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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Islets from male B10.BR mice (H-2k) were isolated by the collagenase technique, handpicked with a Pasteur pipette, and incubated (either intact or after dispersion with Dispase) for 0, 3, 5, 7, 10, or 14 days in tissue culture medium supplemented with either lymphokine supernatants or recombinant murine interferon-gamma. Islets and single cells were examined for IAk molecules by use of indirect immunofluorescence. Ia-positive islet cells were identified on the surface of islets incubated with 5-10% lymphokine for greater than 4 days or with 10, 100, or 1000 ng/ml interferon for greater than 6 days. Islets incubated in unsupplemented medium were Ia negative. Incubation with 5% lymphokine induced Ia expression on 10-40% dispersed islet cells cultured for greater than 9 days. Dual immunofluorescent staining for Ia and insulin revealed that Ia-positive cells included both beta- and non-beta-cells.
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PMID:Interferon-mediated induction of Ia antigen expression on isolated murine whole islets and dispersed islet cells. 309

Islets from male B10.BR mice (H-2k) were isolated by the collagenase technique, hand-picked with a Pasteur pipette, and incubated in tissue culture media supplemented with recombinant murine interferon-gamma (rIFN-gamma), recombinant murine tumor necrosis factor (rTNF), or both. IAk-molecules could be identified on the surface of islets incubated for 5 days with a combination of rIFN-gamma (1, 10, or 100 ng/ml) and rTNF (10 or 50 U/ml) by indirect immunofluorescence. Optimal concentrations of rIFN-gamma and rTNF, 10 ng/ml and 50 U/ml, respectively, were used in all subsequent experiments. Weak Ia-positivity could be identified on the surface of islets cultured with both cytokines for as little as 48 hours; however, the staining appeared most intense after 5 days of culture. Intensely Ia-positive islets were then carefully washed and cultured in media without either cytokine; Ia positivity could be identified on the surface of these islets for up to 1 week. Dispersed islet cells were cultured with rIFN-gamma alone (10 ng/ml), rTNF alone (50 U/ml), or both cytokines for 5 or 10 days. After either 5 or 10 days of culture with both cytokines, intense immunofluorescent staining for Ia could be identified on the surface of greater than 80-90% of the viable islet cells. Culture with IFN alone for 10 days resulted in 15-20% Ia positivity; culture with TNF alone did not cause Ia expression.
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PMID:Tumor necrosis factor enhances interferon-induced Ia antigen expression on murine islet parenchymal cells. 312 60

The immunologic competence of human placental mononuclear cells was compared to that of adult and cord blood mononuclear cells. Mononuclear cells were isolated from fresh placentas by digestion with collagenase and DNase, followed by Ficoll-Hypaque and discontinuous Percoll separation. Placental cells incubated with phytohemagglutinin (PHA) synthesized significantly more interferon-gamma (IFN-gamma) at 2 days (29 +/- 5.5 IU/ml) and 5 days (46 +/- 8.5 IU/ml) than PHA-activated cord cells (3.6 +/- 0.6 IU/ml at 2 days and 2.7 +/- 0.7 IU/ml at 5 days) but less than PHA-activated adult cells (81 +/- 20 IU/ml at 2 days and 270 +/- 161 IU/ml at 5 days). Placental and adult cells, but not cord cells, also synthesized significant quantities of IFN-gamma following incubation with interleukin-2 (IL-2). There was synergism between IL-2 and PHA activation for IFN-gamma production for some cord samples. After a 5- to 7-day incubation with IL-2, the lymphocyte-activated killer (LAK cell) cytotoxicity of placental cells (measured in a 3-hr chromium-release assay at an E:T ratio of 40:1) was enhanced 13-fold against K562 target cells (6 +/- 2% to 77 +/- 4%) compared to a 4-fold increase in cord cells (16 +/- 4% to 68 +/- 3%) and a 2-fold increase in normal adult cells (35 +/- 4% to 65 +/- 3%. Against the natural killer (NK)-resistant Raji target, placental cells increased their LAK cytotoxic activity (3 +/- 1% to 59 +/- 7%) compared to a 7-fold increase with cord cells (6 +/- 1% to 43 +/- 3%) and a 3-fold increase with adult cells (11 +/- 2% to 38 +/- 4%). A notable degree of cytotoxic activity in the absence of IL-2 against Molt targets was noted in 11 of 14 (79%) placental cell samples at 5 days. Only 10 of 24 (42%) adult and 17 of 37 (40%) cord samples showed spontaneous cytotoxic activity equal to or greater than 10%. Some placental samples actually showed an increase in cytotoxic activity when incubated without IL-2. The ability of placental cells to produce significant levels of IFN-gamma, to develop considerable LAK activity, and to maintain or develop cytotoxic activity in the absence of IL-2 suggests a vigorous, active immune system of the placenta compared to the relatively dormant immune system of the neonate. These observations suggest that placental cells may have a primary role in fetal defense.
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PMID:Enhanced interferon production and lymphokine-activated cytotoxicity of human placental cells. 313 Jan 92

