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)

A number of cells, chemotactic factors, and inflammatory mediators are implicated in the complex mechanisms underlying crystal-mediated inflammation. Interleukin-8, released from mononuclear cells that have been exposed to urate and other crystals, is a potent chemotaxin and activator of neutrophils. Experimental and clinical observations suggest that joint movements, local biomechanical factors, and previous joint damage may play a role in influencing the intensity of microcrystalline synovitis and the distribution of articular and periarticular crystal deposits in both calcium pyrophosphate dihydrate crystal deposition disease and gout. There are rare reports of extra-articular calcium pyrophosphate dihydrate crystal deposition in tendons, bursae, dura mater, and ligamentum flavum (with radiculomyelopathy) and of massive "tumoral," tophuslike, periarticular calcium pyrophosphate dihydrate crystal deposits. Synovial fluid levels of ATP, the main substrate for nucleoside triphosphate pyrophosphohydrolase ectoenzyme, which cleaves ATP-releasing inorganic pyrophosphate, are higher in patients with calcium pyrophosphate dihydrate crystal deposition disease than in those with other arthritides, and the levels correlate with inorganic pyrophosphate concentrations. Further reports of acute calcific periarthritis of the first metatarsophalangeal joint (hydroxyapatite pseudopodagra) in young women have been described. The mitogenic response of fibroblasts to stimulation with basic calcium phosphate crystals is accompanied by induction and secretion of collagenase and neutral proteases, implicating a role for the crystals in the pathogenesis of both synovial proliferation and joint damage in chronic basic calcium phosphate crystal-associated arthropathy. Subcutaneous cholesterol crystal deposition with tophus formation is extremely rare and has been described in a patient with scleroderma and calcinosis cutis.
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PMID:Calcium pyrophosphate crystal deposition disease and other crystal deposition diseases. 150 84

We have detected the presence of nuclear 3,5,3'-triiodothyronine (T3) receptors in primary cultures of chick embryo hepatocytes and neurons. Hepatocytes were isolated from livers of embryos of 12, 16 and 19 days by treatment with 0.2% collagenase and hyaluronidase. They were plated at a density of 3-4 x 10(5)/35-mm petri dish in Ham's F-10 medium containing fetal calf serum, tryptose phosphate, and antibiotics. Cells were used for the binding assay at Day 3 of culture. Neurons from 8-day-old embryo brains were cultured in a serum-free medium at a density of 1.2 x 10(6) cells/35-mm petri dish and used for the binding assay after 7 days of culture. Biological activity of hepatocytes was determined by measuring insulin binding, inositol phosphate formation, and 5'-monodeiodinase activity. Neurons or glial cells in culture were identified by immunostaining with anti-neurofilaments and anti-glial fibrillary acidic protein antisera. Binding assay was performed with isolated nuclei and 0.4 M NaCl nuclear extracts. With the latter preparation, the Scatchard analysis showed, in both cells, a single, high-affinity, low-capacity T3 receptor. In the hepatocytes of 12-, 16-, and 19-day-old embryos association constants (Ka) were, respectively, 0.93 +/- 0.02, 0.74 +/- 0.03, and 0.56 +/- 0.04 nM-1, whereas the maximal binding capacities (MBC) were 2.26 +/- 0.2, 2.72 +/- 0.33, and 1.83 +/- 0.19 fmol/microgram DNA (mean +/- SE, n = 3). In neurons Ka was 1.25 +/- 0.53 nM-1 and MBC 0.59 +/- 0.14 fmol/microgram DNA (n = 3). The receptor had a sedimentation coefficient of 3.4 S, an estimated Mr of 59 kDa, and the following relative affinity for thyroid hormone analogues: TRIAC greater than L-T3 greater than L-T4. These data indicate that cultured hepatocytes and neurons of chick embryo contained T3 receptors with properties similar to those described in intact tissues from this and other species. Only the MBC of neurons was 50% lower than that observed in whole brain of embryo, but was comparable to values observed in cultured neurons from other species.
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PMID:Characterization of 3,5,3'-triiodothyronine receptors in primary cultures of hepatocytes and neurons from chick embryo. 160 Dec 52

Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathies that are characterized by synovial proliferation and digestion of articular collagenous structures. BCP crystals are potent mitogens, which may account for this proliferation. The role of collagenase in articular degradation is controversial because, despite the massive loss of collagen, no studies have confirmed collagenolytic activity in synovial fluid, as originally reported. We investigated collagenase messenger RNA induction and enzyme activity in human foreskin fibroblasts proliferating in response to stimulation with BCP crystals, and analyzed the associated secreted proteins. Northern blots revealed a dose-dependent accumulation of collagenase message, evident by 4 hours and continuing to at least 36 hours, in BCP-stimulated cultures. One- and 2-dimensional polyacrylamide gel electrophoresis of conditioned media from BCP crystal-stimulated cultures revealed the selective induction of 2 proteins with molecular weight and pI values consistent with those of collagenase. Increased enzyme activity was also found. Thus, the mitogenic response of fibroblasts to BCP crystals is accompanied by collagenase induction and secretion, supporting the hypothesis that they act as a mediator of joint destruction in BCP crystal-associated arthropathies.
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PMID:The mitogenic response to stimulation with basic calcium phosphate crystals is accompanied by induction and secretion of collagenase in human fibroblasts. 165 Feb 21

