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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent work has established that IL-1beta plays a central role in the inflammation and connective tissue destruction observed in both rheumatoid arthritis and osteoarthritis. These processes result from the ability of this inflammatory cytokine to activate expression of genes for neutral proteases, such as the matrix metalloproteinases. While IL-1beta activates matrix metalloproteinase genes within several hours, it also activates immediate early genes, which are required for the later expression of matrix metalloproteinases and other arthritis-perpetuating genes, are also activated. To identify putative immediate early genes involved in IL-1beta-mediated arthritic disease, a chondrocytic cell line (SW1353) was stimulated with this cytokine for 2 hours, total RNA was isolated, and expressed genes were identified by microarray analysis. This analysis identified alterations in the expression of multiple transcription factors, cytokines, growth factors and their receptors, adhesion molecules, proteases, and signaling intermediates that may contribute to inflammation and cartilage destruction in arthritis. Interestingly, confirmation of the expression of activating protein-1 family members by reverse transcriptase polymerase chain reaction revealed a preferential increase in junB, a known transcriptional antagonist of c-jun. The failure to observe induction of early growth response gene-1, which was detected by reverse transcriptase polymerase chain reaction to be substantially and transiently induced by 1 hour of IL-1 treatment, may be explained by the known instability of the message after early induction. However, this analysis has identified numerous IL-1beta-responsive genes that warrant further investigation as mediators of disease in arthritis.
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PMID:Early response genes induced in chondrocytes stimulated with the inflammatory cytokine interleukin-1beta. 1171 93

Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate molecular and structural changes occurring in four articular cartilage (AC) regions from the knees of anterior cruciate ligament (ACL)-transected rabbits at 3 and 8 weeks post-surgery. Rabbit AC from the lateral and medial femoral condyles (LFC and MFC) as well as from the medial and lateral tibial plateau (MTP and LTP) were processed for histology and for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules (collagen II, aggrecan. biglycan, decorin, fibromodulin, MMP-1, -3, -13, and TIMP-1). While the most severe histological changes were observed in the MTP starting as early as 3 weeks post-ACL transection based on Mankin scores, histological examination demonstrated a progression of osteoarthritic changes in the MFC from 3 to 8 weeks post-surgery. In contrast, very few changes were observed within both the LFC and LTP, and these changes did not worsen with increasing time after surgery. The water content increased significantly in the MFC at 8 weeks post-ACL transection and at both 3 and 8 weeks post-ACL transection in the MTP. Significant decreases in DNA content were observed for the MFC, LTP and MTP at 8 weeks post-ACL transection. Total RNA yields from the MFC and MTP were significantly elevated at 8 weeks post-ACL transection, while in the lateral compartment total RNA was unchanged following ACL transection. Analysis of mRNA levels for a subset of matrix molecules, proteinases and proteinase inhibitors, by RT-PCR demonstrated significant region-specific changes at the mRNA level following ACL transection. These results show that following ACL transection, complex molecular, as well as structural changes occur early in cartilage and that the observed changes are both region-specific and time-dependent.
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PMID:Assessment of specific mRNA levels in cartilage regions in a lapine model of osteoarthritis. 1203 28

Although the complement system is implicated in the inflammatory process in arthritic diseases, a direct interaction between chondrocytes and complement has not been demonstrated. In this study, we investigated expression of the C5a receptor (C5aR) on chondrocytes of cartilage from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and bone fracture as normal controls by reverse transcriptase polymerase chain reaction (RT-PCR), flow cytometry, and immunohistochemistry. The RT-PCR detected mRNA for C5aR in most or all of the tested samples (73% in OA, 100% in RA, 89% in normal). The FACS analysis revealed different expression ratios between individuals varying from 0.7% to 77.1%; however, expression ratios of C5aR were significantly higher in RA than in controls (26.0% in RA, 9.0% in OA, 6.9% in normal). The expression of C5aR was upregulated significantly by addition of IL-1beta in RA and normal samples but not in OA. In addition, the C5aR-positive chondrocytes were confirmed by immunohistochemistry. In conclusion, expression of C5aR and the effect of IL-1beta on the expression were different between RA and OA. The C5aR may contribute to chondrocyte metabolism and the pathogenesis of arthritis differently between in RA and OA.
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PMID:Expression of the anaphylatoxin receptor C5aR (CD88) by human articular chondrocytes. 1207 Jun 75

