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)

We used flow cytometry to measure the expression of human melanoma antigens on cell suspensions dissociated from metastatic masses. The objective was to study the heterogeneity between tumor samples from different patients and between different tumors excised from a single patient. Fifty-three metastases excised from 34 melanoma patients were analyzed with a panel of nine murine monoclonal antibodies (MOABs). Melanoma cells were stained by an indirect fluorescent method and analyzed on a Coulter EPICS C flow cytometer after gating to exclude tumor-infiltrating leukocytes and dead cells. The most consistently and most strongly expressed antigen was the high-molecular-weight proteoglycan (detected by the MOAB 9.2.27), which was expressed on 95% of the melanoma specimens and by a high proportion of cells within each specimen (mean +/- SE, 79.2 +/- 5.5). However, strong expression of this antigen was limited to melanoma cells that had been dissociated mechanically and was markedly diminished by exposure to collagenase. Culture of collagenase-dissociated tumor cells for 24 to 48 h resulted in reexpression of the antigen. The expression of other melanoma-associated antigens was not affected by collagenase treatment, but for these antigens there was more variability between cells from an individual tumor and between tumors from different patients. The percentage of enzyme-dissociated tumors considered positive for MOAB binding (defined as at least 10% of cells positive) and the mean +/- SE of the percentage of positive cells within a tumor were as follows: MOAB ME-9-61 (antigen, p97) = 84% + (41.2 +/- 5.4%); MOAB ME-20.4 (antigen, nerve growth factor receptor) = 40% + (18.7 +/- 5.1%); MOAB ME-24 (antigen, ganglioside GD3) = 84% + (50.8 +/- 4.8%); MOAB ME-311 (antigen, ganglioside 9-O-acetyl-GD3) = 76% + (42.5 +/- 5.1%); MOAB ME-361 (antigen, mainly ganglioside GD2) = 3% + (1.9 +/- 0.8%); MOAB 3F8 (antigen, ganglioside GD2) = 36% (10.5 +/- 3.8%); MOAB 14G2a (antigen, ganglioside GD2) = 86% + (46.0 +/- 6.7%); MOAB L243 (antigen, HLA-DR) = 56% + (22.5 +/- 5.5%). In 19 cases, we were able to compare the antigenic profiles of two tumors excised from the same patient at different times. Analysis by nonindependent t test showed no significant differences in MOAB binding between the paired tumors. Moreover, linear regression analysis indicated that there was a linear relationship, with a slope approximately = 1, between the percentage of positive cells in Tumor 1 versus Tumor 2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Flow cytometric determination of the frequency and heterogeneity of expression of human melanoma-associated antigens. 258 30

Bovine articular and tracheal chondrocytes were cultured at high density in multilayers. Intact or fragmented large aggregating proteoglycans (PG-LA) from cartilage were added to the cultures and the biosynthetic response studied by the incorporation of [3H]-leucine and [35S]-sulfate for proteins and proteoglycans respectively. Incorporated radiolabel and patterns of synthesized macromolecules were compared with control cultures without additives and cultures containing either of the synthetic polymers dextran or dextran sulfate. All proteoglycans and derivatives containing globular protein structures had a stimulatory effect on the biosynthesis of both proteins and proteoglycans as did the highly polyanionic polymer dextran sulfate. Distribution of the radiolabeled material between the cell- and medium pools were however different in the various cultures. A radiolabeled protein, migrating as a triplet band at a position of approximately 140 kDa after reduction, was detected by SDS-PAGE and fluorography. The protein was present in all cell extracts and in the media of cultures stimulated with proteoglycans and proteoglycan fragments, except chondroitin sulfate side chains. The protein was shown to be collagenous in nature by collagenase digestion and identified as procollagen II by immunoprecipitation.
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PMID:Large cartilage proteoglycan (PG-LA) influences the biosynthesis of macromolecules by isolated chondrocytes. 261 94

Osteoarthritis was induced in 12 normal dogs by severing of the anterior cruciate ligament of the right knees, the left knees serving as sham operated controls. The animals were killed at 7 and 14 weeks postsurgery. The total hexuronate, and thus proteoglycan, content of the articular cartilage of operated knees remained unaltered during the period of study. After pretreatment with a highly purified collagenase and in the presence of selected protease inhibitors, a higher proportion of the tissue hexuronate could be extracted from the different topographical areas of osteoarthritic joints under non dissociative conditions (70-75% versus 55-65% for control knees). The nondissociatively recovered osteoarthritic proteoglycans (a-A1 preparations) displayed progressive and consistent changes in their sedimentation profile. First, the size of the fast sedimenting or more saturated aggregates appeared to be reduced in the different regions of osteoarthritic joints at 7 weeks postoperatively. The disappearance of the faster sedimenting mode as well as a dramatic increase in the proportion of monomers were only detected in the topographical zones exhibiting the most severe surface damage and histologic abnormalities at 14 weeks postsurgery. The proteoglycan molecules present as "free" or "nonaggregated" monomers in a-A1 preparations recovered from normal and osteoarthritic cartilage at different time periods after surgery were separated from their corresponding aggregates by rate zonal centrifugation in isokinetic cesium sulfate gradient. Although they were severely depleted in keratan sulfate, the purified "free" and "aggregated" osteoarthritic monomers appeared to be normal in terms of aggregating capacity and size distribution, and were therefore not degraded. This progressive changes in size distribution of proteoglycan aggregates in the early stages of experimental canine osteoarthritis could contribute significantly to the biochemical and biomechanical alterations of osteoarthritic cartilage.
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PMID:Changes in the sedimentation profile of proteoglycan aggregates in early experimental canine osteoarthritis. 263 43