Extracellular, membrane-bound vesicles are widely regarded to be the initial site of calcification in a variety of tissues under normal and pathological conditions. Alkaline phosphatase is believed to play a vital role in this process by hydrolysing ester phosphates or mineral inhibitors, e.g. inorganic phosphates. In the present study, matrix vesicles from normal and rachitic rat growth plates were compared with regard to specific activity of alkaline phosphatase, total vesicle protein and ultrastructural distribution of alkaline phosphatase activity. Matrix vesicles were released from normal or rachitic growth plates by collagenase digestion and isolated by differential centrifugation. Enzyme cytochemical localization involving a cerium capture method was performed on vesicles collected by vacuum filtration on Millipore filters. SDS gels and Western blots on fractions of both normal and rachitic matrix vesicles showed major proteins to be almost identical and confirmed the presence of alkaline phosphatase in both. Total matrix vesicle protein ((mg total matrix vesicle protein/rat) x 10(2)) per rat was significantly greater for the rachitic animals (9.0 +/- 2.0 vs. 4.0 +/- 1.0), P less than 0.0001. Alkaline phosphatase specific activity (units alkaline phosphatase/mg vesicle protein) in the rachitic and normal matrix vesicles was 25.29 +/- 9.36 and 18.78 +/- 3.37, respectively (0.05 less than P less than 0.1). Electron dense cerium phosphate deposits were localized to the outer membrane surface of matrix vesicles derived from both types of rats. This data, the first to quantify the relationship between rickets, matrix vesicle protein and alkaline phosphatase specific activity, suggests that matrix vesicles from rachitic and normal rats have biochemical and morphological similarity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Increased matrix vesicle protein in rachitic rat epiphyseal growth plates. 165 31

The quality of pancreatic islets prepared by an intraductal pancreas collagenase perfusion technique was tested using three independent methods: 31P NMR spectroscopy, an insulin secretion test, and a staining method. The viability of pancreatic islet tissue was evaluated using the ratio of phosphate diester to phosphate monoester (PDE/PME) as a new criterion obtained by 31P NMR spectroscopy. According to this criterion, three types of tissue fragments could be characterized: vital (PDE/PME 0.5-0.9), damaged (PDE/PME less than 0.2), and necrotic (no PDE, no PME). The findings in the three different groups could be correlated to three trends of insulin secretion of the preparations following glucose challenge: good response to the glucose challenge, continuous decrease of insulin production, and no insulin secretion. We feel that 31P NMR spectroscopy offers a rapid and suitable method for classifying the viability of isolated pancreatic islets.
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PMID:31P NMR spectroscopy for in vitro viability testing of porcine pancreatic islets. 170 70

The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGF2 alpha were analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF2 alpha. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. InsP1 and InsP4 were observed after a short (5-sec) lag period. HPLC revealed that PGF2 alpha provoked rapid (5 sec) increases in inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), which was rapidly converted to inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4) and inositol 1,3,4-trisphosphate (Ins 1,3,4-P3). The primary inositol bisphosphate isomer present in PGF2 alpha-stimulated bovine luteal cells was inositol 1,4-bisphosphate (Ins 1,4-P2), with lesser amounts of Ins 1,3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1,4,5-P3 was followed temporally in saponin-permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1,4,5-P3 to form Ins 1,3,4,5-P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphosphorylated inositol phosphates in PGF2 alpha-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1,4,5-P3 during agonist-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF2 alpha-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.
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PMID:Prostaglandin F2 alpha stimulates inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate formation in bovine luteal cells. 184 60

The ultrastructure of crystal formation in vitro associated with extracellular membrane-bound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-beta-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultra-centrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30-200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca) x inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.
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PMID:Matrix vesicles isolated from apical pulp of rat incisors: crystal formation in low Ca x Pi ion-product medium containing beta-glycerophosphate. 196 82

