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)

The trabecular meshwork, a specialized tissue in the anterior chamber of the eye, plays a major role in the regulation of aqueous humor outflow. We studied the effects of ascorbic acid, a significant component in the aqueous humor, on gene expression of type I collagen in cultures of bovine trabecular meshwork cells. These cells were plated for 6 days, exposed to ascorbic acid in concentrations of 100, 250 and 500 micrograms/ml for 3 days and labeled with (3H)proline for the last 24 hrs. Cultures that did not receive ascorbic acid served as controls. Bacterial collagenase assays showed enhanced incorporation of (3H)proline into collagenous proteins in cultures treated with 100 and 250 micrograms/ml of ascorbic acid. Gel electrophoresis and fluorography revealed that ascorbic acid caused a 2.6- to 4.9-fold increase in production of alpha 1 (I) and alpha 2(I) collagen chains by trabecular meshwork cells. Such an increase was found, using a cDNA probe specific for pro alpha 1(I) chains, to be accompanied by an increase in steady-state mRNA levels. Similar findings were also yielded from in situ hybridization experiments. These results, coupled with previously demonstrated ascorbate-induced effects on glycosaminoglycan, fibronectin and laminin synthesis, suggest that ascorbic acid is a key mediator of the extracellular matrix production by trabecular meshwork cells. Fluctuations in its concentration may lead to alterations in the makeup and assembly of matrices underlying the cells.
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PMID:Ascorbic acid modulates collagen type I gene expression by cells from an eye tissue--trabecular meshwork. 130 7

Seeding vascular prostheses with enzymatically harvested endothelial cells can create endothelial linings that improve small-caliber prosthetic patency. But crude bacterial collagenases used for endothelial harvest contain cytotoxic nonspecific proteases and clostridial cell wall debris which might limit their clinical usefulness. We therefore compared the endothelial cell harvest efficiency of crude bacterial collagenase with that of purified bacterial collagenase alone, purified trypsin alone, and combinations of purified bacterial collagenase and trypsin using concentrations of pure collagenase equal in collagenolytic activity to the crude bacterial collagenase material. The efficiency of harvest from human saphenous vein segments was measured by a microtiter well-growth curve assay of the number of living endothelial cells capable of attachment to fibronectin and subsequent growth obtained per unit area of saphenous vein lumen. Whereas pure collagenase and purified trypsin alone both harvested less than 5% of the baseline endothelial cell density on the veins, a combination of purified collagenase and 0.01% w/v purified trypsin was found to harvest 22% +/- 10% (SD) (n = 8 veins) of the approximately 1.3 x 10(5) endothelial cells/cm2 available on normal saphenous veins. This figure was not statistically different from the harvest efficiency of 19% +/- 10% (N = 4 veins) (p greater than 0.05) obtained by use of 0.1% w/v crude collagenase alone. This result suggests that endothelial harvesting can be done with a defined mixture of pure enzymes which would be clinically preferable to presently used crude extracts of clostridial cultures as a standardized preparation for graft seeding.
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PMID:Enzymatic harvesting of adult human saphenous vein endothelial cells: use of a chemically defined combination of two purified enzymes to attain viable cell yields equal to those attained by crude bacterial collagenase preparations. 130 65

In vivo, the extracellular matrix modulates the phenotype of the connective tissue cells both through its biochemical composition and the transfer of mechanical information. In this study, the mechanical effect was investigated in collagen gels populated by skin fibroblasts maintained under tension (bound lattices (BL)) compared with free retracting lattices (FL) and monolayer on plastic. The overall proteins and collagen synthesis of human skin fibroblasts, investigated by isotopic labeling, were decreased respectively by a factor of about 20 and 40 in FL compared with monolayers and increased by a factor of 4 and 6 in BL versus FL. As assayed by the degradation of [3H]collagen type I by trypsin-activated medium conditioned by fibroblasts under the three models of culture, collagenase activity was inversely regulated and increased in lattices when compared with monolayer culture. It was four times higher in FL than in BL. The steady-state level of mRNA coding for procollagen types I, III, and VI polypeptides, fibronectin, elastin, beta-actin, and procollagenase was determined by cDNA hybridization. The mRNA coding for beta-actin as well as for the various extracellular matrix macromolecules were increased in BL when compared with FL while the level of procollagenase mRNA was lower. These data demonstrate the existence of a modulation of the function of the fibroblasts performed by mechanical forces. This regulation operates, at least in part, at a pretranslational level.
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PMID:Pretranslational regulation of extracellular matrix macromolecules and collagenase expression in fibroblasts by mechanical forces. 131 27