Ultracentrifugal polydispersity differential [g(S)] distributions were determined for the proteoglycans of various postmortem human articular cartilage samples extracted from six lateral patellar grooves in nondissociative conditions after mild collagenase digestion of the tissue. The samples consisted of 53 slices (250 microns thick), from normal, mildly fibrillated, and extensively ulcerated knee joints. When statistically analyzed in various subgroupings, the obtained average sedimentation coefficients and polydispersity profiles supported the following conclusions: (a) loss of proteoglycan aggregation and sedimentability is confirmed to be a primary sign of cartilage matrix degradation; (b) higher S values for proteoglycans of the high weight (HW)-bearing areas and lower values for those of the low weight (LW)-bearing areas were a typical finding in normal cartilage samples; (c) inversion of this pattern was indicative of matrix degradation, suggesting that the HW regions are more affected than the LW-bearing areas; (d) the average S value distribution across cartilage thickness tended to resemble the corresponding proteoglycan content versus distance from articular surface; and (e) the deepest cartilage layer had, in most cases, the smallest amount of aggregates while the highest average sedimentability was observed at the middle zone of the normal samples. In the discussion, a role of proteoglycan aggregation for providing a means to "pack" more proteoglycans within the collagen meshwork and to control the generation of osmotic pressure gradients is suggested.
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PMID:Centrifugal characterization of proteoglycans from various depth layers and weight-bearing areas of normal and abnormal human articular cartilage. 270 25

The chemical nature of anionic sites located on both fronts of the endothelial cells (ECs) and in the basement membrane (BM) of mouse brain capillaries was studied using tissue sections embedded in Lowicryl K4M and cationic colloidal gold. Before labelling with cationic probe, the sections were digested with the following enzymes: trypsin, papain, pronase E, proteinase K, collagenase, chondroitinase ABC, hyaluronidase, heparinase, heparitinase, neuraminidase and endoglycosidase H. The results indicate that the negatively charged surface layer on the luminal front differs in chemical nature from that on the abluminal front of the EC. Anionic sites located on the luminal surface of the plasmalemma of the ECs are mainly contributed by sialic acid residues of acidic glycoproteins. On the contrary, the anionic domains on the abluminal front of the EC represent mixed proteoglycan and acid glycopeptides containing hydrophobic amino acids, sialic acid residues, and are rich in heparan sulphate-bearing glycosaminoglycans. The anionic sites of the BM are contributed in a substantial degree by chondroitin and heparan sulphate-rich glycosaminoglycans. The effect of endoglycosidase H suggests that glycopeptides containing oligomannosyl residues linked to N-acetylglucosamine contribute in small degree in maintenance of the negative charge in the BM, but not on the surfaces of the EC. These results show that brain endothelium bears surface anionic domains differing chemically from those described for some fenestrated and continuous endothelia. The distribution of anionic sites indicates that the discrimination against various negatively charged molecules takes place on both fronts of the ECs as well as in the BM of brain micro-blood vessels. The exact role of these domains in the function of the blood-brain barrier remains to be established.
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PMID:Ultracytochemical characterization of anionic sites in the wall of brain capillaries. 274 7

The monoclonal antibody 75d7, specific for type XII collagen (Sugrue, S.P., Gordon, M.K., Seyer, J., Dublet, B., van der Rest, M., and Olsen, B. R. (1989) J. Cell Biol., in press), was used to characterize the intact form of type XII collagen from chick embryo leg tendons. On an immunoblot of a 6% polyacrylamide gel of tendon extracts, one sharp band is recognized by the antibody at Mr = 220,000, while two fuzzy and poorly resolved bands are seen at Mr = 270,000 and Mr = 290,000. By immunoprecipitation of radiolabeled tendon culture media and electrophoresis of the precipitated material, bands with the same mobilities are observed, indicating that type XII collagen is not proteolytically processed in the extracellular space. Type XII collagen was extracted from tendons with 1 M NaCl in a Tris-HCl buffer and partially purified by concanavalin A-Sepharose and gel permeation chromatographies, using dot immunoblots to monitor the purification. Fractions highly enriched in bacterial collagenase-sensitive proteins with the same electrophoretic properties as type XII collagen were obtained. These fractions did not stain with Alcian blue and neither they nor the immunostained type XII collagen were affected by chondroitinase ABC digestion, indicating that type XII collagen is not a proteoglycan. A disulfide-bonded trimeric CNBr peptide was isolated by affinity chromatography on an antibody column and further purified by gel electrophoresis. Its NH2-terminal amino acid sequence was shown to be unique, demonstrating that type XII collagen is a homotrimer [alpha 1 (XII)]3. After bacterial collagenase digestion, both the immunopurified radiolabeled preparation and the purified tendon extract fraction showed by gel electrophoresis the presence of a large disulfide-bonded, 3 x 190-kDa, collagenase-resistant domain. Rotary shadowing and electron microscopy of the purified type XII fraction demonstrated that the molecule has the structure of a cross consisting of a 75 nm collagenase-sensitive tail, a central globule, and three 60 nm arms each ending in a small globule. After heat denaturation and renaturation, only a very large globule can be seen, attached to the triple helical tail. These results show that type XII collagen has a unique structure and is different from the other matrix constituents described so far.
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PMID:The structure of avian type XII collagen. Alpha 1 (XII) chains contain 190-kDa non-triple helical amino-terminal domains and form homotrimeric molecules. 275 5

