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)

The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm-1 shift from 1306 cm-1 in aqueous solution to 1314 cm-1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm-1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm-1; in water, a strong hydrogen-bonding solvent, 1306 cm-1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm-1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.
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PMID:A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase. 388 50

To determine which alpha 2-adrenergic receptor subtypes are present in primary afferent and sympathetic postganglionic neurons we have performed in situ hybridization and immunohistochemistry experiments on rat dorsal root and superior cervical ganglia. Reverse transcriptase polymerase chain reaction was used as a preliminary screen for the presence of mRNA encoding alpha 2-adrenergic subtypes in dorsal root and superior cervical ganglia; polymerase chain reaction primers amplified distinct regions of the rat alpha 2A-(RG20), alpha 2B-(RNG) and alpha 2C-(RG10) adrenergic receptor subtypes in mRNA extracted from lumbar dorsal root and superior cervical ganglia. To localize receptors to cell types in the ganglia, in situ hybridization was performed on cryosections of dorsal root and superior cervical ganglia with oligonucleotide probes designed to distinguish between mRNA encoding for alpha 2-adrenergic receptor subtypes. Immunohistochemistry was performed with a polyclonal antibody against the alpha 2A-adrenergic receptor subtype. Our results with reverse transcriptase polymerase chain reaction indicate that all three alpha 2-adrenergic receptor subtypes are expressed in dorsal root and superior cervical ganglia. Data from the in situ hybridization experiments indicated that the mRNA detected with the reverse transcriptase polymerase chain reaction was present in neuronal cell bodies, except for the mRNA encoding the alpha 2A-adrenergic receptor which was not detectable in dorsal root ganglia. The distribution of mRNA encoding alpha 2B- and alpha 2C-adrenergic receptor subtypes among dorsal root ganglion neurons and alpha 2A-, alpha 2B- and alpha 2C-adrenergic receptor subtypes among superior cervical ganglion neurons suggests that multiple adrenergic receptor subtypes are present in a single neuron. Neuronal cell bodies in both the dorsal root and superior cervical ganglion consistently demonstrated alpha 2A-adrenergic receptor-like immunoreactivity. The apparent co-expression of multiple alpha 2-adrenergic receptor subtypes in dorsal root and superior cervical ganglion neurons enables a single transmitter to produce a number of effects in the same neuron; which receptors are functionally active may vary with the presence of nerve injury, inflammation or other physiological and pathophysiological conditions.
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PMID:Alpha 2-adrenergic receptor subtypes in rat dorsal root and superior cervical ganglion neurons. 906 29

Induction of hepatic phase I/II detoxification enzymes and alternative excretory pumps may limit hepatocellular accumulation of toxic biliary compounds in cholestasis. Because the nuclear xenobiotic receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate involved enzymes and transporters, we aimed to induce adaptive alternative pathways with different CAR and PXR agonists in vivo. Mice were treated with the CAR agonists phenobarbital and 1,4-bis-[2-(3,5-dichlorpyridyloxy)]benzene, as well as the PXR agonists atorvastatin and pregnenolone-16alpha-carbonitrile. Hepatic bile acid and bilirubin-metabolizing/detoxifying enzymes (Cyp2b10, Cyp3a11, Ugt1a1, Sult2a1), their regulatory nuclear receptors (CAR, PXR, farnesoid X receptor), and bile acid/organic anion and lipid transporters (Ntcp, Oatp1,2,4, Bsep, Mrp2-4, Mdr2, Abcg5/8, Asbt) in the liver and kidney were analyzed via reverse-transcriptase polymerase chain reaction and Western blotting. Potential functional relevance was tested in common bile duct ligation (CBDL). CAR agonists induced Mrp2-4 and Oatp2; PXR agonists induced only Mrp3 and Oatp2. Both PXR and CAR agonists profoundly stimulated bile acid-hydroxylating/detoxifying enzymes Cyp3a11 and Cyp2b10. In addition, CAR agonists upregulated bile acid-sulfating Sult2a1 and bilirubin-glucuronidating Ugt1a1. These changes were accompanied by reduced serum levels of bilirubin and bile acids in healthy and CBDL mice and by increased levels of polyhydroxylated bile acids in serum and urine of cholestatic mice. Atorvastatin significantly increased Oatp2, Mdr2, and Asbt, while other transporters and enzymes were moderately affected. In conclusion, administration of specific CAR or PXR ligands results in coordinated stimulation of major hepatic bile acid/bilirubin metabolizing and detoxifying enzymes and hepatic key alternative efflux systems, effects that are predicted to counteract cholestasis.
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PMID:CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice. 1602 8