A protein doublet (M(r) = 68,000) that copurifies with chicken cardiac collagen types I and III is purified and characterized in the present study. Peptide mapping and amino terminus sequencing for both 68-kDa polypeptides show they have similar structures. This is supported by amino terminus sequencing of a 39-kDa proteolytic fragment of each polypeptide. The 68-kDa polypeptides appear at pI 6.7-6.8 in two-dimensional gels. Under nonreducing, electrophoretic conditions, the doublet appears as a large multimer or aggregate. Amino acid sequencing of the protein shows that its amino terminus contains a heptapeptide (VCLXXGK) that appears in the heparin/fibrin-binding domain of fibronectin and the collagen-binding domain of laminin. Cardiac myocytes synthesize and secrete the protein in vitro onto cell surfaces and onto the substratum. Indirect immunofluorescence shows the protein first appears in the chicken subepicardium at approximately 10 days following fertilization. As collagen accumulates in the subepicardium and the volume of the subepicardial space increases, the 68-kDa protein is found predominantly at the interface between myocardial cells and the connective tissue and between epicardial cells and the connective tissue. In adult hearts, the protein is also present at lower concentrations in endomysial connective tissue. The 68-kDa protein is also present in the skeletal muscle endomysium of embryonic chickens. Electron microscopic immunocytochemistry shows the 68-kDa protein is located at the surface of subepicardial collagen fibers. In addition, a direct interaction between the 68-kDa protein and collagen are indicated by: 1) equilibrium gel filtration of the 68-kDa protein in the presence of gelatin, 2) gelatin affinity chromatography of the 68-kDa protein, and 3) comigration of type I collagen and the 68-kDa protein during gel filtration under reducing conditions. The 68-kDa protein exhibits no collagenase activity under native conditions or in zymograms. Together, the data indicate that the 68-kDa protein is a novel collagen-associated protein appearing in late epicardial development.
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PMID:Identification and distribution of a novel, collagen-binding protein in the developing subepicardium and endomysium. 132 25

We have screened six human squamous carcinoma cell lines for their ability to invade connective tissue by using the experimentally modified chorioallantoic membrane of a chick embryo as an in vivo model of invasion. In confirmation of our earlier studies, all the invasive cell lines expressed high levels of surface-bound urokinase type plasminogen activator (uPA). However, some cell lines expressing this activity were not invasive, suggesting that surface uPA, although necessary, was not sufficient. Since in addition to fibronectin, that can be degraded by uPA or plasmin, chorioallantoic membrane connective tissue contains collagen, we examined the profile of collagenases secreted by the various cell lines in search for an activity that would coincide with the invasive phenotype. We found, using gelatin substrate gels, that type IV gelatinase was produced by all six cell types tested, three cell types produced the M(r) 92,000 gelatinase, and three a lower-molecular-weight activity, which we identified by immunoprecipitation with specific antibodies, and by a direct assay of activity, as interstitial collagenase. Only the latter cells were found to be highly invasive. We showed previously that continuous culture in vitro of one of the carcinoma cell lines, HEp3, led to a gradual extinction of their malignant phenotype. To confirm the correlation between invasion and the production of interstitial collagenase, we examined these two functions in cells freshly isolated from a HEp3 tumor and intermittently during passage in vitro. We found that, although the surface uPA activity was slightly diminished in the in vitro grown cultures, it was still within the range of values found in highly malignant cells, suggesting that it is not the reason for the decrease in invasiveness. In contrast, the reduction in interstitial collagenase closely followed the loss of the invasive phenotype; after 30 in vitro passages the cells were almost completely devoid of interstitial collagenase and unable to invade. The decrease in collagenase activity was not the result of an increased tissue inhibitor of metalloproteinases production.
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PMID:Invasion of connective tissue by human carcinoma cell lines: requirement for urokinase, urokinase receptor, and interstitial collagenase. 133 82

