Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe an in vitro assay for measuring the ability of tumour cells to permeabilize basement membranes, using transwell chambers coated with the reconstituted basement membrane, matrigel. Unlike previous matrigel-based procedures which quantified passage of tumour cells across a matrigel barrier, the new assay measures the ability of tumour cells to degrade the basement membrane and increase the diffusion rate of fluorescent (FL) dextran through the barrier. The procedure has the major advantage that permeability can be rapidly and accurately quantified, either by fluorometry or by the use of radiolabelled dextran, thus avoiding tedious and subjective scoring methods. Optimal conditions for the assay are described. In addition, it is demonstrated that the assay can clearly discriminate between metastatic and non-metastatic tumour cell lines, metastatic tumours permeabilizing the basement membrane and non-metastatic counterparts failing to do so. A range of enzyme inhibitors suggested that the increase in basement-membrane permeability caused by the metastatic mammary adenocarcinoma 13762 MAT is probably dependent upon the synergistic action of several degradative enzymes, namely proteases, type-IV collagenase, and heparanase. Furthermore, the ability to permeabilize the basement membrane was dependent upon intact tumour cells; tumour cell extracts, lysates and supernatants were inactive.
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PMID:A basement-membrane permeability assay which correlates with the metastatic potential of tumour cells. 139 13

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.
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PMID:A multiwell format assay for heparanase. 1292 26

The specific role of solid extracellular matrix components in opposing development of pulmonary interstitial edema was studied in adult anesthetized rabbits by challenging the lung parenchyma with an intravenous injection of a bolus of collagenase or heparanase. In 10 rabbits, pulmonary interstitial pressure (Pip) was measured by micropuncture in control and up to 3 h after collagenase or heparanase intravenous injection. With respect to control (Pip= -9.3 +/- 1.5 cmH2O, n = 10), both treatments caused a significant increase of Pip and of the wet weight-to-dry weight lung ratio. However, while tissue matrix stiffness was maintained after 60 min of collagenase, as indicated by the attainment of a positive Pip peak (Pip= 4.5 +/- 0.3 cmH2O, n = 5), this mechanical response was lost with heparanase (Pip= -0.6 +/- 1.3 cmH2O, n = 5). Biochemical analysis performed on a separate rabbit group (n = 15) showed an increased extraction of uronic acid with both enzymes, indicating a progressive matrix fragmentation. Gel chromatography analysis of the proteoglycan (PG) families showed that 60 min of both enzymatic treatments left the large-molecular-weight PGs (versican) essentially unaffected. However, the heparan-sulfate PG fraction was significantly cleaved, as indicated by a significant increase of the smaller PG fragments with heparanase, but not with collagenase. Hence, present data suggest that the integrity of the heparan-sulfate PGs is required to maintain the three-dimensional architecture of the pulmonary tissue matrix and in turn to counteract tissue fluid accumulation in situations of increased fluid filtration.
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PMID:Differential degradation of matrix proteoglycans and edema development in rabbit lung. 1621 13

During mammalian follicle development, a fluid-filled antrum develops in the avascular centre of the follicle. We investigated the hypothesis that follicular fluid contains osmotically-active molecules, sufficiently large so as to not freely escape the follicular fluid. Such molecules could generate an osmotic differential and thus recruit fluid from the surrounding vascularised stroma into the antrum. Follicular fluid was collected from bovine follicles classified histologically as healthy (n = 4 pools) or atretic (n = 4 pools). Dialysis of the follicular fluid at 300 kDa or 500 kDa resulted in a reduction in colloid osmotic pressure of 35% and 60%, respectively, in fluid from healthy follicles and 29% and 80% from atretic follicles. Digestion of follicular fluid with Streptomyces hyaluronidase, chondroitinase ABC or DNase 1 followed by dialysis resulted in reductions in osmotic pressure of 43%, 53% and 43% respectively for fluids from healthy follicles and 34%, 20% and 31% for atretic follicles. Digestion with collagenase I, proteinase K, heparanase 1 or keratanase had no significant effect on the osmotic pressure of follicular fluid of healthy follicles. Ion exchange and size exclusion, Western blotting and ELISA identified the proteoglycans versican and inter-alpha trypsin inhibitor and the glycosaminoglycan hyaluronan in follicular fluid. We conclude that these molecules or aggregates of them are of sufficient size to contribute to the osmotic potential of follicular fluid and thus recruit fluid into the follicular antrum. DNA may also contribute but it is probably not a component that is regulated for this role.
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PMID:Formation of ovarian follicular fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans. 1681 38