Articular chondrocytes cultured in the presence of recombinant human interleukin 1 alpha (rhIL-1 alpha) or recombinant human tumor necrosis factor alpha (rhTNF alpha) caused increased production of the latent metalloproteinase (collagenase and caseinase) and the proteoglycan release from cartilage. The existences of IL-1 and TNF alpha in the chondrocytes of human articular cartilage were also shown by immunohistochemical staining using polyclonal antibodies. Furthermore, chondrocyte was found to be a producer of interleukin 6 (IL-6), known as a pleiotropic cytokine and thought to be an important mediator of the cell interactions in arthritis. In addition, the production of IL-6 was also shown to be stimulated by rhIL-1 alpha or rhTNF alpha. From our findings, we suggest there exists a very complicated autocrine control system of chondrolysis by the chondrocyte itself.
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PMID:The role of cytokines in chondrocyte mediated cartilage degradation. 281 Feb 94

Using EDTA extraction and collagenase digestion, cancellous bone of the femoral heads from 10 normal and 9 osteoporotic subjects were analyzed for their contents of collagen, sialoprotein, proteoglycan and carbohydrate. The percentage of extracted matrix proteins of the osteoporotic bone in EDTA was significantly decreased, as was the collagenase-resistant fraction (p less than 0.05). The sialic acid level in osteoporotic bone matrix was lower than in controls (p less than 0.05). The alterations found in bone matrix constituents in osteoporotic bone relative to controls suggest that in osteoporosis and fractures, not only bone mass changes, but also bone quality changes play a role in bone strength.
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PMID:Studies on EDTA extracts and collagenase digests from osteoporotic cancellous bone of the femoral head. 282 Jun 17

We have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast-deposited matrix component, possibly heparan sulfate-proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B-16 melanoma cells have been demonstrated. Binding of 3H-heparin and 35S-heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H-heparin yielded a Kd of 2-5 x 10(-8) M and a Bmax of 0.5 x 10(7) heparin molecules bound per cell. Binding of 35S-heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S-heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H-heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H-heparin to transblots of the detergent extract of the B-16 cell membranes indicated that at least part of the binding activity is a 14,000-dalton protein.
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PMID:Heparin and heparan sulfate binding sites on B-16 melanoma cells. 284 Apr 40

Neutral metalloproteinases degrade components of the extracellular matrix, including collagen types I-V, fibronectin, laminin and proteoglycan. However, their ability to degrade intact glomerular basement membrane (GBM) has not previously been investigated. Incubation of [3H]GBM (50,000 c.p.m.; pH 7.5; 24 h at 37 degrees C) with purified gelatinase or stromelysin (2 units) resulted in significant GBM degradation: gelatinase, 46 +/- 2.2; stromelysin, 59 +/- 5.8 (means +/- S.E.M.; percentage release of non-sedimentable radioactivity; n = 4). In contrast, 2 units of collagenase released only 5.6 +/- 0.52% (n = 3) of the [3H]GBM radioactivity compared with 2.0 +/- 0.15% (n = 7) released from [3H]GBM incubated alone. Sephadex G-200 gel chromatography of supernatants obtained from incubations of [3H]GBM with either gelatinase or stromelysin confirmed the ability of these enzymes to degrade GBM and revealed both high-(800,000) and relatively low-(less than 20,000) Mr degradation products for both enzymes. GBM degradation by gelatinase and stromelysin was dose-dependent (range 0.02-2.0 units), near maximal between pH 6.0 and 8.6, and was completely inhibited (greater than 95%) by 2 mM-o-phenanthroline. Collagenase (2 units) did not enhance the degradation of GBM by either gelatinase (0.02 or 0.2 unit) or stromelysin (0.02 or 0.2 unit). Our results indicate that metalloproteinase-mediated GBM degradation by neutrophils and glomeruli may be attributable to gelatinase (neutrophils) and/or stromelysin (glomeruli) and suggest an important role for these proteinases in glomerular pathophysiology.
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PMID:Degradation of glomerular basement membrane by purified mammalian metalloproteinases. 284 58


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