The TodS and TodT proteins form a previously unrecognized and highly specific two-component regulatory system in which the TodS sensor protein contains two input domains, each of which are coupled to a histidine kinase domain. This system regulates the expression of the genes involved in the degradation of toluene, benzene, and ethylbenzene through the toluene dioxygenase pathway. In contrast to the narrow substrate range of this catabolic pathway, the TodS effector profile is broad. TodS has basal autophosphorylation activity in vitro, which is enhanced by the presence of effectors. Toluene binds to TodS with high affinity (Kd = 684 +/- 13 nM) and 1:1 stoichiometry. The analysis of the truncated variants of TodS reveals that toluene binds to the N-terminal input domain (Kd = 2.3 +/- 0.1 microM) but not to the C-terminal half. TodS transphosphorylates TodT, which binds to two highly similar DNA binding sites at base pairs -107 and -85 of the promoter. Integration host factor (IHF) plays a crucial role in the activation process and binds between the upstream TodT boxes and the -10 hexamer region. In an IHF-deficient background, expression from the tod promoter drops 8-fold. In vitro transcription assays confirmed the role determined in vivo for TodS, TodT, and IHF. A functional model is presented in which IHF favors the contact between the TodT activator, bound further upstream, and the alpha-subunit of RNA polymerase bound to the downstream promoter element. Once these contacts are established, the tod operon is efficiently transcribed.
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PMID:The TodS-TodT two-component regulatory system recognizes a wide range of effectors and works with DNA-bending proteins. 1670 39

The dinuclear RuII arene complexes [{(eta6-arene)RuCl}2(mu-2,3-dpp)](PF6)2, arene=indan (1), benzene (2), p-cymene (3), or hexamethylbenzene (4) and 2,3-dpp=2,3-bis(2-pyridyl)pyrazine, have been synthesized and characterized. Upon irradiation with UVA light, complexes 1 and 2 readily underwent arene loss, while complexes 3 and 4 did not. The photochemistry of 1 was studied in detail. In the X-ray structure of [{(eta6-indan)RuCl}2(mu-2,3-dpp)](PF6)2 (1), 2,3-dpp bridges two RuII centers 6.8529(6) A apart. In water, aquation of 1 in the dark occurs with replacement of chloride with biexponential kinetics and decay constants of 100+/-1 min-1 and 580+/-11 min-1. This aquation was suppressed by 0.1 M NaCl. UV or visible irradiation of 1 in aqueous or methanolic solution led to arene loss. The fluorescence of the unbound arene is approximately 40 times greater than when it is complexed. Irradiation of 1 also had a significant effect on its interactions with DNA. The DNA binding of 1 is increased after irradiation. The non-irradiated form of 1 preferentially formed DNA adducts that only weakly blocked RNA polymerase, while irradiation of 1 transformed the adducts into stronger blocks for RNA polymerase. The efficiency of irradiated 1 to form DNA interstrand cross-links was slightly greater than that of cisplatin in both 10 mM NaClO4 and 0.1 M NaCl. In contrast, the interstrand cross-linking efficiency of non-irradiated 1 in 10 mM NaClO4 was relatively low. An intermediate amount of cross-linking was observed when the sample of DNA already modified by non-irradiated 1 was irradiated. DNA unwinding measurements supported the conclusion that both mono- and bifunctional adducts with DNA can form. These results show that photoactivation of dinuclear RuII arene complexes can simultaneously produce a highly reactive ruthenium species that can bind to DNA and a fluorescent marker (the free arene). Importantly, the mechanism of photoreactivity is also independent of oxygen. These complexes, therefore, have the potential to combine both photoinduced cell death and fluorescence imaging of the location and efficiency of the photoactivation process.
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PMID:Dual triggering of DNA binding and fluorescence via photoactivation of a dinuclear ruthenium(II) arene complex. 1749 48

