Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:009431 (fibronectin)
 32,104 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two kinds of gelatinases (or type IV collagenases), 90-kDa and 64-kDa gelatinases, were purified in a tissue inhibitor of metalloproteinases (TIMP)- or TIMP-2-free form from the serum-free conditioned medium of human schwannoma YST-3 cells, and their activities on extracellular matrix proteins were compared. Sequential chromatographies on a gelatin-Sepharose column, an LCA-agarose column, and a gel filtration column in the presence of 5 M urea yielded 600 micrograms of the 64-kDa enzyme and 45 micrograms of the 90-kDa enzyme from 2.8 liters of the conditioned medium. The purified enzymes showed high gelatinolytic activities without activation by p-aminophenyl mercuric acetate (APMA), indicating that 5 M urea used in the final chromatography not only dissociated the inhibitors from the progelatinases but also activated the proenzymes. The inhibitor-free gelatinases showed a much higher activity than the APMA-activated inhibitor-bound enzymes. The specific activity of the 90-kDa enzyme was nearly 25 times higher than that of the 64-kDa enzyme. The 90-kDa gelatinase hydrolyzed type I collagen as well as native and pepsin-treated type IV collagens at 30 degrees C, while at 37 degrees C it potently hydrolyzed types I, III, and IV collagens but not fibronectin or laminin. The 64-kDa gelatinase showed a similar substrate specificity to that of the 90-kDa enzyme, except that it did not hydrolyze type I collagen and native type IV collagen at 30 degrees C.
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PMID:Comparison of extracellular matrix-degrading activities between 64-kDa and 90-kDa gelatinases purified in inhibitor-free forms from human schwannoma cells. 131 81

To determine if singlet oxygen (O2(1 delta g)) is produced by neutrophils (PMNs) during the process of phagocytosis, glass beads were coated with a specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA). Singlet oxygen, but not other reactive oxygen species, reacts rapidly with DPA at a rate of kr = 1.3 x 10(6) M-1 s-1 to form a stable product, DPA-endoperoxide (Corey, E. J., and Taylor, W. C. (1964) J. Am. Chem. Soc. 86, 3881-3882; Wasserman, H. H., Scheffer, J. R., and Cooper, J. L. (1972) J. Am. Chem. Soc. 94, 4991-4996; Turro, N. J., Chow, M.-F., and Rigaudy, J. (1981) J. Am. Chem. Soc. 103, 7218-7224). The production of DPA-endoperoxide was determined by ultraviolet spectroscopy as a decrease in DPA absorbance at 355 nm. The absorbance of DPA was normalized to the absorbance of perylene, which was included in the coating on the beads as a nonreactive, internal standard. In the present study, DPA- and perylene-coated beads were initially allowed to adhere to fibronectin-coated coverslips. PMNs were then added to the bead-coated coverslips and allowed to adhere and phagocytose the beads for 1 h at 37 degrees C. In some experiments, 4B-phorbol-12-myristate-13-acetate (PMA) (1 ng/2.5 x 10(7) cells/ml), a known activator of the PMN NADPH-oxidase, was added as a co-stimulant. The amount of O2(1 delta g) produced by phagocytically stimulated PMNs was calculated to be 11.3 +/- 4.9 nmol of O2(1 delta g)/1.25 x 10(6) cells. Low dose PMA co-stimulation increased the production of O2(1 delta g) to 14.1 +/- 4.1 nmol/1.25 x 10(6) cells. Averaged together these amounts represent approximately 19 +/- 5.0% of the total oxygen consumed by PMNs in response to DPA- and perylene-coated beads. The specificity of the DPA reaction with O2(1 delta g) was confirmed by warming to 120 degrees C, which releases O2(1 delta g) from the DPA-endoperoxide, regenerating the parent DPA compound (Wasserman et al., 1972; Turro et al., 1981) and the absorbance at 355 nm. In addition, beta-carotene, an avid quencher of O2(1 delta g), was included in the coating of some bead preparations; assays in which these beads were used showed no change in the absorbance at 355 nm. Singlet oxygen production by myeloperoxidase was also measured using the coated bead assay and the results suggest that this is a major pathway by which singlet oxygen is generated in phagocytically stimulated PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap. 132 20

