Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory diseases. In the present study, we investigated the potential role of NO in an ocular model of inflammation, endotoxin-induced uveitis (EIU), in Lewis rats. Injection of LPS in one footpad induces severe uveitis after 16 h, which is accompanied by an increase of NO in the aqueous and vitreous humors, as evaluated by nitrite assay. Reverse transcriptase-PCR experiments reveal a large increase of inducible NO synthase (iNOS) mRNA in the iris/ciliary body, from 2 to 24 h after LPS treatment. In the retina, maximal increase of iNOS mRNA was detected 16 h after LPS treatment. Two i.p. injections of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), which inhibits nitrite release in the aqueous and vitreous humors, profoundly reduce clinical and histologic inflammation in EIU rats. These results implicate the NO pathway in the pathogenesis of EIU and demonstrate the possibility of modulating this inflammatory disease by injection of a NOS inhibitor.
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PMID:Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase. 753 24

Hereditary C3 deficiency in a 22-year-old woman was studied. Previous works indicated that C3 could not be detected in the serum of such a patient by enzyme immunoassay. In this study, we demonstrated that C3 genes of this patient and her parents have no gross structural aberration. However, C3 mRNA was almost not detectable in the skin fibroblasts of this patient. The activity of the patient's C3 upstream regulatory elements was tested and showed no functional abnormality. Using reverse-transcriptase polymerase chain reaction (RT-PCR) to amplify RNA from LPS-stimulated patient's fibroblasts, two shorter cDNAs within C3 exon 8 to exon 12 were noted. DNA sequence analysis of the RT-PCR products revealed that one deleted 116 nucleotides of the full exon 10 and the other deleted 34 nucleotides in the 3' region of exon 10. A single base substitution (G to T) in the splice donor site of intron 10 was identified. This aberrant splicing involving exon 10 could result in translational pretermination at exon 11. Thus, this mutation provided the molecular basis for the deficiency of C3 in the patient.
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PMID:A hereditary C3 deficiency due to aberrant splicing of exon 10. 792 34

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.
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PMID:Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts. 847 46

C-reactive protein (CRP) is well characterized as one of the serum acute phase proteins, the levels of which increase dramatically after infection. CRP has been shown to be involved in multiple immunoregulatory functions. For example, it activates the classical complement cascade, opsonizes bacteria for phagocytosis, and stimulates phagocytic cells. Although CRP is predominantly produced and secreted by hepatocytes, other cells including subsets of lymphocytes, Kupffer cells, and blood monocytes have been shown to synthesize this protein as well. We hypothesized that CRP may be produced in the lung, and therefore it could function directly in pulmonary host defense. Western blot analysis showed that CRP was present in the lung tissue, lung lavage, and alveolar macrophages. This result was further confirmed by immunohistochemical staining of lung sections that showed the localization of CRP in alveolar macrophages. The CRP mRNA was detected subsequently by reverse-transcriptase PCR (RT-PCR), and a single amplified product was obtained from alveolar macrophages as well as from whole lung tissue. Both were the same size as the amplified product obtained from liver mRNA. Furthermore, in situ hybridization with CRP riboprobe demonstrated specific staining of alveolar macrophages both in lung sections and isolated cells. In addition, in situ hybridization showed that CRP mRNA levels in isolated alveolar macrophages were up-regulated by in vitro LPS stimulation. In summary, these results indicate that CRP is produced by alveolar macrophages, and suggest that CRP may be involved in the pulmonary immune response.
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PMID:Expression of C-reactive protein by alveolar macrophages. 864 29

Human peritoneal mesothelial cells (HPMC) respond to tumor necrosis factor alpha (TNF alpha) by releasing various cytokines that may activate the endothelium and induce recruitment of leukocytes during peristonitis. We characterized the receptors for TNF on HPMC to elucidate their functions in peritonitis. Scatchard analysis determined the presence of 70 x 10(3) TNF receptors/cell with a kDa of 0.44 nM. TNF receptor 1 (TNF-R1, p55) and TNF-R2 (p75) mRNA were demonstrated by reverse-transcriptase-PCR (RT-PCR). TNF-R1 protein was solely detected by flow cytometry (FCM). Interleukin-1 alpha (IL-1 alpha) induced down-regulation of TNF-R1. This was concomitant with accumulation of soluble TNF-R1 (sTNF-R1) detected by specific ELISA. LPS had a lower TNF-R1-shedding activity while TNF alpha did not induce shedding. The IL-1-induced-sTNF-R1-shedding was suppressed by the protein-kinase-A (PKA) inhibitor, H-8, or by H-7, the inhibitor of both PKC and PKA, but not by the specific PKC inhibitor GF. These experiments suggest a role for PKA in the IL-1-shedding signal. No change in TNF-R1 mRNA levels was observed after IL-1 alpha or TNF alpha stimulation while TNF-R2 (p75) mRNA basal levels transiently increased three to fivefold, reaching a peak after four hours followed by an accumulation of sTNF-R2 in the supernatant. Our data suggest that the main receptor expressed on HPMC is TNF-R1. Down-regulation and shedding of TNF-R1 induced by IL-1, and the transient expression of TNF-R2 induced by IL-1 and TNF, may regulate the responses to TNF by HPMC. These results may be important in understanding the inflammatory process of peritonitis were TNF plays a major role.
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PMID:TNF-receptors on human peritoneal mesothelial cells: regulation of receptor levels and shedding by IL-1 alpha and TNF alpha. 880 91

