified the cis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous beta-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication.
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PMID:A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo. 1007 7

The anticancer drug, nitracrine, a 1-nitro-9-aminoalkyl derivative of acridine exhibits potent cytotoxic effects which are due to its metabolic activation, followed by covalent binding to macromolecules--DNA being the target for the drug. The renaturable fraction of DNA from L-1210 cells pretreated with nitracrine is assayed by means of ethidium bromide fluorescence assay and chromatography on hydroxyapatite column. The effect of the drug was compared with furocoumarins of different DNA crosslinking potencies. The existence of crosslinks in DNA upon incubation of cells with nitracrine (1-4 microM) have been confirmed with two different methods under the conditions where 8-methoxypsoralen, a classic crosslinking agent induced the renaturation. The DNA preparation isolated from the drug pretreated cells exhibited decreased transcriptional template activity with E. coli DNA-dependent RNA polymerase.
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PMID:Crosslinking of cellular DNA by nitracrine and furocoumarin derivatives. 1035 34

RNA replication of all positive-strand RNA viruses is closely associated with intracellular membranes. Brome mosaic virus (BMV) RNA replication occurs on the perinuclear region of the endoplasmic reticulum (ER), both in its natural plant host and in the yeast Saccharomyces cerevisiae. The only viral component in the BMV RNA replication complex that localizes independently to the ER is 1a, a multifunctional protein with an N-terminal RNA capping domain and a C-terminal helicase-like domain. The other viral replication components, the RNA polymerase-like protein 2a and the RNA template, depend on 1a for recruitment to the ER. We show here that, in membrane extracts, 1a is fully susceptible to proteolytic digestion in the absence of detergent and thus, a finding consistent with its roles in RNA replication, is wholly or predominantly on the cytoplasmic face of the ER with no detectable lumenal protrusions. Nevertheless, 1a association with membranes is resistant to high-salt and high-pH treatments that release most peripheral membrane proteins. Membrane flotation gradient analysis of 1a deletion variants and 1a segments fused to green fluorescent protein (GFP) showed that sequences in the N-terminal RNA capping module of 1a mediate membrane association. In particular, a region C-terminal to the core methyltransferase homology was sufficient for high-affinity ER membrane association. Confocal immunofluorescence microscopy showed that even though these determinants mediate ER localization, they fail to localize GFP to the narrow region of the perinuclear ER, where full-length 1a normally resides. Instead, they mediate a more globular or convoluted distribution of ER markers. Thus, additional sequences in 1a that are distinct from the primary membrane association determinants contribute to 1a's normal subcellular distribution, possibly through effects on 1a conformation, orientation, or multimerization on the membrane.
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PMID:Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes. 1171 27

The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E. coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E. coli RNA polymerase ("abortive initiation"); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter phi 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G x C) specificity (actinomycin D), the non-intercalating, d(A x T) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.
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PMID:Effects of anticancer drugs on transcription in vitro. 1172

A susceptibility of promoter DNA for adaptive conformational transitions has been studied using a cationic surfactant dodecyltrimethylammonium bromide (C(12)TAB) as a model DNA-binding ligand. DNAse 1 and KMnO(4) were utilized as structure-specific reagents. Both reagents revealed ligand-induced perturbations in the double helix of promoters T7A1 and T7D. These conformational transitions appeared to be strongly associated with pyrimidine-purine steps, which have non-random distribution within RNA polymerase contact region of the promoter DNA and are present in the binding sites for a majority of transcription regulation proteins. Potential flexibility of these elements creates therefore a specific media for transcription complex formation. Molecular mechanism of DNA interaction with C(12)TAB is discussed.
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PMID:Flexible elements in the structure of promoter DNA as probed by cationic surfactant binding. 1192 45

Drug resistance is a major cause of the failure of anticancer chemotherapy. Multidrug resistance is often caused by overexpression of the P-glycoprotein (Pgp) or the multidrug resistance-related protein (MRP). In the present study, we compared daunorubicin (DNR) accumulation, subcellular distribution, and the effect of modulators on drug accumulation and subcellular distribution in the Pgp-expressing K562 cell line and the MRP-expressing HL60 cell line using reverse-transcriptase polymerase chain reaction, MTT (3-[4, 5-dimethylthiazol-z-yl]-2,5-diphenyltetrazolium bromide) drug cytotoxicity assay, fluorocytometry, and confocal laser scanning microscopy. The 2 resistant cell lines exhibit similar levels of resistance to DNR and decreased drug accumulation. Altered drug subcellular distribution in the resistant cell lines compared to that in the sensitive cell lines was shown and, moreover, differences in drug distributions between the 2 resistant cell lines were found. DNR fluorescence in the resistant HL60 cell line was distributed into punctate regions in the cytoplasm; the nucleus and other cytoplasm were almost negative. In contrast, the resistant K562 cells showed a bright fluorescent signal located in the peripheral cytoplasm and perinuclear region; the nucleus and other cytoplasmic regions showed no signal. Use of the modulator verapamil increased drug accumulation and restored the altered subcellular distribution of the drug in the 2 resistant cell lines. The Golgi apparatus inhibitor brefeldin A had similar action in the resistant HL60 line but had little effect in the resistant K562 line. Therefore, our study suggested that there were differences between the 2 resistant cell lines in the compartments sequestering DNR.
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PMID:Comparison of Pgp- and MRP-mediated multidrug resistance in leukemia cell lines. 1193 61

Lipid/DNA complexes or Lipoplexes have been characterized by various biochemical and biophysical methods to understand the physical basis of transfection. Here we have addressed the effect of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), on transcription of DNA templates in vitro. Transcriptional activity of DNA-dependent RNA polymerase at DNA templates complexed with the cationic lipid varied as a function of charge ratio of lipid/DNA. At low charge ratios of 0.3:1 lipid/DNA and up to 1:1, we observed stimulation in transcription, while at higher charge ratios of lipid/DNA 3:1, complete inhibition in the activity occurred. Cetyl tri-methyl ammonium bromide (CTAB), a cationic detergent, and polyethylenimine (PEI), a cationic polymer, also bring about similar changes although to a lesser extent. The stimulation in transcription motivated us to probe into the molecular nature of the lipid/DNA interactions by absorbance spectroscopy and circular dichroism (CD). Upon interaction with lipids, hyperchromicity and susceptibility to micrococcal nuclease has increased, which suggests that the DNA was partially denatured. On complexation with the cationic lipid (DOTAP), the magnitude of the positive band in CD spectra decreased, accompanied with a red shift, as a function of charge ratio. Results from spectroscopic and enzyme assays suggest that at low charge ratios DNA may be partially unwound.
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PMID:Structural changes in DNA mediated by cationic lipids alter in vitro transcriptional activity at low charge ratios. 1249 16

Reverse transcriptase-polymerase chain reaction (RT-PCR) is a powerful, sensitive, and rapid method to monitor small amounts of nucleic acids. This is of particular interest for small amounts of cells, as in cartilage. We present here two protocols to isolate total RNA and a protocol to study matrix metalloproteinase and type II collagen gene expression from chondrocytes of human origin. Specific gene expression is revealed on an ethidium bromide-containing agarose gel on an ultraviolet plate and normalized to that of a housekeeping gene.
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PMID:Semiquantitative analysis of gene expression in cultured chondrocytes by RT-PCR. 1528 May 88


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