Although the M(r) 72,000 type IV collagenase (matrix metalloproteinase 2) has been implicated in a variety of normal and pathogenic processes, its activation mechanism in vivo is unclear. We have found that fibroblasts from normal and neoplastic human breast, as well as the sarcomatous human Hs578T and HT1080 cell lines, activate endogenous matrix metalloprotease 2 when cultured on type I collagen gels, but not on plastic, fibronectin, collagen IV, gelatin, matrigel, or basement membrane-like HR9 cell matrix. This activation is monitored by the zymographic detection of M(r) 59,000 and/or M(r) 62,000 species, requires 2-3 days of culture on vitrogen to manifest, is cycloheximide inhibitable, and correlates with an arborized morphology. A similar activation pattern was seen in these cells in response to Concanavalin A but not transforming growth factor beta or 12-O-tetradecanoylphorbol-13-acetate. The interstitial matrix may thus play an important role in regulating matrix degradation in vivo.
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PMID:Collagen-induced activation of the M(r) 72,000 type IV collagenase in normal and malignant human fibroblastoid cells. 132 93

Administration of amiodarone, although often lifesaving, is associated with pulmonary side effects. Patients with amiodarone pulmonary toxicity can present with either a chronic disorder that suggests pulmonary fibrosis or a more acute process. Mechanisms of acute pulmonary injury resulting from amiodarone are unclear. Previous studies have demonstrated that the drug is preferentially concentrated in alveolar macrophages. In the present study, the authors examined whether in vitro exposure to amiodarone resulted in alteration of rat alveolar macrophage superoxide, leukotriene B4, or fibronectin release. In addition, the authors assessed whether macrophages were ultrastructurally altered by in vitro amiodarone exposure. Twenty four hour exposure to therapeutic tissue concentrations of amiodarone resulted in enhancement of phorbol myristate acetate-stimulated macrophage superoxide release. In addition, 48 hours exposure to amiodarone caused a dose-dependent inhibition of spontaneous fibronectin release by macrophages. Macrophages exposed to 48 hours of 10 micrograms/ml amiodarone were ultrastructurally abnormal, containing lamellar inclusions and demonstrating a large degree of vacuolization. The authors concluded that alveolar macrophages are very sensitive to therapeutic tissue concentrations of amiodarone. Alteration of macrophage mediator release by amiodarone may be one mechanism for lung damage induced by the drug.
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PMID:Effects of in vitro amiodarone exposure on alveolar macrophage inflammatory mediator production. 133 29

The inflammatory response requires the localization of monocytic cells to sites of tissue injury through adherence to extracellular matrix molecules such as fibronectin (Fn), a nonimmune opsonin, which binds to collagen, fibrin, heparin and cell surfaces. Adherence to this molecule of two myeloid cell lines differing in their stage of differentiation, was studied. In the baseline state, U937 monocytic cells bound specifically to matrix-bound Fn, while HL-60 promyelocytic cells bound minimally. Exposure to Phorbol myristate acetate (PMA) dramatically increased binding of both U937 and HL-60 cells to Fn with plateau effects at 10 ng/ml for both cell lines and at 30 and 60 minutes for U937 and HL-60, respectively. Treatment with metabolic inhibitors suggests that PMA stimulation depends at least in part on intact energy metabolism, protein synthesis and cytoskeletal components. This system should help elucidate the early molecular and biochemical events involved in monocyte adherence to the extracellular matrix.
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PMID:Induction of monocytic cell adherence to matrix-bound fibronectin by phorbol ester. 134 May 4

Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover, PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.
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PMID:Phorbol 12-myristate 13-acetate induces resistance of human melanoma cells to natural-killer- and lymphokine-activated-killer-mediated cytotoxicity. 137 27