1. Induction of nitric oxide synthase (iNOS) results in overproduction of nitric oxide (NO), which may be a principal cause of the massive vasodilatation and hypotension observed in septic shock. Since NO-induced vasorelaxation is mediated via the soluble isoform of guanylate cyclase (sGC), the regulation of sGC activity during shock is of obvious importance, but yet poorly understood. The aim of the present study was to investigate the activation of sGC by sodium nitroprusside (SNP) before and after exposure of rat aortic smooth muscle cells to endotoxin (LPS) or interleukin-1 beta (IL-1 beta). 2. Exposure of rat aortic smooth muscle cells to SNP (10 microM) elicited up to 200 fold increases in cyclic GMP. This effect was attenuated by 30-70% in IL-1 beta- or LPS-pretreated cells, in a pretreatment time-and IL-1 beta- or LPS-concentration-dependent manner. When, however, cells were exposed to IL-1 beta or LPS and then stimulated with the particulate guanylate cyclase activator, atriopeptin II, no reduction in cyclic GMP accumulation was observed. 3. Pretreatment of rats with LPS (5 mg kg-1, i.v.) for 6 h led to a decrease in aortic ring SNP-induced cyclic GMP accumulation. 4. The IL-1 beta-induced reduction in SNP-stimulated cyclic GMP accumulation in cultured cells was dependent on NO production, as arginine depletion abolished the downregulation of cyclic GMP accumulation in response to SNP. 5. Reverse-transcriptase-polymerase chain reaction analysis revealed that the ratio of steady state mRNA for the alpha, subunit of sGC to glyceraldehyde phosphate dehydrogenase was decreased in LPS- or IL-1 beta-treated cells, as compared to vehicle-treated cells. 6. Protein levels of the alpha 1 sGC subunit remained unaltered upon exposure to LPS or IL-1 beta, suggesting that the early decreased cyclic GMP accumulation in IL-1 beta- or LPS-pretreated cells was probably due to reduced sGC activation. Thus, the observed decreased responsiveness of sGC to NO stimulation following cytokine or LPS challenge may represent an important homeostatic mechanism to offset the extensive vasodilatation seen in sepsis.
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PMID:Downregulation of nitrovasodilator-induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin-1 beta. 883 57

Effects of uromodulin (URO) and Tamm-Horsfall protein (THP), the most abundant proteins in the urine of pregnant and normal women, respectively, on the induction of TNF-alpha secretion and tissue factor (TF) expression of human monocytes were studied. THP, URO, and its fragments stimulated human mononuclear cells to proliferate and secrete TNF-alpha. The release of URO and THP-induced TNF-alpha in monocytes was dependent upon protein tyrosine kinase activation that results in tyrosine phosphorylation. URO and THP also induced TF expression of human monocytes and monocytic cell line U937 in a dose-dependent manner. TF expression was transient, reached its peak at 6 h and declined toward basal levels by 24 h. Reverse transcriptase-PCR and dot-blot analysis confirmed the induction of TF mRNA synthesis. URO and THP-induced TF expression were inhibited by actinomycin D and pentoxifylline further supporting the requirement of de novo TF mRNA synthesis. The possibility of LPS contamination of URO and THP was excluded because: 1) URO and THP-induced TF expression were inhibited by specific Ab; 2) URO was less capable of inducing TF in HUVEC as compared with LPS; 3) polymyxin B blocked the induction of Limulus clotting by LPS but not by URO and THP; 4) both LPS-sensitive (C3H/HeN) and -resistant (C3H/HeJ) mice produced little or no TNF-alpha after URO challenge. Therefore, our findings suggest that URO and THP play a significant role in the innate immunity of the urinary system and that the immunostimulatory activity of URO is potentially useful for immunotherapy.
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PMID:Uromodulin and Tamm-Horsfall protein induce human monocytes to secrete TNF and express tissue factor. 912 Mar 6