Nitric oxide (NO) is a messenger molecule that is produced from L-arginine by NO synthase (NOS). Some NOS isoforms are present in cells constitutively, whereas others can be induced by cytokines. Recent evidence suggests that NO inhibits intracellular pH regulation by the vacuolar H(+)-adenosinetriphosphatase (ATPase) in macrophages, which contain an inducible form of NOS. The vacuolar H(+)-ATPase is involved in proton secretion in intercalated cells in the collecting duct. We have therefore examined the effect of NO on bafilomycin-sensitive H(+)-ATPase activity in individual cortical collecting ducts (CCD) microdissected from collagenase-treated kidneys of normal rats using a fluorometric microassay. Incubation of CCD with the NO donors, sodium nitroprusside (0.1 and 1 mM) or 3-morpholino-sydnonimine hydrochloride (SIN-1, 30 microM), caused a dose-dependent decrease in H(+)-ATPase activity. Incubation of CCD with lipopolysaccharide (LPS) and interferon-gamma, which induces NOS in macrophages, decreased H(+)-ATPase activity by 85%. This effect was prevented by simultaneous incubation with N omega-nitro-L-arginine, a competitive inhibitor of NOS, indicating that the decrease in H(+)-ATPase activity was caused by NO production. Incubation with 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP) also inhibited H(+)-ATPase activity, suggesting that NO may exert its effect in the CCD via activation of guanylyl cyclase and production of cGMP. Immunohistochemistry using antibodies to the macrophage-type NOS revealed strong labeling of intercalated cells in the CCD, confirming the presence of NOS in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nitric oxide inhibits bafilomycin-sensitive H(+)-ATPase activity in rat cortical collecting duct. 752 55

Nitric oxide is a highly reactive mediator released in the liver by hepatocytes, Kupffer cells and endothelial cells during endotoxin-induced inflammation. In this study we determined whether Ito cells also produce nitric oxide after exposure to endotoxin. For induction of endotoxemia, rats were injected intravenously with Escherichia coli lipopolysaccharide (2.5 mg/kg). Ito cells were isolated from the animals 48 hr later by means of in situ perfusion of the liver with protease and collagenase followed by purification on an arabinogalactan gradient. Ito cells from untreated and endotoxemic rats were found to produce low levels of nitric oxide in response to interferon-gamma. In both cell types, this response depended on L-arginine and was blocked by NG-monomethyl-L-arginine, a specific nitric oxide synthase inhibitor. Cells from rats treated with endotoxin produced significantly more nitric oxide than did cells from untreated animals; this was due, at least in part, to increased expression of protein for an inducible form of nitric oxide synthase. These cells also responded to stimulation with lipopolysaccharide in vitro, as well as the combination of interferon-gamma and lipopolysaccharide, which was synergistic in stimulating nitric oxide production. Tumor necrosis factor-alpha and macrophage colony-stimulating factor were also found to stimulate nitric oxide production by Ito cells from endotoxemic rats. In addition, in these cells, tumor necrosis factor-alpha synergized with interferon-gamma in inducing nitric oxide production. The combination of interferon-gamma and lipopolysaccharide was also found to inhibit Ito cell DNA synthesis, as measured on the basis of [3H]-thymidine uptake.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of hepatic Ito cell nitric oxide production after acute endotoxemia. 752 4

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin. 752 31

Matrix metalloproteinases (MMPs) are a family of inducible enzymes that degrade extracellular matrix components, allowing cells to traverse connective tissue structures efficiently. Specific tissue inhibitors (TIMPs) function as physiologic inhibitors of MMP activity. Because neovascularization may require various proteinases, we characterized the profile of metalloenzyme production by microvascular endothelial cells (MEC) and the modulation of expression by phorbol esters (PMA) and by the physiologically relevant cytokines tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor, and interferon-gamma. MMP expression by MEC and large-vessel human umbilical vein endothelial cells (HUVEC) was determined by enzyme-linked immunosorbent assay, immunoprecipitation, Northern hybridization, and transfection assays. Constitutive expression of MMPs by endothelial cells was low. PMA stimulated the production of collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 in both endothelial cell types. TIMP-2 was constitutively expressed by MEC and HUVEC, but was down-regulated by PMA. TNF-alpha induced an endothelial-cell-specific up-regulation of collagenase with a concomitant inhibition of PMA-induced TIMP-1 up-regulation, a response that is distinct from that of fibroblasts. Interferon-gamma up-regulated TIMP-1 production by MEC and blocked PMA and TNF-induced up-regulation of collagenase. Northern hybridization assays showed pretranslational control of PMA-, basic fibroblast growth factor-, and TNF-alpha-induced MMP expression. Collagenase-promoter CAT constructs containing 2.28 kb of the 5' region of the collagenase gene demonstrated transcriptional regulation. The potential physiologic relevance of such regulation was shown in an in vitro migration assay. MEC were stimulated to migrate by wounding and exposure to TNF-alpha. Collagenase mRNA was prominently expressed by the migrating cells, as shown by in situ hybridization. In sum, MEC have a unique profile of MMP expression and regulation compared with other cell types, which may be important for wound healing and angiogenesis, particularly during the early phase of migration.
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PMID:Human dermal microvascular endothelial cells produce matrix metalloproteinases in response to angiogenic factors and migration. 754 47