We found previously that two fibrinolytic enzymes (jararafibrases I and II) purified from Bothrops jararaca venom displayed a haemorrhagic activity. To elucidate the mechanisms involved and the role of the enzymatic activity in haemorrhage, the enzymatic properties of the purified enzymes were examined. The substrate specificity of the enzymes was determined using type I collagen, type IV collagen, gelatin, laminin and fibronectin as substrates. The enzymes degraded type IV collagen, gelatin, laminin and fibronectin into smaller fragments, but degraded type I collagen only partially in a non-specific manner. The specific activities of jararafibrase I for type IV collagen and gelatin were 172 +/- 5 units/mg protein and 1315 +/- 177 units/mg protein, respectively. The specific activities of jararafibrase II for type IV collagen and gelatin were 9.2 +/- 0.6 units/mg protein and 143 +/- 15 units/mg protein, respectively. It was evident that the enzymes had rather broad substrate specificities and degraded basement membrane components including type IV collagen. The number of type IV collagen units of bacterial collagenase which gave the minimal haemorrhagic dose was 191.4, while the numbers of type IV collagenase units of jararafibrases I and II which gave the minimal haemorrhagic dose were 1.5 and 0.25, respectively. It is suggested that the broad substrate specificity of the enzymes is essential for inducing haemorrhage with a single enzyme.
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PMID:Broad substrate specificity of snake venom fibrinolytic enzymes: possible role in haemorrhage. 133 30

We have isolated undulin, an extracellular matrix protein associated with the surface of collagen fibrils, from chicken embryos. The protein showed a molecular mass of about 600 kDa and is composed of three 210-kDa subunits linked by reducible as well as non-reducible bonds. In contrast to human undulin which reportedly is devoid of collagenous sequences, the chicken protein contained a short triple-helical segment that was sensitive to digestion by bacterial collagenase. Screening of an expression library with affinity-purified antibodies yielded two cDNA clones specific for chicken undulin. Analysis of the amino acid sequence deduced from the nucleotide sequence of these clones showed that the human and the chicken protein shared 71% sequence identity. At the amino-terminus both polypeptides contained several similar repeats related to the type III modules found in fibronectin. Towards the carboxyl terminus, however, the two sequences diverged substantially from each other. While the human sequence terminated in a proline-rich segment, the chicken sequence continued with a domain related to von Willebrand factor, with a domain similar to the noncollagenous domain NC4 of type IX collagen and with a typical collagenous triple helix. A short segment of this sequence was found to be identical with the published sequence of a bovine peptide derived from type XIV collagen. Our protein must therefore represent chicken type XIV collagen. One way to explain these results is the possibility that undulin exists in at least two alternatively spliced variants, one lacking the collagenous domain, as described initially for human undulin, and one containing the triple-helical domain, as found in type XIV collagen.
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PMID:Type XIV collagen is a variant of undulin. 133 49

Polyhydroxyethylmethacrylate (PHEMA) based microcarriers with different bulk structures were prepared by a phase inversion polymerization technique. PHEMA surfaces were further modified chemically by glow-discharge treatment, and biologically by covalent attachment of fibrinogen and collagen. Hepatocytes were isolated from young male Wistar rats using an in situ portal vein collagenase perfusion technique. Freshly isolated hepatocytes were seeded at 6 x 10(5) cells/mL and microcarrier concentration was 10 g/L. Stationary microcarrier cultures were carried out in standard (nontissue culture) polystyrene petri dishes in a humidified 5% CO2 incubator at 37 +/- 0.5 degrees C. Cell attachment was followed by light microscopy by taking samples from the culture medium every 30 min. Urea and protein syntheses by microcarrier-attached hepatocytes were determined by standard techniques. Nonswellable (highly cross-linked) hydrophilic PHEMA microcarriers did not support cell attachment and viability. However, swellable (low cross-linked) PHEMA microcarriers (pretreated in FBS) allowed high attachment and cell spreading. PHEMA microcarriers treated in dimethylaminoethylmethacrylate (DMAEMA) glow-discharge plasma also improved the cell attachment characteristics of the PHEMA microcarriers. The highest attachment efficiencies (immobilization yields) were observed with the biologically modified PHEMA microcarriers, especially modified with fibronectin. Metabolic activity, as estimated by urea and protein syntheses, was also higher in these microcarriers.
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PMID:Hepatocyte immobilization on PHEMA microcarriers and its biologically modified forms. 134 12