The objective of the current study was to determine whether the balance of interleukin-1 and intracellular interleukin-1 receptor antagonist in chondrocytes in osteoarthritic human joints favors agonist action. Chondrocytes were isolated from cartilage specimens taken at the time of joint arthroplasty. Interleukin-1alpha, interleukin-1beta, and intracellular interleukin-1 receptor antagonist messenger ribonucleic acids were assessed by reverse transcriptase-polymerase chain reaction, and chondrocyte lysates were analyzed by enzyme-linked immunosorbent assay for the respective proteins. Type I intracellular interleukin receptor antagonist transcripts were the only intracellular variant detected in osteoarthritis chondrocytes. In cartilage graded as advanced osteoarthritis both interleukin proteins in chondrocyte lysates decreased, correlating with decreased interleukin-1alpha and beta messenger ribonucleic acids. Interleukin-1 receptor antagonist exceeded interleukin-1alpha in chondrocyte lysates by one order of magnitude except that in moderate osteoarthritis, antagonist was only two- to fourfold in excess. Interleukin-1alpha and interleukin-1beta proteins were correlated closely in individual lysates, with interleukin-1beta exceeding interleukin-1beta by one order of magnitude. In moderately degenerated cartilage, intracellular antagonist may not be sufficiently abundant to block postulated intracellular functions of precursor interleukin-1alpha. Furthermore, if stored interleukin-1alpha, interleukin-1beta, and interleukin receptor antagonist are released from chondrocytes, the localized antagonist would be insufficient to prevent signaling through cell surface receptors. Chondrocyte-derived interleukin-1alpha and interleukin-1beta may locally overwhelm inhibition by interleukin receptor antagonist to promote the early degenerative changes in osteoarthritis.
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PMID:Intracellular interleukin-1 receptor antagonist in osteoarthritis chondrocytes. 1267 14

Osteoarthritis is a significant, debilitating disease that afflicts millions of Americans, yet its etiology is poorly understood. However, there is substantial evidence that biomechanical factors play a role in the development and progression of osteoarthritis. Previous work has demonstrated that biomechanical factors such as an acute insult or the cumulative effects of repetitive loads can induce degenerative changes in joints, cartilage explants, and isolated chondrocytes. Nevertheless, all of these studies suffer from the limitation that the precise nature of the mechanical loads experienced by individual cells is not well defined. Implementation of a single-cell approach, employing existing cell mechanics methodologies and molecular techniques such as single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), offers an exciting new means to identify which biomechanical factors precipitate pathological changes in chondrocytes indicative of osteoarthritis. This article reviews the particular methods used in mechanical studies of single cells with emphasis on techniques that have been used to investigate chondrocytes and similar anchorage-dependent cell types.The fundamentals of RT-PCR and its application at the single-cell level are also discussed.
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PMID:Biomechanics of single chondrocytes and osteoarthritis. 1273 53

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.
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PMID:Detection of oncofetal h19 RNA in rheumatoid arthritis synovial tissue. 1293 31

Osteoarthritis (OA) is a heterogeneous disease which rheumatologists consider to be noninflammatory. However, recent studies suggest that, at least in certain patients, OA is an inflammatory disease and that patients often exhibit inflammatory infiltrates in the synovial membranes (SMs) of macrophages and activated T cells expressing proinflammatory cytokines. We report here that the expression of CD3zeta is significantly decreased in T cells infiltrating the SMs of patients with OA. The CD3zeta chain is involved in the T-cell signal transduction cascade, which is initiated by the engagement of the T-cell antigen receptor and which culminates in T-cell activation. Double immunofluorescence of single-cell suspensions derived from the SMs from nine patients with OA revealed significantly increased proportions of CD3epsilon-positive (CD3epsilon+) cells compared with the proportions of CD3zeta-positive (CD3zeta+) T cells (means +/- standard errors of the means, 80.48% +/- 3.92% and 69.02% +/- 6.51%, respectively; P = 0.0096), whereas there were no differences in the proportions of these cells in peripheral blood mononuclear cells (PBMCs) from healthy donors (94.73% +/- 1.39% and 93.79% +/- 1.08%, respectively; not significant). The CD3zeta+ cell/CD3epsilon+ cell ratio was also significantly decreased for T cells from the SMs of patients with OA compared with that for T cells from the PBMCs of healthy donors (0.84 +/- 0.17 and 0.99 +/- 0.01, respectively; P = 0.0302). The proportions of CD3epsilon+ CD3zeta+ cells were lower in the SMs of patients with OA than in the PBMCs of healthy donors (65.04% +/- 6.7% and 90.81% +/- 1.99%, respectively; P = 0.0047). Substantial proportions (about 15%) of CD3epsilon+ CD3zeta-negative (CD3zeta-) and CD3epsilon-negative (CD3epsilon-) CD3zeta- cells were found in the SMs of patients with OA. Amplification of the CD3zeta and CD3delta transcripts from the SMs of patients with OA by reverse transcriptase PCR consistently exhibited stronger bands for CD3delta cDNA than for CD3zeta cDNA The CD3zeta/CD3delta transcript ratio in the SMs of patients with OA was significantly lower than that in PBMCs from healthy controls (P < 0.0001). These results were confirmed by competitive MIMIC PCR. Immunoreactivities for the CD3zeta protein were detected in the SMs of 10 of 19 patients with OA, and they were of various intensities, whereas SMs from all patients were CD3epsilon+ (P = 0.0023). The decreased expression of the CD3zeta transcript and protein in T cells from the SMs of patients with OA relative to that of the CD3epsilon transcript is suggestive of chronic T-cell stimulation and supports the concept of T-cell involvement in OA.
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PMID:Decreased expression of the CD3zeta chain in T cells infiltrating the synovial membrane of patients with osteoarthritis. 1471 68