In this study, the P2 receptor-mediated modulation of [3H]glutamate and [3H]noradrenaline release were examined in rat spinal cord slices. Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and 2-methylthioadenosine 5'-diphosphate (2-MeSADP) decreased the electrical stimulation-evoked [3H]glutamate efflux with the following order of potency: ADP>2-MeSADP>ATP. The effect of ATP was antagonized by suramin (300microM), the P2Y12,13 receptor antagonist 2-methylthioadenosine 5'-monophosphate (2-MeSAMP, 10microM), and partly by 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS, 30microM) and the P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-diphosphate (MRS 2179, 10muM). ATP, ADP and 2-MeSADP also decreased evoked [3H]noradrenaline outflow; the order of agonist potency was ADP> or =2-MeSADP>ATP. The effect of ATP was reversed by 2-MeSAMP (10microM), and partly by MRS 2179 (10microM). By contrast, 2-methylthioadenosine-5'-triphosphate (2-MeSATP, 10-300microM) increased resting and electrically evoked [3H]glutamate and [3H]noradrenaline efflux, and this effect was prevented by the P2X1 receptor selective antagonist 4,4',4'',4'''-[carbonylbis[imino-5,1,3-benzenetriyl bis (carbonyl-imino)]] tetrakis (benzene-1,3-disulfonic acid) octasodium salt (NF449, 100nM). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that mRNAs encoding P2Y12 and P2Y13 receptors are expressed in the brainstem, whereas P2Y13 but not P2Y12 receptor mRNA is present in the dorsal root ganglion and spinal cord. P2Y1 receptor expression in the spinal cord is also demonstrated at the protein level. In conclusion, inhibitory P2Y and facilitatory P2X1-like receptors, involved in the regulation of glutamate (P2Y13 and/or P2Y1) and noradrenaline (P2Y13 and/or P2Y1, P2Y12) release have been identified, which provide novel target sites for analgesics acting at the spinal cord level.
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PMID:Modulation of neurotransmitter release by P2X and P2Y receptors in the rat spinal cord. 1806

A bacterium designated strain BD-a59, able to degrade all six benzene, toluene, ethylbenzene, and o-, m-, and p-xylene (BTEX) compounds, was isolated by plating gasoline-contaminated sediment from a gasoline station in Geoje, Republic of Korea, without enrichment, on minimal salts basal (MSB) agar containing 0.01% yeast extract, with BTEX as the sole carbon and energy source. Taxonomic analyses showed that the isolate belonged to Pseudoxanthomonas spadix, and until now, the genus Pseudoxanthomonas has not included any known BTEX degraders. The BTEX biodegradation rate was very low in MSB broth, but adding a small amount of yeast extract greatly enhanced the biodegradation. Interestingly, degradation occurred very quickly in slurry systems amended with sterile soil solids but not with aqueous soil extract. Moreover, if soil was combusted first to remove organic matter, the enhancement effect on BTEX biodegradation was lost, indicating that some components of insoluble organic compounds are nutritionally beneficial for BTEX degradation. Reverse transcriptase PCR-based analysis of field-fixed mRNA revealed expression of the tmoA gene, whose sequence was closely related to that carried by strain BD-a59. This study suggests that strain BD-a59 has the potential to assist in BTEX biodegradation at contaminated sites.
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PMID:Influence of soil components on the biodegradation of benzene, toluene, ethylbenzene, and o-, m-, and p-xylenes by the newly isolated bacterium Pseudoxanthomonas spadix BD-a59. 1883 99