IL-12, a 75-kDa heterodimeric cytokine composed of two chains (p35 and p40), is a central regulator of immune responses and may be implicated in the pathogenesis of certain inflammatory diseases of the central nervous system (CNS). We have examined the capacity of two CNS APC, microglia and astrocytes, to produce IL-12 upon stimulation with cytokines, LPS, or a neurotropic virus. In purified microglial cultures from neonatal mouse brains, expression of IL-12 p35 and p40 mRNA is induced by LPS and is stimulated maximally by combined IFN-gamma/LPS treatment, as detected by semiquantitative reverse-transcriptase PCR. LPS induces secretion of IL-12 p40, but not of IL-12 p75, as detected by specific ELISA. Combined stimulation with IFN-gamma/LPS enhances IL-12 p40 secretion and induces IL-12 p75 secretion by microglia. Conversely, mouse astrocytes do not express IL-12 p35 mRNA and do not secrete IL-12 p75 under any condition tested. IL-12 production by activated microglia is inhibited by IL-10, PGE2, and cAMP-elevating agents. Coculture of microglia with astrocytes or exposure of microglia to astrocyte-conditioned medium also results in marked reduction of IL-12 p75 and p40 secretion by IFN-gamma/LPS-stimulated microglia, indicating a regulatory role of astrocytes on IL-12 production. This novel mechanism of IL-12 regulation may play an important role in the control of immune responses during infection or in Th1 cell-mediated autoimmune diseases of the CNS.
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PMID:IL-12 production by central nervous system microglia is inhibited by astrocytes. 925 19

Four p38 mitogen-activated protein kinases (p38alpha, beta, gamma, delta) have been described. To understand the role of p38 family members in inflammation, we determined their relative expression in cells that participate in the inflammatory process. Expression was measured at the level of mRNA by reverse-transcriptase PCR and protein by Western blot analysis. p38alpha was the dominant form of p38 in monocytes; expression of p38delta was low and p38beta was undetected. In macrophages, p38alpha and p38delta were abundant, but p38beta was undetected. p38alpha and p38delta were also expressed by neutrophils, CD4+ T cells, and endothelial cells. Again, p38beta was not detected in neutrophils, although low amounts were present in CD4+ T cells. In contrast, p38beta was abundant in endothelial cells. p38gamma protein was not detected in any cell type, although p38gamma mRNA was present in endothelial cells. Immunokinase assays showed a strong activation of p38alpha and a lesser activation of p38delta in LPS-stimulated macrophages. Abs specific for mono- and dual-phophorylated forms of p38 suggested that LPS induces dual phosphorylation of p38alpha, but primarily mono-phosphorylation of p38delta. IL-1beta activated p38alpha and p38beta in endothelial cells. However, p38alpha was the more activated form based on kinase assays and phosphorylation analysis. Expression and activation patterns of p38alpha in macrophages and endothelial cells suggest that p38alpha plays a major role in the inflammatory response. Additional studies will be needed to define the contribution of p38delta to macrophage, neutrophil, and T cell functions, and of p38beta to signaling in endothelial cells and T cells.
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PMID:Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages. 1020 54

Murine experimental meningitis models induced by either Escherichia coli LPS, live Streptococcus pneumoniae, or Listeria monocytogenes were used to study the origin and potential function of soluble CD14 (sCD14) in the brain during bacterial meningitis. Whereas intracerebral infection caused only a minor and/or transient increase of sCD14 levels in the serum, dramatically elevated concentrations of sCD14 were detected in the cerebrospinal fluid. Reverse-transcriptase PCR and FACS analysis of the leukocytes invading the subarachnoid compartment revealed an active amplification of CD14 transcription and concomitant surface expression. These findings were confirmed by in situ hybridization and immunohistochemical analysis. In contrast, parenchymal astrocytes and microglial cells were shown not to significantly contribute to the elevated levels of sCD14. Simultaneous intracerebral inoculation of rsCD14 and S. pneumoniae resulted in a markedly increased local cytokine response. Taken together, these data provide the first evidence that sCD14 can act as an inflammatory co-ligand in vivo. Thus, during bacterial meningitis, sCD14 is massively released by intrathecal leukocytes, and the sCD14 found in the cerebrospinal fluid can play an important role in the pathogenesis of this disease.
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PMID:The origin and function of soluble CD14 in experimental bacterial meningitis. 1020 18


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