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)

It has previously been demonstrated that the epidermis is a rich source of proinflammatory cytokines and growth factors and that complex interactions between these factors may affect inflammatory responses in skin. To investigate whether IL-10 (cytokine synthesis inhibitory factor) is part of this complex process, RNA was extracted from normal epidermis at various times after application of various chemicals to murine skin and mRNA signals for IL-10 were sought using a quantitative reverse-transcriptase-polymerase chain reaction technique. IL-10 signal strength was normalized to that of beta-actin in each sample. IL-10 mRNA signals were occasionally identified in normal epidermis but were uniformly enhanced 4 h after hapten application, and were maximal after 12 h. Contact allergens induced IL-10 mRNA signals whereas vehicles and irritants did not. Depletion of Langerhans cells, Thy-1+ dendritic epidermal cells, and T lymphocytes demonstrated that keratinocytes were the main source of IL-10 mRNA. IL-10 signals were also detected in mRNA derived from PAM 212 (spontaneously transformed keratinocyte) cells. IL-10 protein could be detected by immunoprecipitation, with an IL-10 mAB, of supernatants obtained 16 h after cultured epidermal cells were coupled with hapten. This study demonstrates that murine keratinocytes are capable of producing IL-10 mRNA and protein, and that signal strength of IL-10 mRNA is enhanced by hapten application.
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PMID:Identification and induction of keratinocyte-derived IL-10. 160 65

The production of enzymatically active proteinase by lactococci requires the joint presence of a proteinase gene, prtP, and a gene encoding a maturation protein, prtM. A 32-kDa protein produced by Escherichia coli upon expression of the prtM gene under the direction of the T7 RNA polymerase promoter was purified and used to obtain PrtM-specific antibodies. With these antibodies, immunogold labeling of lactococcal cells revealed that PrtM was associated with the lactococcal cell envelope. Western blot (immunoblot) analysis of whole lactococcal cells and isolated membrane vesicles indicated that PrtM was a membrane-associated protein. Radiolabeling of Lactococcus lactis with [3H]palmitic acid showed that PrtM was a lipoprotein. Partial secretion of PrtM into the culture medium was observed after Cys-24, the target residue for lipid modification, was replaced by an Ala residue by means of site-directed mutagenesis. This mutation did not affect proteinase activity.
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PMID:Lactococcal proteinase maturation protein PrtM is a lipoprotein. 190 66

In alphavirus-infected cells the four virus-specific nonstructural proteins (nsP1-nsP4) are located on modified endosomes and lysosomes known as type I cytopathic vacuoles. In this paper, we show that when nsP1 was expressed alone in HeLa cells with the aid of the recombinant T7 RNA polymerase vaccinia (vTF7-3) virus system, it was tightly associated with intracellular smooth membranes. The membrane association may be due to acylation, since nsP1 could be labeled with [3H]palmitic acid in both Semliki Forest virus-infected and nsP1-transfected HeLa cells. Release of the 3H-label by alkaline methanolysis suggests that the palmitate was associated with nsP1 via an ester bond. Pulse-chase experiments done on nsP1-transfected cells revealed that this protein was rapidly associated with the membranes. After synchronizing the synthesis of the nsP1 gene product in transfected cells, nsP1 appeared first at the plasma membrane and thereafter on vesicles, many of which contained the endosomal transferrin receptor marker. Later, nsP1 appeared on large vacuoles, which contained the lysosome specific h-lamp-1 protein. Membrane association of nsP1, and its affinity to endosomes and lysosomes, suggest a role of this protein in the biogenesis of the alphavirus-specific RNA replication complex.
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PMID:The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles. 774 33

Ferrochelatase (EC 4.99.1.1), a mitochondrial inner membrane-bound protein, is the terminal enzyme of heme biosynthesis. The cDNA encoding the human mature ferrochelatase was placed under transcriptional control of T7 RNA polymerase in an Escherichia coli expression system. The bacteria produced large amounts of 42 kDa protein which reacted with anti-ferrochelatase antibodies. Expressed ferrochelatase exhibited iron- and zinc-chelating activities, and was found as a soluble protein. The recombinant enzyme has been purified to apparent homogeneity with a high yield, by one-step purification involving Blue-Sepharose chromatography. The purified enzyme which showed a molecular weight of about 40,000 by gel-filtration, functioned in a monomeric form. Km value for both mesoporphyrin IX and protoporphyrin IX with zinc was 12.5 microM. Km values for iron and zinc with mesoporphyrin IX were 6.7 microM and 11.8 microM, respectively. Zinc-chelating activity was markedly stimulated by palmitic acid, but iron-chelating activity remained unchanged. The above results were similar to those reported previously for mammalian ferrochelatase. The overexpression and the simple purification of a functional ferrochelatase exhibiting the same properties as natural enzyme will allow us to elucidate the mechanism of the enzyme reaction and structural changes of the mutated enzyme.
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PMID:Overexpression in Escherichia coli, and one-step purification of the human recombinant ferrochelatase. 803 31

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E. coli haemolysin (HlyA). The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA.
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PMID:Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro. 897 51