Growth plate cartilage from normal and vitamin D-phosphate deficient (-VDP) rats was cultured to study the production of collagenase and tissue inhibitor of metalloproteinases (TIMP) in vitro. All tissues secreted latent collagenase into the medium at a constant rate during the 5 days in culture. Microdissected-VDP growth plates, containing predominatly hypertrophic cells, released up to 8-fold more collagenase into the medium than either intact-VDP or normal growth plates. TIMP was also secreted during the culture, but its rate of production was not as dependent on tissue type as collagenase. The tissue level of collagenase and TIMP before culture was compared with that found in conditioned medium and remnant tissue after culture. During the 5 day culture period microdissected-VDP growth plates, containing predominatly hypertrophic cells, produced 3-times more collagenase/microgram DNA over the starting level than either intact-VDP or normal growth plates. TIMP was never found in tissues after they had been cultured, but was present in all tissues before culture except those containing predominatly hypertrophic cells. The amount of TIMP required to block collagenase was calculated. Growth plates in culture produced enough TIMP to block all collagenase found in the medium and remnant tissue, while extracts of uncultured intact -VDP growth plates, and those divided to contain hypertrophic cells, had an excess of collagenase over TIMP. The results suggest that hypertrophic cells produce far more collagenase than other cells in the growth plate, but all cell types have about the same capacity to synthesize TIMP. As a result, increased collagenase synthesis by hypertrophic cells may surpass increases in TIMP synthesis and lead to collagen removal. This would allow for thinning of the longitudinal septa and expansion of the hypertrophic cells.
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PMID:Production of collagenase and tissue inhibitor of metalloproteinases (TIMP) by rat growth plates in culture. 196 14

The silicon dioxide mineral, cristobalite (CRS) induces inflammation involving both alveolar cells and connective tissue compartments. In this study, we compared lung cells recovered by whole lung lavage and by digestion of lung tissue from rats at varying times after 8 days of exposure to aerosolized CRS. Control and exposed rats were examined between 2 and 36 wk after exposure. Lavaged cells were obtained by bronchoalveolar lavage with phosphate-buffered saline. Lung wall cells were prepared via collagenase digestion of lung tissue slices. Cells from lavage and lung wall were separated by Percoll density centrifugation. The three upper fractions, containing mostly macrophages, were cultured, and the conditioned medium was assayed for effect on lung fibroblast growth and for activity of the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase. Results demonstrated that the cells separated from the lung walls exhibited different reaction patterns compared with those cells recovered by lavage. The lung wall cells exhibited a progressive increase in the number of macrophages and lymphocytes compared with a steady state in cells of the lung lavage. This increase in macrophages apparently was due to low density cells, which showed features of silica exposure. Secretion of a fibroblast-stimulating factor was consistently high by lung wall macrophages, whereas lung lavage macrophages showed inconsistent variations. The secretion of NAG was increased in lung lavage macrophages, but decreased at most observation times in lung wall macrophages. No differences were found among cells in the different density fractions regarding fibroblast stimulation and enzyme secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of lung alveolar and tissue cells in silica-induced inflammation. 198 84

Previous studies identified synapsin I as a potential substrate for a newly discovered growth factor-sensitive, proline-directed protein kinase originally isolated from rat pheochromocytoma. The present study describes the site-specific phosphorylation of synapsin I by highly purified preparations of proline-directed protein kinase. The incorporation of [32P]phosphate into bovine brain synapsin I was dependent upon both the amount of kinase present and the time of incubation. The maximum stoichiometry of phosphorylation approached 1 mol of phosphate/mol of synapsin I protein. When analyzed by sodium dodecyl sulfate-gel electrophoresis and autoradiography, [32P]phosphate was found to be incorporated into both synapsin Ia and Ib. Phosphoamino acid analysis demonstrated that serine residues were phosphorylated exclusively. Digestion of phosphorylated synapsin I with trypsin followed by high performance liquid chromatography (HPLC) phosphopeptide analysis indicated that the tryptic peptide containing the major phosphorylation site eluted as a single peak at approximately 17% acetonitrile. The primary structure of this phosphopeptide, determined by gas-phase sequencing, was found to be Gln-Ser-Arg-Pro-Val-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Pro-Ala-Thr-Arg-Pro-Pro- Ala-Ser-Pro-Ser-Pro-Gln-Arg. Sequential Edman degradation of this HPLC-purified tryptic phosphopeptide revealed that serine 20 of this peptide was the major phosphorylated residue. This phosphoacceptor site is immediately flanked by a carboxyl-terminal proline residue, an observation that further verifies the proline-directed nature of this protein kinase. The tryptic phosphopeptide corresponds exactly to a sequence in the collagenase-sensitive, proline-rich "tail" region of bovine synapsin I. This novel phosphorylation site is close to but distinct from phosphorylation sites 2 and 3, which are known to be phosphorylated by calcium/calmodulin-dependent protein kinase II and are considered to be of regulatory importance.
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PMID:Phosphorylation of synapsin I at a novel site by proline-directed protein kinase. 210 63


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