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)

Monocyte chemotactic protein-1 (MCP-1) is a proinflammatory cytokine that attracts and activates specific types of leucocytes. The purpose of this work was to analyse the generation of MCP-1 and mRNA transcript in a model of chronic inflammation using a granulomatous tissue induced by potassium permanganate (KMnO4; water soluble crystals). The data presented here shows that MCP-1 is generated in granuloma tissue and its level was strongly increased by i.p. injections of lipopolysaccharide (LPS) and inhibited in rats treated with injections of dexamethasone, 18 hr before the animals were killed. In histological studies LPS and dexamethasone increased and decreased, respectively, the recruitment of mononuclear cells in the granuloma tissue compared with the control granulomas from phosphate-buffered saline (PBS)-treated animals. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for mRNA extraction and cDNA synthesis. mRNA MCP-1 was significantly produced in the granuloma tissue of untreated animals, an effect increased by LPS and inhibited by dexamethasone, compared with the controls. Moreover, MCP-1 protein was found in the supernatant from homogenized granuloma tissues and the levels of MCP-1 were higher in the LPS-treated animals, while they were lower in the dexamethasone group, compared with the granulomas from the PBS-treated groups (control). The generation of MCP-1 was also found in minced granuloma tissue incubated for 18 hr (overnight) from treated (LPS or dexamethasone) and untreated (PBS) rats. When LPS was added in vitro for 18 hr to the controls and treated animals the production of MCP-1 was further increased except in the dexamethasone group (P > 0.05). Analysing blood serum from LPS, dexamethasone or PBS-treated rats, we found that MCP-1 was also present. The level was higher in the LPS group and lower in the dexamethasone group, compared with the control (PBS). In these studies we show for the first time that MCP-1 transcript and translation is generated in chronic experimental inflammatory tissue, an effect inhibited by dexamethasone.
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PMID:Augmentation of monocyte chemotactic protein-1 and mRNA transcript in chronic inflammatory states induced by potassium permanganate (KMnO4) in vivo. 941 40

1. The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. 2. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. 4. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. 5. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6-22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. 6. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in PC12 cells and possibly other oxygen-sensitive cells during hypoxia.
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PMID:Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor. 949 Aug 23

We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >>+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.
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PMID:HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. 950 Dec 1

1. Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization-activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N-methyl-D-glucamine) or by addition of 10 mM caesium or barium ions. 2. The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl- > Br- > I- > cyclamate. All substituted permeant anions also blocked the current. 3. The current was blocked by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), 9AC (anthracene-9-carboxylic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) but was unaffected by SITS (4-acetamido-4'-isothiocyanatostilbene- 2,2'-disulphonic acid) and niflumic acid. The effective blockers were voltage dependent; DIDS and NPPB were more effective at depolarized potentials while 9AC was more effective at hyperpolarized potentials. 4. The current was enhanced by extracellular acidification and reduced by extracellular alkalinization. Reducing external osmolarity was without effect in conventional whole-cell recording but enhanced current amplitude in those perforated-patch recordings where little current was evident in control external solution. 5. The current in SCG neurons was blocked by external cadmium and zinc. ClC-2 chloride currents expressed in Xenopus oocytes were also sensitive to block by these divalent ions and by DIDS but the sensitivity of ClC-2 to block by cadmium ions was lower than that of the current in SCG neurons. 6. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed the presence of mRNA for ClC-2 in SCG neurons but not in rat cerebellar granule cells which do not possess a hyperpolarization-activated Cl- current. 7. The data suggest that ClC-2 may be functionally expressed in rat SCG neurons. This current may play a role in regulating the internal chloride concentration in these neurons and hence their response to activation of GABAA receptors.
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PMID:Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons. 950 29

Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify sequences for delayed rectifier potassium (drk) channel transcripts in Xenopus laevis inner ear and brain. We used degenerate primers that spanned a region between the N-terminal cytoplasmic portion and a region located between the S2 and S3 transmembrane domains of the potassium channel protein. When inner ear total RNA or brain mRNA was used as a template for RT-PCR, a unique product of the expected size (approximately 560 bp) was observed as a single band after electrophoresis on agarose gels. The PCR product from reactions using X. laevis genomic DNA as template was similarly sized, indicating a lack of introns in this region. The RT-PCR products from inner ear and brain were isolated, cloned, and sequenced. Sequence analysis showed that the X. laevis inner ear and brain clones were identical. Sequence alignments of the cloned RT-PCR products with posted GenBank sequences established that the drk sequences from X. laevis inner ear and brain share highest identity with larval X. laevis brain, mouse, rat, and human Kv2 sequences. Positive signals were obtained from inner ear and brain mRNA in Northern dot blots hybridized with digoxigenin labeled probes from the inner ear clone. Taken together, results provide evidence for the expression of Kv2 sequences in the X. laevis inner ear and brain.
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PMID:Detection of transcripts for delayed rectifier potassium channels in the Xenopus laevis inner ear. 964 25

The most potent promoters in the ddlB-ftsA region of the dcw cluster have been analysed for sigmaS-dependent transcription. Only the gearbox promoter ftsQ1p was found to be transcribed in vitro by RNA polymerase holoenzyme coupled to sigmaS (EsigmaS). This dependency on sigmaS was also found in vivo when single-copy fusions to a reporter gene were analysed in rpoS and rpoS+ backgrounds. Although ftsQ1p can be transcribed by RNA polymerase containing either sigmaD or sigmaS, there is a preference for EsigmaS when the assay conditions include potassium glutamate and supercoiled templates, a property shared with the bolA1p gearbox promoter. The rest of the promoters assayed, ftsQ2p and ftsZ2p3p4p, similarly to the control bolA2p promoter, were preferentially transcribed by EsigmaD, the housekeeper polymerase. The ftsQ1p and the bolA1p promoters also share the presence of AT-rich sequences upstream of the - 35 region and the requirement for an intact wild-type alpha-subunit for a proficient transcription, allowing their joint classification as gearboxes.
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PMID:The ftsQ1p gearbox promoter of Escherichia coli is a major sigma S-dependent promoter in the ddlB-ftsA region. 979 Nov 85

We have studied the role of the C-terminal domain of the alpha subunit (alphaCTD) of Escherichia coli RNA polymerase during transcription initiation at promoters lacking an UP-element. The temperature requirement for open complex formation was used as an indication of the kinetics of this process. We have previously shown that alphaCTD is required for transcription initiation at low temperature at the galP1 promoter, a promoter containing an UP-element. DNase I footprinting has been used to reveal the structure of open promoter complexes and the temperature requirement for open complex formation has been determined using potassium permanganate as a probe. In this work we show that, although alphaCTD is not absolutely required for transcription initiation at promoters lacking an UP-element, it does play a role during transcription initiation. This role is independent of the sequence of the promoter upstream from the -35 region and does not require stable alphaCTD-DNA interactions as determined by DNase I footprinting. The role of alphaCTD at promoters lacking an UP-element is discussed.
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PMID:Open complex formation during transcription initiation at the Escherichia coli galP1 promoter: the role of the RNA polymerase alpha subunit at promoters lacking an UP-element. 1019 40

