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)

Several reports have implicated an association between the development of connective tissue disorders and exposure to silicone in breast implant patients. These connective tissue disorders include local or systemic scleroderma-like syndrome and have been characterized by fibrosis as well as the presence of circulating autoantibodies. Whether silicone does potentiate the development of a scleroderma-like syndrome is still to be defined. The purpose of the present study was to determine the effect of silicone on the tight skin (TSK/+) mouse which develops a scleroderma-like syndrome and on its normal pa/pa TSK/- littermate. Groups of six TSK and five of their normal pa/pa littermates were injected subcutaneously with low molecular weight silicone (LMW-PDMS), high molecular weight silicone gel (HMW-PDMS), incomplete Freunds' adjuvant (IFA) or Hank's balanced salt solution (HBSS) in the dorsal neck area and ventrally in the upper chest region. Serum was obtained prior to and 1 month after injection of silicone, IFA, or control HBSS. Antibody levels to bovine serum albumin (BSA), RNA polymerase (RNAP) and topoisomerase I were determined. Mice were then euthanized and strips of skin from the injection sites as well as samples of kidney and liver were studied histologically. No significant pathological changes were observed in TSK/- mice 1 month following injection with HBSS, LMW-PDMS or HMW-PDMS. Skin samples from TSK/+ mice which received LMW-PDMS showed hyperplasia of the dermis and peri-panniculus carnosus tissue and infiltrates of macrophages containing lipid-like vacuolated materials. Lipid vacuoles were observed throughout the deeper dermis as multiple loculated vacuoles. TSK mice which received HMW-PDMS showed similar thickening of the dermis and the peri-panniculus carnosus connective tissue. There were no significant differences in the histologic characteristics of the silicone-injected TSK/+ mice compared to those that received HBSS or IFA. No detectable changes in the kidney, spleen, or liver samples taken from TSK/+ or TSK/- mice injected with HBSS, IFA or silicone (LMW-PDMS, HMW-PDMS) preparations were noted. Baseline circulating antibody levels to BSA, RNAP and topoisomerase were significantly higher in TSK/+ mice compared to the control TSK/- littermates. Administration of silicone (LMW-PDMS or HMW-PDMS) did not significantly alter circulating antibody levels to BSA, RNAP and topoisomerase in either the TSK/+ or the TSK/- mice. The results of this study indicate that silicone administration does not potentiate the development of the scleroderma-syndrome characterized by skin lesions and presence of circulating antibodies in the TSK/+ model.
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PMID:Silicone does not potentiate development of the scleroderma-like syndrome in tight skin (TSK/+) mice. 886 22

Transcription and replication of the influenza virus RNA genome take place in the nuclei of infected cells. Ribonucleoprotein (RNP) complexes consisting of viral RNA, RNA polymerase, and nucleocapsid protein (NP) are proven to be the catalytic unit for RNA synthesis, while it has been indicated that the viral RNA polymerase activity is modulated by host-derived nuclear factors. Here we have identified such host factors present in nuclear extracts prepared from uninfected HeLa cells with biochemical complementation assays using the in vitro RNA synthesis system. The stimulatory activity was not absorbed to phosphocellulose but was tightly bound to Q-Sepharose. The eluate recovered from Q-Sepharose was able to stimulate the RNA synthesis catalyzed by both RNP complexes and purified RNA polymerase and NP. The stimulatory activity was further separated into two distinct fractions, designated RAF-1 (RNA polymerase activating factor-1) and RAF-2 fractions, through phenyl-Sepharose column chromatography. When these fractions were fractionated through a gel filtration column, RAF-1 and RAF-2 activities were recovered in fractions corresponding to the molecular mass of 350 kDa and 60 kDa, respectively. Furthermore, the RAF-2 fraction was shown to contain an inhibitory activity, tentatively designated RIF-1 (RNA polymerase inhibitory factor-1). RIF-1 sedimented as fast as bovine serum albumin in glycerol density gradient centrifugation. Roles of these host factors are discussed in the context of viral RNA transcription and replication.
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PMID:Identification of host factors that regulate the influenza virus RNA polymerase activity. 915 Aug 91