Tumor necrosis factor-alpha (TNF-alpha) is a potent inhibitor of connective tissue formation. The cellular effects of TNF-alpha are mediated by two distinct cell-surface receptors, TNF-R55 and TNF-R75, both present on various types of cells, including fibroblasts. In this study we wanted to elucidate the role of TNF-R55 as a mediator of the connective tissue effects of TNF-alpha by using a mutant, TNF-R55-specific form of human TNF-alpha. This mutant TNF-alpha markedly induced collagenase and stromelysin-1 gene expression in dermal fibroblasts, the maximal activation (up to 42-fold) being 65%-89% of that noted with wild-type human TNF-alpha. In addition, TNF-R55-specific TNF-alpha suppressed type I collagen mRNA levels as potently as wild-type TNF-alpha (by 60%). The enhancement of collagenase gene expression by TNF-R55-specific TNF-alpha was augmented by simultaneous treatment of normal and scleroderma skin fibroblasts with interferon-gamma, indicating specific enhancement of TNF-R55 signaling pathway by interferon-gamma. These results show that stimulation of the TNF-R55 signaling pathway is sufficient for the inhibitory effects of TNF-alpha on extracellular matrix formation by dermal fibroblasts. It is conceivable that due to reduced systemic toxicity, TNF-R55-specific forms of human TNF-alpha may prove to be feasible in the therapy of fibrotic disorders.
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PMID:TNF-R55-specific form of human tumor necrosis factor-alpha induces collagenase gene expression by human skin fibroblasts. 763 1

Matrix metalloproteases (MMP) constitute a family of proteolytic enzymes degrading extracellular matrix components. Their activity is inhibited by tissue inhibitors of metalloproteases (TIMP). Previous studies have demonstrated that various cytokines can modulate MMP and TIMP gene expression. In this study, we demonstrate that interferon-gamma coordinately upregulates MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) gene expression in cultured keratinocytes, as determined at the mRNA steady-state levels, and this effect is dependent on on-going protein synthesis. In contrast, there was no effect on TIMP-1 gene expression. Enhanced MMP-1 expression by IFN-gamma was also demonstrated at the protein level by Western analysis. Transient transfections with MMP-1 and MMP-3 promoter/reporter gene constructs revealed no response to IFN-gamma, whereas incubation of keratinocytes with this cytokine appeared to stabilize the MMP-1 mRNA, resulting in reduced turnover of the transcript. These data suggest that IFN-gamma enhances MMP gene expression at the post-transcriptional level. The altered MMP expression by IFN-gamma without concomitant effect on TIMP gene expression potentially leads to imbalance between these proteases and their inhibitors, and enhanced proteolytic activity may play a role in the remodeling of cutaneous tissue involving inflammatory processes, such as wound healing.
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PMID:Interferon-gamma coordinately upregulates matrix metalloprotease (MMP)-1 and MMP-3, but not tissue inhibitor of metalloproteases (TIMP), expression in cultured keratinocytes. 786 Oct 7

Treatment of rats with bacterially derived lipopolysaccharide (LPS), a condition that mimics acute endotoxemia, results in a significant increase in the number of endothelial cells and macrophages in the liver. This is correlated with the release of proinflammatory and cytotoxic mediators that induce liver damage. In the present studies, we analyzed the effects of various inflammatory mediators released during the pathogenesis of hepatic injury on proliferation of liver nonparenchymal cells. To induce acute endotoxemia female Sprague-Dawley rats were injected intravenously with 5 mg/kg LPS. Endothelial cells and macrophages were isolated 48 h later by combined collagenase and pronase perfusion of the liver followed by centrifugal elutriation. Interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) had no effect on proliferation of either endothelial cells or macrophages. In contrast, whereas interleukin-1 beta (IL-1 beta) inhibited the proliferation of endothelial cells from untreated rats, this cytokine stimulated the growth of cells from endotoxemic rats. The colony-stimulating factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), also markedly enhanced the proliferation of endothelial cells, as well as macrophages from endotoxemic rats. Macrophages from endotoxemic rats were more sensitive to the colony-stimulating factors than cells from untreated rats. In contrast, the inflammatory mediators LPS and interferon-gamma (IFN-gamma) inhibited endothelial cell and macrophage growth, an effect that was partially blocked in endothelial cells by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). This suggests that growth inhibition in these cells is mediated, in part, by nitric oxide. Interestingly, in both endothelial cells and macrophages from endotoxemic rats, GM-CSF, M-CSF, and IL-1 beta synergized with LPS and IFN-gamma to induce nitric oxide production. This was correlated with a further inhibition of proliferation that was partially reversed by L-NMMA in endothelial cells but not macrophages. Taken together these data demonstrate that endothelial cell and macrophage proliferation in the liver is controlled by a variety of mediators released during endotoxemia; however, the mechanisms regulating growth in the two cell types are distinct.
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PMID:Regulation of hepatic endothelial cell and macrophage proliferation and nitric oxide production by GM-CSF, M-CSF, and IL-1 beta following acute endotoxemia. 814 21


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