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)

Psoriasis is histologically characterized by hyperkeratosis and papillomatosis with elongated vessels in the upper dermis. In order to evaluate the role of gelatinases in remodelling psoriatic skin in this study we examined the production of the 72-kDa (gelatinase A), 92-kDa collagenase (gelatinase B) and their tissue inhibitors TIMP-2 and TIMP-1. A total of 19 patients affected by different types of psoriasis were included in this study. An immunohistochemical study on cryosections was performed using antibodies to 72-kDa gelatinase, 92-kDa gelatinase, TIMP-1, TIMP-2, laminin, collagen types I, III, IV, VII. mRNA expression for gelatinases and their inhibitors were also analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). In 14 of 19 patients there was a positivity in 92-kDa protein expression in keratinocytes. The 92-kDa gelatinase protein was also present in the upper dermis with prevalence around blood vessels. In 15 of 19 patients the 72-kDa was localized in the upper dermis, almost exclusively in the papillary dermis but absent in epidermis. TIMP-1 and TIMP-2 were both negative in all cases in immunoperoxidase and RT-PCR. Using RT-PCR we show that the 72-kDa mRNA is expressed exclusively in the dermis, on the contrary the 92-kDa was present in epidermis and dermis. Type I, III, IV and VII collagens did not show any alteration or disruption. Overexpression and production of gelatinases without inhibitory effects suggest a role of these proteins in remodelling the psoriatic skin probably inducing the typical histological pattern of papillomatosis.
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PMID:The 72-kDa and the 92-kDa gelatinases, but not their inhibitors TIMP-1 and TIMP-2, are expressed in early psoriatic lesions. 941 21

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain-containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase-polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis.
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PMID:Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1. 977 30

Lipodermatosclerosis refers to skin induration of the lower extremities and is associated with patients preceding venous ulcerations. To better understand the pathogenesis of ulcer formation we investigated the expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in lipodermatosclerosis. By preparing biopsies from healthy skin and liposclerotic lesions, MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were analyzed by using reverse transcriptase-polymerase chain reaction, western blot, zymography, hydrolysis of [3H]labeled collagens, and immunohistochemistry. Our investigations provide evidence that mRNA and protein expression of MMP-1, MMP-2, and TIMP-1 were significantly increased in lipodermatosclerosis, whereas the total amount of MMP-9 and TIMP-2 mRNA and protein was not altered. Western blot of liposclerotic lesions revealed an inactive proMMP-1-TIMP-1 complex, whereas MMP-2 was prominent as an active 66 kDa band. Increased proteolytic activity of MMP-2 could be proven in lesional in comparison with healthy skin by zymography and [3H] collagen degradation. Increased diffuse staining was found for MMP-1 in the epidermis and dermis in comparison with controls. In lipodermatosclerosis, MMP-2 was predominantly localized in the basal and suprabasal layers of the epidermis, in perivascular regions, and in the reticular part of the dermis. Furthermore, MMP-2 was imbalanced by locally reduced expression of TIMP-2 in the basement membrane zone of lesional skin. Our findings indicate lipodermatosclerosis to be characterized by elevated matrix turnover.
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PMID:Lipodermatosclerosis is characterized by elevated expression and activation of matrix metalloproteinases: implications for venous ulcer formation. 1038 52