Increasingly, molecular biological tools, most notably quantitative polymerase chain reaction (qPCR), are being employed to provide a more comprehensive assessment of bioremediation of petroleum hydrocarbons and fuel oxygenates. While qPCR enumeration of key organisms or catabolic genes can aid in site management decisions, evaluation of site activities conducted to stimulate biodegradation would ideally include a direct measure of gene expression to infer activity. In the current study, reverse-transcriptase (RT) qPCR was used to monitor gene expression to evaluate the effectiveness of an oxygen infusion system to promote biodegradation of BTEX and MTBE. During system operation, dissolved oxygen (DO) levels at the infusion points were greater than 30 mg/L, contaminant concentrations decreased, and transcription of two aromatic oxygenase genes and Methylibium petroleiphilum PM1-like 16S rRNA copies increased by as many as 5 orders of magnitude. Moreover, aromatic oxygenase gene transcription and PM1 16s rRNA increased at downgradient locations despite low DO levels even during system operation. Conversely, target gene expression substantially decreased when the system was deactivated. RT-qPCR results also corresponded to increases in benzene and MTBE attenuation rates. Overall, monitoring gene expression complemented traditional groundwater analyses and conclusively demonstrated that the oxygen infusion system promoted BTEX and MTBE biodegradation.
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PMID:Monitoring gene expression to evaluate oxygen infusion at a gasoline-contaminated site. 2068 21

To optimize the antitumor activity of oncrasin-1, a small molecule RNA polymerase II inhibitor, we evaluated 69 oncrasin-1 analogues for their cytotoxic activity against normal human epithelial cells and K-Ras mutant tumor cells. About 40 of those compounds were as potent as or more potent than oncrasin-1 in tumor cells and had a minimal cytotoxic effect on normal cells. Structure-activity relationship analysis revealed that most of the active compounds contained either a hydroxymethyl group or an aldehyde group as a substitute at the 3-position of the indole. Both electron-donating and electron-withdrawing groups in the benzene ring were well tolerated. The hydroxymethyl compounds ranged from equipotent with to 100 times as potent as the corresponding aldehyde compounds. We tested three active analogues' effect on RNA polymerase phosphorylation and found that they all inhibited phosphorylation of the C-terminal domain of RNA polymerase II, suggesting that the active compounds might act through the same mechanisms as oncrasin-1.
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PMID:Analogues and derivatives of oncrasin-1, a novel inhibitor of the C-terminal domain of RNA polymerase II and their antitumor activities. 2144 18

A cornerstone of Synthetic Biology is the engineering of gene regulatory networks. Construction of such biological circuits has been used not only to elucidate the dynamics of gene expression but also for designing whole-cell biosensors that translate environmental signals into quantifiable outputs. To this end, distinct components of given regulatory systems are rationally rewired in a way that translates an external stimulus (for instance, the presence of one chemical species) into a measurable readout typically fluorescence or luminescence. Various biosensors for BTEX (a mixture of benzene, toluene, ethylbenzene and xylenes) are based on XylR, the main transcriptional regulator of the TOL pathway of Pseudomonas putida mt-2. In the presence of its natural effectors (e.g., m-xylene, toluene or 3-methylbenzylalcohol), XylR triggers expression of the upper pathway genes by means of the Pu promoter. Available biosensors combine the xylR gene and a direct fusion between the cognate Pu promoter and the luxCDABE operon, all components stably integrated in the chromosome of P. putida. A versatile development of the same biosensing concept is described, aimed at increasing the sensitivity of the genetic circuit toward XylR inducers. The new platform utilizes mini-transposon vectors tailored for engineering an artificial expression cascade that operates as an amplifier of the signal/response ratio of the biosensor. This strategy was applied to the construction of a strain that carries a transcriptional fusion between the Pu promoter and T7 RNA polymerase (which becomes under the control of XylR and its effectors), along with a T7 promoter controlling expression of the luxCDABE operon. This simple regulatory architecture produced a dramatic increase of bioluminescence emission in respect to the strain that carries only the direct fusion between the Pu promoter and the luxCDABE reporter.
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PMID:Engineering whole-cell biosensors with no antibiotic markers for monitoring aromatic compounds in the environment. 2214 65


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