Transcription is the fundamental process by which RNA is synthesized by RNA polymerases on double-stranded DNA templates. One structurally simple RNA polymerase is encoded by bacteriophage T7. T7 RNA polymerase is an excellent candidate for studying structural aspects of transcription, because unlike the eucaryotic and bacterial RNA polymerases, it is a single subunit enzyme and does not require additional factors to carry out the entire process of transcription from start to finish. An important advantage of studying transcription using this enzyme is that the high-resolution crystal structure of T7 RNA polymerase has been solved. However, a cocrystal structure of the polymerase complexed with promoter has not yet been published. Here, we have used cross-linking techniques to understand the interaction of promoter with T7 RNA polymerase. We constructed promoters that were substituted with the photo-cross-linkable nucleotide 5-iodo uracil at every dT in the promoter from -17 to -1. This substitution replaces the 5-methyl in dT with an iodine atom. The substituted promoters were photo-cross-linked to T7 RNAP, and the efficiency of cross-linking was quantitated at every position. In the melting domain, the strongest contacts occurred at -3 and at -1 on the template strand while very weak cross-linking was seen at -2 and at -4 on the nontemplate strand. In the binding domain, the strongest contacts were seen at -16, -15, and -13 and at -10 on the template strand while at -17 and -14 on the nontemplate strand very weak cross-linking was observed. Cross-linking was poor in the intervening region between the binding and the melting domains. These results suggested that, in the T7 RNA polymerase-promoter complex, the polymerase molecule mainly contacts the template bases in the TATA box while the upstream contacts are used as an anchor for DNA binding. For a systematic study designed to probe the nature of base-specific interactions in the polymerase-promoter complex, we used neutral salts from the Hofmeister series. In general, the order of perturbation was sulfate > citrate > acetate for anions and ammonium > magnesium > potassium for cations. Using acrylamide, a neutral hydrophobic agent to probe for nonionic contacts, we observed that at -2, -4, and -17 the contacts had a hydrophobic component, while at many other positions there was no significant effect, suggesting that the contacts in the promoter-polymerase complexes were predominantly ionic but at certain positions nonionic interactions also existed. To localize a specific interaction in the melting domain, we proteolyzed the cross-linked T7 RNAP and analyzed the fragments using gel electrophoresis, mass spectrometry, and amino acid composition. High-resolution mapping indicated that amino acid residues 614-627 may be in the vicinity of the melting domain. Specifically, Y623 may contact -3 on the template strand.
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PMID:Probing the interaction of T7 RNA polymerase with promoter. 1021 99

The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T(2)) of sporulation in wild-type cells and in spoIIIG (SigG(-)) and spoIVCB (SigK(-)) mutants but not in spoIIAC (SigF(-)) and spoIIGAB (SigE(-)) mutants. The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced from T(2) of sporulation in a B. subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein. Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yaaH mutant spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination. These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T(2) of sporulation and that it is required for the L-alanine-stimulated germination pathway.
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PMID:The Bacillus subtilis yaaH gene is transcribed by SigE RNA polymerase during sporulation, and its product is involved in germination of spores. 1041 57

Cultured sympathetic neurones are depolarized and release noradrenaline in response to extracellular ATP, UDP and UTP. We examined the possibility that, in neurones cultured from rat thoracolumbar sympathetic ganglia, inhibition of the M-type potassium current might underlie the effects of UDP and UTP. Reverse transcriptase-polymerase chain reaction indicated that the cultured cells contained mRNA for P2Y(2)-, P2Y(4)- and P2Y(6)-receptors as well as for the KCNQ2- and KCNQ3-subunits which have been suggested to assemble into M-channels. In cultures of neurones taken from newborn as well as from 10 day-old rats, oxotremorine, the M-channel blocker Ba(2+) and UDP all released previously stored [(3)H]-noradrenaline. The neurones possessed M-currents, the kinetic properties of which were similar in neurones from newborn and 9 - 12 day-old rats. UDP, UTP and ATP had no effect on M-currents in neurones prepared from newborn rats. Oxotremorine and Ba(2+) substantially inhibited the current. ATP also had no effect on the M-current in neurones prepared from 9 - 12 day-old rats. Oxotremorine and Ba(2+) again caused marked inhibition. In contrast to cultures from newborn animals, UDP and UTP attenuated the M-current in neurones from 9 - 12 day-old rats; however, the maximal inhibition was less than 30%. The results indicate that inhibition of the M-current is not involved in uracil nucleotide-induced transmitter release from rat cultured sympathetic neurones during early development. M-current inhibition may contribute to release at later stages, but only to a minor extent. The mechanism leading to noradrenaline release by UDP and UTP remains unknown.
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PMID:M-type K+ currents in rat cultured thoracolumbar sympathetic neurones and their role in uracil nucleotide-evoked noradrenaline release. 1068 96


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