Membrane-type matrix metalloproteinases (MT-MMP) activate the zymogen form of MMP-2/Gelatinase A on cell surfaces and are expressed in invasive tumors. We sought to identify and characterize MT-MMP in a non-malignant cell type that undergoes a physiologic and reversible invasive phenotype during angiogenesis. Human dermal microvascular endothelial cells (HDMEC) were isolated from neonatal tissue and purified by anti-CD31 (PECAM) affinity beads. MT-MMP-1 and -3 transcripts were amplified by reverse transcriptase-polymerase chain reaction and northern blots showed a single 4.5 kB mRNA for MT-MMP-1 that was modulated by angiogenic factors and phorbol ester. Immunoblotting of reduced cellular extracts with different MT-MMP-1 antibodies showed the presence of the 63-65 kDa and 57-60 kDa forms, as well as additional forms at lower molecular weights. HDMEC membranes extracted with Triton X114 were incubated with gelatin-sepharose purified MMP-2 and MMP-9 to show activation of proenzymes. Pre-incubation of HDMEC with anti-MT-MMP-1 antibodies decreased proMMP-2 conversion activity only. The movement of HDMEC and the formation of tubule-like structures in three-dimensional collagen gels was markedly delayed by preincubation with the same anti-MT-MMP-1 antibodies. These results demonstrate the presence of MT-MMP in cutaneous microvascular cells in vitro. Modulation of these cell surface proteinases by angiogenic factors, demonstration of multiple processed forms, and specific attenuation of HDMEC morphogenetic patterns in three-dimensional collagen gels implicate their potential roles in the formation of new blood vessels in the skin.
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PMID:Membrane-type matrix metalloproteinases in human dermal microvascular endothelial cells: expression and morphogenetic correlation. 985 32

Our aim was to investigate the collagenolytic potential and localization of matrix metalloproteinase-2 (MMP-2) in relation to its regulatory proteins membrane type MT1-MMP and tissue inhibitor of metalloproteinases-2 (TIMP-2) in rheumatoid arthritis (RA). For this purpose, we have used purification of MMP-2, MMP-8, MMP-9 and interstitial type I, II and III collagens; SDS-PAGE/densitometric collagenase activity assay; zymography; Western blotting; reverse transcriptase polymerase chain reaction; in situ hybridization; and immunofluorescence, ABC, ABC-APAAP double immunostainings. MMP-2 degraded human type II collagen almost as effectively as MMP-8, whereas MMP-9 did not cleave type II collagen. In synovial tissue, MT1-MMP, TIMP-2 and MMP-2 were found in synovial lining in fibroblast- and macrophage-like cells, in stromal cells and in vascular endothelium. MT1-MMP, TIMP-2 and MMP-2 were strongly expressed in the pannocytes of the invasive pannus at the interface, but staining was weak and/or there were few positive cells both "above" and "below" the soft-to-hard tissue (cartilage and/or bone) interface. Rheumatoid synovial tissue extract contained proteolytically active 62/59 kDa MMP-2 and 43 kDa MT1-MMP, but no free TIMP-2. These results indicate that components of the ternary MT1-MMP/TIMP-2/MMP-2 complex are coexpressed in the normal synovial lining and in its pathological extension on the hyaline articular cartilage. MMP-2 may participate in the remodeling of the normal lining and also seems to be localized/focalized to pannocytes at a site critical for tissue destruction in arthritis.
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PMID:New collagenolytic enzymes/cascade identified at the pannus-hard tissue junction in rheumatoid arthritis: destruction from above. 992 52

Irreversible congenital heart block (CHB) and the transient rash of neonatal lupus are strongly associated with maternal antibodies to SSA/Ro and SSB/La proteins; however, the precise mechanism by which these antibodies mediate organ-specific injury is not yet defined. Culturing of keratinocytes has provided critical insights. Accordingly, successful culturing of human fetal cardiac myocytes at high yield would constitute a powerful tool to directly examine conditions that promote expression of the target autoantigens. To accomplish this aim, fetal cardiac myocytes from 18- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusion of the aorta with collagenase in a Langendorff apparatus. After preplating to decrease fibroblast contamination, cardiocytes were grown in flasks and slide chambers. Staining with monoclonal anti-sarcomeric alpha-actinin revealed the expected striations typical of cardiac myocytes in 70-90% of the cells after 4 d in culture. Furthermore, the cells were observed to beat at rates varying between 25-75 beats per minute (bpm) after the addition of 1.8 mM CaCl2. An average yield of 45-60 x 10(6) cells was obtained from a 3- to 5-g heart. Cellular localization of SSA/Ro and SSB/La by indirect immunofluorescence and demonstration of mRNA expression by reverse transcriptase polymerase chain reaction supports the feasibility of cultured cardiac myocytes for the study of congenital heart block. In contrast to the increased expression of SSA/Ro reported for keratinocytes, incubation of cultured human cardiac myocytes with either 17beta-estradiol or progesterone did not alter mRNA expression or cellular localization of 48 kD SSB/La, 52 kD SSA/Ro, or 60 kD SSA/Ro. In summary, we describe a novel method to successfully culture human fetal cardiac myocytes that should provide a valuable resource for investigation of the molecular mechanism(s) contributing to the development of congenital heart block. Differential constitutive and estradiol-induced expression of 52 and 60 kD SSA/Ro in human cardiac myocytes compared with keratinocytes may be a factor contributing to the marked discordance of clinically detectable injury in these two target tissues.
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PMID:mRNA and protein expression of SSA/Ro and SSB/La in human fetal cardiac myocytes cultured using a novel application of the Langendorff procedure. 1002