Endoglin is a glycoprotein expressed predominantly on human endothelial cells. It was first identified with mAb 44G4, produced against the pre-B acute lymphoblastic HOON cell line. We now report that four mAbs independently produced against human umbilical vein endothelial cells (HUVECs), chronic myelogenous leukemia in blast crisis, or U-937 pro-monocytic cells stimulated with phorbol myristate acetate also react with endoglin. High levels of reactivity of all mAbs were observed with HUVEC, while intermediate levels were seen with HOON and U-937 cells. By sequential immunoprecipitation from HUVEC and U-937 cell extracts, it was established that RMAC8, HEC-19, 8E11, and 1G2 mAbs react with the same protein as 44G4. Three distinct epitopes recognized by 44G4, RMAC8, and 1G2 mAbs were identified by competitive radioimmunoassay and flow cytometry. The HEC-19 epitope is spatially related to the 44G4 epitope, whereas the 8E11 epitope is most closely related to the 1G2 epitope. Western blot analysis showed that all antibodies react with the endoglin dimer (Mr = 170,000) purified from placenta. Immunostaining of sections of full-term placenta revealed reactivity not only with fetal vessels but also with the syncytiotrophoblast, the fetal cell layer which interfaces with maternal blood. When HUVEC monolayers were treated with the different mAbs to endoglin, prior to incubation with U-937 cells, a 5- to 10-fold stimulation of adhesion was observed. A fibronectin hexapeptide containing RGD, but not the corresponding RGE peptide, was capable of inhibiting the increased adhesion, when tested with mAb 44G4 and RMAC8. However, the same peptides had no effect on the binding of any of the five anti-endoglin mAbs to cells. Since 44G4 and RMAC8 recognize two distinct epitopes of endoglin, and since all five mAbs stimulated adhesion, the results suggest that a signal has been triggered through endoglin on HUVECs. Endoglin might be implicated either directly, by binding to a specific integrin-like ligand, or indirectly, by regulating the level of adhesion between certain integrins and their receptors.
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PMID:Identification of distinct epitopes of endoglin, an RGD-containing glycoprotein of endothelial cells, leukemic cells, and syncytiotrophoblasts. 137 94

Most mononuclear leukocytes and cell lines express the integrin alpha 4 beta 1 (VLA-4) heterodimer. In this study we have used Northern blotting and immunoprecipitation experiments to demonstrate that a B lymphoblastoid cell line (JY) expressed the integrin beta 7 subunit in association with alpha 4. These alpha 4 beta 7-positive JY cells bound poorly or not at all to VLA-4 ligands (soluble form of vascular cell adhesion molecule 1 (sVCAM-1) and the CS1 region of fibronectin). In contrast, a beta 1-positive variant of JY cells (selected to express a mixture of alpha 4 beta 1 and alpha 4 beta 7) bound avidly to VLA-4 ligands, and this binding was completely inhibitable by anti-alpha 4 and anti-beta 1 monoclonal antibodies. Thus, beta 1 expression appears to be a critically important component of VLA-4-mediated binding to its ligands. After either JY or JY-beta 1 cells were stimulated for 15 min with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, the majority of adhesion to VCAM or fibronectin remained alpha 4- and beta 1-dependent, but a low amount of adhesion to sVCAM-1 or fibronectin became alpha 4-dependent, beta 1-independent, thus suggesting a role for alpha 4 beta 7. In summary, we have found (i) that alpha 4 beta 7 makes little or no contribution to fibronectin or VCAM-1 binding on unstimulated JY cells, (ii) that alpha 4 beta 7 perhaps makes a minor contribution to ligand binding on 12-O-tetradecanoyl-phorbol-13-acetate-stimulated cells, and (iii) that alpha 4 beta 1 is the functionally dominant VCAM-1 and fibronectin receptor even when expressed in relatively low amounts compared to alpha 4 beta 7.
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PMID:Adhesion to vascular cell adhesion molecule 1 and fibronectin. Comparison of alpha 4 beta 1 (VLA-4) and alpha 4 beta 7 on the human B cell line JY. 137 25

In testing the hypothesis that the stimulation of the release of fibronectin (FN) by 12-O-tetradecanoylphorbol 13-acetate (TPA) from human lung fibroblasts in culture is the result of activation of protein kinase C (PKC), we found that the PKC inhibitor sphingosine strongly inhibited FN release in presence and even in absence of TPA. However, a different PKC inhibitor, calphostin C, despite almost complete inhibition of PKC, had no effect on FN release. We concluded that sphingosine is a potent inhibitor of FN release from the cell surface, independent of its inhibition of PKC; and that TPA stimulates release of FN by a pathway other than activation of PKC. We found that the activation of PKC by TPA was accompanied by inhibition of the cAMP-dependent protein kinase (PKA). When PKA was inhibited by an antagonist (H8, a cAMP analogue) at a concentration specific for PKA inhibition, the release of FN was stimulated similar to the stimulation with TPA. Activation of PKA with forskolin resulted in decreased FN release. In conclusion, we have shown that: (1) sphingosine had a robust effect inhibiting the release of FN from fibroblasts, independent of its action on PKC; (2) TPA treatment of these cells resulted in inhibition of PKA; (3) inhibition of PKA stimulated FN release whereas its activation decreased this release. It is possible that PKA, by phosphorylating a protein, may function, directly or indirectly, in keeping FN attached to the cell surface of fibroblasts.
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PMID:The effect of sphingosine and phorbol ester on the signal transduction enzymes and fibronectin release in cell culture. 137 51

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
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PMID:Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells. 137 48


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