MMP-9 or Gelatinase B, a member of the matrix metalloproteinase family (MMPs), plays important roles in physiological events such as tissue remodeling and in pathological processes that lead to destructive bone diseases, including osteoarthritis and periodontitis. In addition to its effect on the increase of total bone mass, statin (an HMG-CoA reductase inhibitor) suppresses the expression of MMPs. In this study, we proposed that simvastatin reduces MMP-9 expression in osteoblasts and HT1080 fibrosarcoma cell line. Gelatin zymography, Western blot analysis and reverse transcriptase-PCR were used to investigate the effects of simvastatin on MMP-9 in primary calvaria cells, U2-OS osteosarcoma cells, and HT1080 fibrosarcoma cells. The results from gelatin zymography and Western blot analysis revealed that simvastatin suppressed MMP-9 activity in these cells in concentration- and time-dependent manners. The effective concentrations of simvastatin were 100 - 500 nM, 5 - 15 microM, and 2.5 - 10 microM in primary calvaria, U2-OS, and HT1080 cells, respectively. Collectively, these results suggest that simvastatin is a potent drug for inhibition of MMP-9 expression in osteoblastic cells and HT1080 fibrosarcoma cells.
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PMID:Simvastatin, an HMG-CoA reductase inhibitor, reduced the expression of matrix metalloproteinase-9 (Gelatinase B) in osteoblastic cells and HT1080 fibrosarcoma cells. 1510 80

Hyaluronan is a major molecule in joint fluid and plays a crucial role in joint motion and the maintenance of joint homeostasis. The concentration and average molecular weight of hyaluronan in the joint fluids are reduced in osteoarthritis and rheumatoid arthritis. To elucidate the underlying mechanism, we analyzed the message expression of three isoforms of hyaluronan synthase and hyaluronidase from knee synovium, using real-time reverse transcriptase polymerase chain reaction. Synovia were obtained from 17 patients with osteoarthritis, 14 patients with rheumatoid arthritis, and 20 healthy control donors. The message expression of hyaluronan synthase-1 and -2 in the synovium of both types of arthritis was significantly less than in the control synovium, whereas that of hyaluronidase-2 in the synovium of both arthritides was significantly greater than in the control synovium. The decreased expression of the messages for hyaluronan synthase-1 and -2 and/or the increased expression of the message for hyaluronidase-2 may be reflected in the reduced concentration and decreased average molecular weight of hyaluronan in the joint fluids of patients with osteoarthritis and rheumatoid arthritis.
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PMID:Expression analysis of three isoforms of hyaluronan synthase and hyaluronidase in the synovium of knees in osteoarthritis and rheumatoid arthritis by quantitative real-time reverse transcriptase polymerase chain reaction. 1553 29

In the present study, we have investigated the presence of pro-opiomelanocortin C-terminal fragment derived-peptides in human articular cartilage and cultured chondrocytes. beta-Lipotropin and beta-endorphin were monitored in different cell cultures and biopsies using different techniques. Biopsies were taken from patients undergoing total knee arthroplasty due to osteoarthritis. Both fresh tissue sections and chondrocytes cultured in monolayer were used in the study. Immunohistochemistry, immunocytochemistry, reverse transcriptase-polymerase chain reaction and qualitative Western blots were carried out. The results of the reverse transcriptase-polymerase chain reaction showed transcription of a truncated-form of mRNA for pro-opiomelanocortin in native cartilage and cultured chondrocytes. There was no detection of endogenous production of beta-lipotropin or beta-endorphin in human articular chondrocytes, either in situ or in vitro. Whether pro-opiomelanocortin-derived peptides of non-cartilaginous origin are present in articular cartilage itself still remains unclear.
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PMID:Detection of mRNA transcripts of truncated opiate precursor (POMC) in human cartilage. 1589 26


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