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of fibrous gingival overgrowth. The purpose of this study was to investigate the effect of CsA on the type I collagen metabolism in the gingiva of rats fed a powdered diet either containing or lacking CsA. Immunohistochemical analysis revealed that type I collagen was more prevalent in the connective tissue of CsA-treated gingiva than in those of control rats on days 15, 30, and 55 after the start of feeding. Total RNAs were isolated from mandibular molar gingiva on days 0, 3, 8, 15, 30, and 55. Quantitative analysis of mRNA by reverse transcriptase-polymerase chain reaction revealed that the CsA-treated groups showed a gradual decrease in expression of type I collagen and collagenase mRNAs, 0.4% and 18.0% on day 55 compared with those on day 0, respectively. In the control groups, type I collagen and collagenase mRNAs also decreased to 19.7% and 63.0%, respectively, however, both mRNA expressions were significantly lower in the CsA-treated group than in the controls. An electron microscopic analysis of fibroblasts was performed to count the number of cells with collagen fibrils in the cytoplasm, a marker of phagocytosis of collagen by fibroblasts. The collagen fibrils were detected in 4.7% +/- 2.7% and 24.3% +/- 13.7% of fibroblasts in the overgrown gingiva treated with CsA rat for 8 days and 30 days, but in 57.0% +/- 5.3% and 81.3% +/- 9.2% of fibroblasts in the each control group gingiva, respectively. Furthermore, in vitro analysis was performed to measure the phagocytosis of cultured fibroblasts by flow cytometry using collagen-coated latex beads. Fibroblasts isolated from CsA-treated gingiva on day 8 and day 30 contained 5.7% +/- 0.6% and 9.9% +/- 1.5% phagocytic cells, whereas control fibroblasts contained 50.3% +/- 5.5% and 33.3% +/- 4.9% phagocytic cells, respectively. The inhibition rate of phagocytic activity was similar between in vivo and in vitro assays. These findings suggest that the decrease of the collagen degradation due to the lower phagocytosis and the lower collagenase mRNA expression are closely associated with the increase of type I collagen accumulation in CsA-treated rat gingiva.
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PMID:Cyclosporin A decreases the degradation of type I collagen in rat gingival overgrowth. 1065 1