Sulfated polysaccharides (SP) such as heparin are known to exhibit a wide range of biological activities, e.g., anticoagulant, anti-inflammatory, and antimetastastic effects. However, since the anticoagulant activity of heparin is dominating, its therapeutic use for other medical indications is limited due to an associated risk of bleeding. Further disadvantages of heparin are its animal origin, the shortage of resources, and its complex and variable composition. However, SP without these limitations may represent a substance class with good prospects for applications other than anticoagulation. In this study, the in vitro pharmacological profiles of two nonanimal-derived SP were investigated in comparison with unfractionated heparin. One is the natural SP fraction from the red algae Delesseria sanguinea (D.s.-SP). The other one is the chemically defined PS3, a semisynthetic beta-1,3-glucan sulfate with proven in vivo anti-inflammatory and antimetastatic activities. All three polysaccharides were examined in vitro for their inhibitory effects on the coagulation and complement system, polymorphonuclear neutrophil elastase, hyaluronidase, matrix metalloproteinase-1, heparanase, and p-selectin-mediated cell adhesion. Compared with heparin, the nonanimal-derived polysaccharides have a four times weaker anticoagulant activity, but mostly exhibit stronger (1.4-224 times) effects on test systems investigating targets of inflammation or metastasis. According to their different structures, PS3 and D.s.-SP differ in their pharmacological profile with PS3 being the strongest inhibitor of heparanase and cell adhesion and D.s.-SP being the strongest inhibitor of hyaluronidase and complement activation. Considering both pharmacological profile and pharmaceutical quality parameters, PS3 represents a candidate for further development as an anti-inflammatory or antimetastatic drug whereas D.s.-SP might have perspectives for cosmetic applications.
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PMID:Pharmacological profiles of animal- and nonanimal-derived sulfated polysaccharides--comparison of unfractionated heparin, the semisynthetic glucan sulfate PS3, and the sulfated polysaccharide fraction isolated from Delesseria sanguinea. 1910 33

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.
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PMID:Acute UV irradiation increases heparan sulfate proteoglycan levels in human skin. 2237 42

Plant products in general and soybeans in particular can challenge the function and health of the intestinal tract. Salmonids develop an intestinal inflammation when fed diets containing soybean meal (SBM) and certain other legume ingredients. In the present study a 44K oligonucleotide salmonid microarray, qPCR and histology were used to investigate early response mechanisms in the distal intestine of Atlantic salmon (Salmo salar L.) during the first week of oral exposure to a diet containing 20% extracted SBM. The distal intestine transcriptome was profiled on days 1, 2, 3, 5 and 7 and compared to a control group fed fishmeal as the sole protein source. Histological evaluation of the distal intestine revealed the first signs of inflammation on day 5. The most prominent gene expression changes were seen on days 3 and 5. Up-regulation in immune-related genes was observed during the first 5 days, including GTPase IMAP family members, NF-kB-related genes and regulators of T cell and B cell function. Many functional genes involved in lipid metabolism, proteolysis, transport, metabolism and detoxification were initially up-regulated on days 1-3, possibly as an attempt by the tissue to compensate for the initiating immune response. Cell repair and extracellular matrix remodeling genes were up-regulated (heparanase, collagenase) on days 3 and 5. Down regulation of genes related to endocytosis, exocytosis, detoxification, transporters and metabolic processes from day 5 indicated initiation of dysfunction of digestive and metabolic functions that may occur as a result of inflammation or as a response to the introduction of soybean meal in the diet. This is the first study conducting transcriptomic profiling to characterize early responses during the development of SBMIE. Switching Atlantic salmon from a fishmeal to a 20% SBM diet resulted in rapid changes to the intestinal transcriptome, indicating an immune reaction with subsequent impaired epithelial barrier function and other vital intestinal functions.
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PMID:Early response of gene expression in the distal intestine of Atlantic salmon (Salmo salar L.) during the development of soybean meal induced enteritis. 2324 10

Total joint arthroplasty (TJA) of the hip or knee (THA and TKA) is the primary surgical intervention for individuals with degenerative joint disease (DJD). Although it is commonly thought that shear force on the joint causes the degradation of articular cartilage, it is possible that there are other factors that contribute to the progression of DJD. It is plausible that specific enzymes that degrade the joint are upregulated, or conversely, there is downregulation of enzymes critical for joint lubrication. The aim of this study is to profile collagenase-1, elastase, heparanase, and lubricin levels in patients undergoing TJA in order to determine potential preexisting dysregulation that contributes to the pathogenesis of DJD. Deidentified blood samples were obtained from patients undergoing TJA 1 day pre- and 1 day postoperatively. Plasma samples were analyzed using enzyme-linked immunosorbent assay kits for elastase, collagenase-1, heparanase, and lubricin. In comparison to healthy controls, there were significant increases in circulating collagenase-1, elastase, and lubricin levels in both the preoperative and postoperative samples. There were no significant differences in heparanase levels in the preoperative or postoperative samples. Comparing the preoperative versus postoperative patient samples, only lubricin demonstrated a significant change. The results of this study confirm that patients undergoing TJA have preexisting alterations in the levels of matrix-degrading enzymes and lubricin. The alterations observed in this study may provide insight into the pathogenesis of DJD.
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PMID:Levels of Matrix-Degrading Enzymes and Lubricin in Patients With Degenerative Joint Disease Requiring Arthroplasty. 2887 7