Previous studies have demonstrated the feasibility of implantation of human blood cells or tissues in lethally irradiated mice or rats, radioprotected with SCID mouse bone marrow cells: The Trimera system. In the present study, we describe the development of a mouse Trimera model for human hepatitis B virus (HBV) infection. In this model, viremia is induced by transplantation of ex vivo HBV-infected human liver fragments. Engraftment of the human liver fragments, evaluated by hematoxylin-eosin staining and human serum albumin mRNA expression, was observed in 85% of the transplanted animals 1 month postimplantation. Viremia levels were determined in these mice by measuring serum HBV DNA using polymerase chain reaction (PCR), followed by dot-blot hybridization. HBV DNA is first detected 8 days after liver transplantation. Viremia attains a peak between days 18 and 25 when HBV infection is observed in 85% of the transplanted animals. The HBV-Trimera model was used to evaluate the therapeutic effects of human polyclonal anti-HBs antibodies (Hepatect) and of two reverse-transcriptase inhibitors, lamivudine (3TC) and beta-L-5-fluoro-2',3'-dideoxycytidine (beta-L-5FddC). Treatment of HBV-Trimera mice with these drugs effectively reduced both the percentage of infected animals and the viral load in their sera. Treatment cessation resulted in rebound of viral load, indicating HBV replication upon drug withdrawal. These results show that the HBV-Trimera model represents a novel experimental tool for simulating human HBV infection and evaluating potential anti-HBV therapeutic agents.
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PMID:The hepatitis B virus-trimera mouse: a model for human HBV infection and evaluation of anti-HBV therapeutic agents. 991 35

The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies. A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6x His were added in reverse primers used to amplify the corresponding cDNAs. The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl-beta-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni-nitrilotriacetic acid agarose column. This method yielded 20-40 mg of highly pure and very stable proteins per liter of cell culture. Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6x His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions.
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PMID:Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus. 1116 5

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326-338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.
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PMID:Biogenesis of the Semliki Forest virus RNA replication complex. 1126 76

Thyroglobulin (Tg), has recently been identified as a transcriptional regulator of thyroid-restricted genes. The extrathyroidal expression of several of these genes (including the transcription factor Pax-8) together with the occurrence of specific Tg binding sites suggests a secondary role for Tg as a circulating hormone. In this study, we demonstrate using Northern analysis that Pax-8 is expressed in the mouse mesangial cell, and that its transcript levels are suppressed by Tg. These cells also express an asialoglycoprotein receptor, a receptor involved in Tg endocytosis in the thyroid, and a Tg transcript smaller than the 8.3-kb thyroidal form. Reverse transcriptase PCR showed that suppression of Pax-8 by Tg is correlated with reduced expression of bcl-2 apoptosis suppressor. Tg, but not triiodothyronine (T(3)) significantly increased MC proliferation above control as determined by DNA content of MC cultures. The effect of Tg on proliferation was not duplicated by either bovine serum albumin, gamma-globulins, lactoferrin, or the ASGPR-specific ligand,orosomucoid. These results suggest a possible endocrine role for Tg in regulating both Pax-8 related gene transcription and cell division in the mesangial cell.
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PMID:Thyroglobulin increases cell proliferation and suppresses Pax-8 in mesangial cells. 1145 62

Dysregulated renal water handling is a cardinal feature of nephrotic syndrome that has been shown in animal models of experimental nephrosis to mediate renal aquaporin (AQP) expression. However, data on the effect of proteinuria on the proximal tubule, which is heavily vested with AQP1 and therefore may participate in water homeostasis, are limited. To investigate this, we exposed primary human proximal tubular epithelial cells (PTECs) to two key proteinuric components shown to perturb tubule function: human serum albumin and transferrin. Using reverse-transcriptase polymerase chain reaction and immunocytochemical techniques, PTECs in the quiescent state were found to express AQP3 in addition to AQP1 gene and protein, which was also validated in a human proximal tubule cell line, HK-2. Immunohistochemical staining localized AQP1 synthesis to the apical and basolateral membranes and AQP3 synthesis to the basolateral membrane of proximal tubule epithelium. Transferrin in doses reaching nephrotic range upregulated PTEC transcription and translation of both AQP1 and AQP3 in a time- and dose-dependent manner. After 24 hours of stimulation, transferrin led to a 2.4- and 2.2-fold increase in AQP1 and APQ3 messenger RNA expression, whereas protein synthesis surged by 40.7% +/- 2.48% and 24.2% +/- 0.9% compared with control, respectively. These effects were not observed with albumin challenge and were not caused by osmolality fluctuation with transferrin treatment. In summary, our novel finding of AQP3 in PTECs indicates a role for AQP3 in proximal tubule water reabsorption. The pathophysiological significance of heightened AQP1 and AQP3 expression in PTECs on protein challenge as occurs in the nephrotic state requires further investigation.
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PMID:In vitro studies of aquaporins 1 and 3 expression in cultured human proximal tubular cells: upregulation by transferrin but not albumin. 1147 58