The interstitial collagenase gene (CLG), one of the main candidates in severe generalized recessive epidermolysis bullosa dystrophica (SGREBD), is closely linked to the stromelysin-1 (STMY1) and stromelysin-2 (STMY2) genes. These three loci map on chromosome 11 (q21-q22.3), where they constitute a cluster of genes coding for metalloproteinases involved in the degradation of the extracellular matrix (ECM). A recessive form of cerebellar ataxia of post-puberal onset (CLA1) has also been assigned to chromosome 11 (q14-q21). Since useful restriction fragment length polymorphisms (RFLPs) for the CLG gene are not available, we have studied the inheritance of the marker TaqI RFLP of the STMY1 gene in a North Italian family with a child affected by SGREBD, and his two sisters showing cerebellar ataxia (CA) of post-puberal onset. We have also studied the MspI RFLP of the fibronectin gene (FN1), which is located on chromosome 2q34-q36, and which codes for non-collagenous matrix proteins. Since we did not observe the segregation of the pathological phenotypes with STMY1 and FN1 RFLPs, we excluded the involvement of these genes in both the SGREBD and CA present in this family. The exclusion of the STMY1 gene indicates that the mutation causing SGREBD cannot be located in the CLG and/or STMY2 genes because of their proximity to the STMY1 locus. These data also indicate that the CA form here reported is not attributable to alterations in regions close to the collagenase cluster on chromosome 11.
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PMID:Exclusion of stromelysin-1, stromelysin-2, interstitial collagenase and fibronectin genes as the mutant loci in a family with recessive epidermolysis bullosa dystrophica and a form of cerebellar ataxia. 135 52

One of the mechanisms by which normal hematopoietic progenitor cells remain localized within the bone marrow microenvironment is likely to involve adhesion of these cells to extracellular matrix (ECM) proteins. For example, there is evidence that uncommitted, HLA-DR-negative progenitor cells and committed erythroid precursors (BFU-E) bind to fibronectin. However, fibronectin is not known to mediate binding of committed myeloid (granulocyte-macrophage) progenitors, raising the possibility that other ECM proteins may be involved in this process. We investigated the binding of the MO7 myeloid cell line to a variety of ECM proteins and observed significant specific binding to collagen type I (56% +/- 5%), minimal binding to fibronectin (18% +/- 4%) or to laminin (19% +/- 5%), and no binding to collagen type III, IV, or V. Similarly, normal bone marrow myeloid progenitor cells (CFU-GM) demonstrated significant specific binding to collagen type I (46% +/- 8% and 47% +/- 12% for day 7 CFU-GM and day 14 CFU-GM, respectively). The ability of collagen to mediate binding of progenitor cells was not restricted to the myeloid lineage, as BFU-E also showed significant binding to this ECM protein (40% +/- 10%). The binding of MO7 cells and CFU-GM was collagen-mediated, as demonstrated by complete inhibition of adherence after treatment with collagenase type VII, which was shown to specifically degrade collagen. Binding was not affected by anti-CD29 neutralizing antibody (anti-beta-1 integrin), the RGD-containing peptide sequence GRGDTP, or divalent cation chelation, suggesting that collagen binding is not mediated by the beta-1 integrin class of adhesion proteins. Finally, mature peripheral blood neutrophils and monocytes were also found to bind to collagen type I (25% +/- 8% and 29% +/- 6%, respectively). These data suggest that collagen type I may play a role in the localization of committed myeloid and erythroid progenitors within the bone marrow microenvironment.
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PMID:Myeloid and erythroid progenitor cells from normal bone marrow adhere to collagen type I. 137 Jun 40


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