Plasma cholesterol levels increase after birth, and to a greater extent in breast-fed versus formula-fed infants. This increase is believed to be due to the high fat and cholesterol content of the infant diet, but little is known about the effects of early diet on the expression of proteins involved in regulating cholesterol metabolism. This study examined changes in the expression of hepatic proteins regulating cholesterol metabolism during development. Newborn piglets were fed sow milk or one of four formulas for 18 days. The formulas had similar levels of palmitic acid (16:0) as in milk, supplied as palm olein oil with 16:0 esterified predominantly to the sn-1,3 position or as synthesized triglyceride (TG) with 16:0 esterified mainly to the sn-2 position of glycerol, each with no cholesterol (<0.10 mmol/L) or 0.65 mmol/L cholesterol added. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA levels was used to assess the effects of diet on hepatic hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, low-density lipoprotein (LDL) receptor, and 7alpha-hydroxylase (C7H). LDL receptor mRNA levels showed no appreciable difference between milk- and formula-fed piglets. However, the levels of HMG-CoA reductase and C7H mRNA were higher (P < .05) in all formula-fed versus milk-fed piglets, irrespective of the formula TG source or cholesterol content. The lower levels of HMG-CoA reductase and C7H mRNA in milk-fed piglets were accompanied by higher (P < .05) plasma total, high-density lipoprotein (HDL), and apolipoprotein (apo) B-containing cholesterol. These studies show that the levels of hepatic HMG-CoA reductase and C7H mRNA, but probably not LDL receptor mRNA, are altered by early diet.
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PMID:Early diet influences hepatic hydroxymethyl glutaryl coenzyme A reductase and 7alpha-hydroxylase mRNA but not low-density lipoprotein receptor mRNA during development. 944 Apr 72

In this paper, we borrow the idea of the receiver operating characteristic (ROC) from clinical medicine and demonstrate its application to sequence comparison. The ROC includes elements of both sensitivity and specificity, and is a quantitative measure of the usefulness of a diagnostic. The ROC is used in this work to investigate the effects of scoring table and gap penalties on database searches. Studies on three families of proteins, 4Fe-4S ferredoxins, lysR bacterial regulatory proteins, and bacterial RNA polymerase sigma-factors lead to the following conclusions: sequence families are quite idiosyncratic, but the best PAM distance for database searches using the Smith-Waterman method is somewhat larger than predicted by theoretical methods, about 200 PAM. The length independent gap penalty (gap initiation penalty) is quite important, but shows a broad peak at values of about 20-24. The length dependent gap penalty (gap extension penalty) is almost irrelevant suggesting that successful database searches rely only to a limited degree on gapped alignments. Taken together, these observations lead to the conclusion that the optimal conditions for alignments and database searches are not, and should not be expected to be, the same.
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PMID:Use of receiver operating characteristic (ROC) analysis to evaluate sequence matching. 1671 63

The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) stavudine (d4T) can induce mild to severe liver injuries such as steatosis (i.e. triglyceride accumulation), steatohepatitis and liver failure. NRTI-induced toxicity has been ascribed to the inhibition of mitochondrial DNA (mtDNA) replication causing mtDNA depletion and respiratory chain dysfunction. This can secondarily impair the tricarboxylic acid cycle and fatty acid oxidation (FAO), thus leading to lactic acidosis and hepatic steatosis. However, NRTIs could also impair mitochondrial function and induce hepatic steatosis through other mechanisms. In this study, we sought to determine whether d4T could inhibit mitochondrial FAO and induce triglyceride accumulation through a mtDNA-independent mechanism. Since human tumoral and non-tumoral hepatic cell lines were unable to efficiently oxidize palmitic acid, the effects of d4T on mitochondrial FAO were assessed on cultured rat hepatocytes. Our results showed that 750 microM of d4T significantly inhibited palmitic acid oxidation after 48 or 72 h of culture, without inducing cell death. Importantly, high concentrations of zidovudine and zalcitabine (two other NRTIs that can induce hepatic steatosis), or beta-aminoisobutyric acid (a d4T metabolite), did not impair FAO in rat hepatocytes. D4T-induced FAO inhibition was observed without mtDNA depletion and lactate production, and was fully prevented with l-carnitine or clofibrate coincubation. l-carnitine also prevented the accretion of neutral lipids within rat hepatocytes. High concentrations of d4T were unable to inhibit FAO on freshly isolated liver mitochondria. Moreover, a microarray analysis was performed to clarify the mechanism whereby d4T can inhibit mitochondrial FAO and induce triglyceride accumulation in rat hepatocytes. The microarray data, confirmed by quantitative real-time PCR analysis, showed that d4T increased the expression of sterol regulatory element-binding protein-1c (SREBP1c) and reduced that of microsomal triglyceride transfer protein (MTP). Finally, d4T-induced alteration of SREBP1c and MTP expression was partially prevented by l-carnitine. Thus, short-term incubation with high concentrations of d4T can rapidly induce accumulation of neutral lipids within rat hepatocytes, which can be fully prevented by l-carnitine. Furthermore, our investigations suggested that lipid accumulation could be the consequence of a dual mechanism, namely a mtDNA-independent impairment of mitochondrial FAO and a reduction of lipid export from the hepatocytes.
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PMID:High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes. 1829 83

The tumor suppressor p53 has critical roles in regulating lipid metabolism, but whether and how p53 regulates cardiolipin (CL) de novo biosynthesis is unknown. Here, we report that p53 physically interacts with histone deacetylase SIRT6 in vitro and in vivo, and this interaction increases following palmitic acid (PA) treatment. In response to PA, p53 and SIRT6 localize to chromatin in a p53-dependent manner. Chromatin p53 and SIRT6 bind the promoters of CDP-diacylglycerol synthase 1 and 2 (CDS1 and CDS2), two enzymes required to catalyze CL de novo biosynthesis. Here, SIRT6 serves as a co-activator of p53 and effectively recruits RNA polymerase II to the CDS1 and CDS2 promoters to enhance CL de novo biosynthesis. Our findings reveal a novel, cooperative model executed by p53 and SIRT6 to maintain lipid homeostasis.
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PMID:p53 cooperates with SIRT6 to regulate cardiolipin de novo biosynthesis. 3023 40