Adynamic bone disease and elevated serum levels of advanced glycation end products (AGEs) often are found in patients with renal failure caused by diabetic nephropathy. To clarify the role of AGEs in adynamic bone disease, we investigated the effect of these substances on cultured human osteoblasts and parathyroid cells. After 72 hours of incubation with AGEs-bovine serum albumin (BSA) (1,000 microgram/mL), there was significant inhibition of the synthesis of type I collagen and osteocalcin in response to stimulation with 10(-10) to 10(-8) M of 1,25-dihydroxycholecalciferol. In a human osteoblastic cell line (MG 63), AGEs-BSA did not affect human osteocalcin promoter activity. In human parathyroid cells, a receptor for AGEs was detected by reverse-transcriptase polymerase chain reaction. Incubation with AGEs-BSA for 48 hours significantly inhibited parathyroid hormone secretion in response to a low calcium concentration of 0.81 mM (P < 0.01). In HEK-293 cells, expressing calcium-sensing receptors, the same AGE concentration caused a significant potentiation of the extracellular Ca(2+) induced-intracellular calcium concentration after 24 and 48 hours of incubation (P < 0.05 and P < 0.01). These data suggest that AGEs are involved in the pathogenesis of adynamic bone disease by inhibiting osteoblastic activity and by inhibiting parathyroid hormone secretion in response to hypocalcemia.
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PMID:Role of advanced glycation end products in adynamic bone disease in patients with diabetic nephropathy. 1157 45

Both innate and adaptive immune systems are thought to participate in the pathogenesis of rheumatoid arthritis in adults and children. The experiments reported here were undertaken to examine how immune complexes, potent stimulators of inflammation, may regulate cells of the adaptive immune system. Human T cells were prepared from peripheral blood by negative selection and incubated with bovine serum albumin (BSA)-anti-BSA immune complexes that were formed in the presence or absence of human C1q. C1q-bearing immune complexes, but not unopsonized complexes, elicited both TNF-alpha and IFN-gamma secretion from human T cells. Secretion of both cytokines was time- and dose-dependent. Cross-linking C1q on the cell surface of T cells produced the same results. Cytokine secretion was not inhibited by blocking the C3b receptor (CR1, CD35) on T cells prior to incubation with immune complexes. Reverse transcriptase polymerase chain reaction (RT-PCR) of immune complex-stimulated cells revealed accumulation of both TNF-alpha and IFN-gamma mRNA within 2 h post-stimulation. IL-2 was not detected in cell culture supernatants, but IL-2 receptor alpha chain (CD25) was detected in low density on a small proportion of T cells activated by C1q-bearing immune complexes. Secretion of both cytokines was inhibited partially, but not completely, by IL-10. These experiments show that immune complexes, potent inflammatory mediators, may activate T cells through a novel mechanism. These findings have implications for chronic inflammatory diseases in humans.
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PMID:T cell activation by soluble C1q-bearing immune complexes: implications for the pathogenesis of rheumatoid arthritis. 1251 87

The DNA of bacteriophage SP8, when denatured, yields two components differing in buoyant density in cesium chloride gradients and separable by chromatography on a column of methylated bovine serum albumin and kieselguhr. The denser of the two strands (H) contains more pyrimidines and fewer purines than the lighter (L) strand. Only the H strand forms hybrids with the RNA synthesized by the infected host. The L strand is capable of annealing with complementary RNA synthesized in vitro with it as primer in reactions catalyzed by RNA polymerase. During the vegetative development of phage, host-specific messenger RNA is also synthesized.
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PMID:TRANSCRIPTION IN VIVO OF DNA FROM BACTERIOPHAGE SP8. 1405 3


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