Calbindin D28k has been reported to be involved in the transcellular calcium transport along the rat distal tubule. It has also been shown that chronic metabolic acidosis (CMA) induces significant hypercalciuria. The present study investigated whether CMA affects the mRNA and the protein expression of calbindin D28k along isolated distal tubule (DT) of rats. The animals were made acidotic by adding 0.28 mol/L NH4Cl to the drinking water for 7 d. This maneuver was associated with an increase in plasma ionized calcium. Inulin clearance experiments demonstrated that metabolic acidosis did not affect GFR, but it significantly increased both total and fractional urinary calcium excretion. To define the role of calbindin D28k, total RNA was extracted from DT, identified, and microdissected from collagenase-treated kidneys. cDNA was synthesized from RNA using reverse transcriptase and oligo(dT)(12-18) primers. Calbindin D28k mRNA abundance was semiquantified by a competitive reverse transcription-PCR, using an internal standard of cDNA that differed from the wild-type calbindin D28k by a deletion of 86 bp. The reverse transcription-PCR was performed starting from the same amount of total RNA. For each set of experiments, control and acidotic rats were studied in parallel. The identity of the DT was further verified by the presence of the thiazide-sensitive NaCl cotransporter (rTSC1) mRNA. Calbindin D28k mRNA abundance was 0.89 +/- 0.21 amol/ng total RNA in DT of CMA rats (n = 5) compared with 0.30 +/- 0.12 amol/ng total RNA of control rats (n = 5) (P < 0.05). Using specific rabbit polyclonal anti-calbindin D28k antibody, Western blotting was performed starting from thin slices of outer cortex. Densitometric analysis revealed that in acidotic rats (n = 7) there was a 17 +/- 5% (P < 0.05) increase in calbindin D28k protein abundance compared with controls (n = 7). These results indicate that in the rat, ammonium chloride loading induces an increase in filtered ionized calcium load that is associated with a significant upregulation of calbindin D28k both at the mRNA and protein level. These last effects will help to reduce the concomitant hypercalciuria, thus mitigating the consequence of CMA on calcium metabolism.
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PMID:Effect of chronic metabolic acidosis on calbindin expression along the rat distal tubule. 1066 27

The aim of this study was to examine the effects of intraarticular administration of hyaluronan (HA) on cartilage degradation. Using a partial menisectomy model of osteoarthritis (OA) in the rabbit knee, the authors investigated the catabolic and anabolic changes induced by intraarticular injection of HA. To analyze anabolic changes, the authors assessed cell proliferation by measuring [3H] thymidine uptake, and proteoglycan biosynthesis by noting [35S] sulfate incorporation. For catabolic changes, messenger ribonucleic acid (mRNA) expression of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in cartilage and synovium were detected with reverse transcriptase polymerase chain reaction (RT-PCR). Of significance for blocking the development of early OA in chondrocytes was the finding that total proteoglycan synthesis in the HA treatment group was significantly higher than in the controls. At the mRNA level in cartilage and synovium, HA inhibited MMP-3 and TIMP-1 production in the same way in the HA treatment group, while not affecting MMP-1 production. Thus it can be concluded that HA affects cartilage catabolism and anabolism to prevent the progress of OA.
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PMID:Effects of sodium hyaluronate on experimental osteoarthritis in rabbit knee joints. 1068 73

Hereditary gingival fibromatosis (HGF) is characterized by an excess accumulation of extracellular matrix (ECM) resulting in a generalized and fibrotic enlargement of the gingiva. To investigate some of the regulatory features of this condition, gingival fibroblasts from normal gingiva (NG) and HGF were examined for the expression and production of matrix metalloproteinases (MMPs) and their inhibitors, tissue matrix metalloproteinases inhibitor (TIMPs). Our results, obtained from 2 different assays, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography, clearly demonstrated that the expression and production of MMP-1 and MMP-2 was significantly lower in fibroblasts from HGF than from NG. Interestingly, TIMP-1 and TIMP-2 expression from NG cells was shown to be slightly higher to those from HGF. Addition of antibodies against transforming growth factor-beta 1 (TGF-beta 1), which is produced in greater amounts by HGF fibroblasts, resulted in a slight increase in MMP-1 and a decrease in MMP-2 expression, whereas TIMP-1 and TIMP-2 expressions were unaffected. These patterns of expression and production suggest that enhanced TGF-beta 1 production reduce the proteolytic activities of HGF fibroblasts, which favor the accumulation of ECM.
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PMID:Alteration in expression of MMP-1 and MMP-2 but not TIMP-1 and TIMP-2 in hereditary gingival fibromatosis is mediated by TGF-beta 1 autocrine stimulation. 1069 2


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