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)

During bone remodeling, activation of resorption is followed by a cycle of formation and this ordered sequence of events has long suggested that local interactions between osteoclasts and osteoblasts are an important regulatory mechanism in bone metabolism. To study this phenomenon, we have prepared bone cells containing primarily osteoclasts by brief digestion of mice calvariae in collagenase, overnight attachment to polystyrene tissue culture flasks in serumless medium supplemented with OB (osteoblast) cell conditioned medium and subsequent growth in low serum. These OC (osteoclast) cells were found to be highly enriched in acid phosphatase activity and expressed cAMP responses to PTH (parathyroid hormone) and prostaglandin E2 but exhibited no PTH-stimulated hyaluronate synthesis in contrast to prostaglandin E2. PTH effects on hyaluronate, however, could be restored upon coculture of OC cells with OB cells (noncontact) or with OB cell conditioned medium, thereby suggesting that OB cells regulate OC cell PTH responsiveness and/or differentiation by soluble cell products secreted into the medium.
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PMID:Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone. 632 52

Deflazacort is a new synthetic glucocorticoid which is an oxazoline derivative of prednisolone. In previous studies, it was shown that deflazacort, depending on the test model used, not only showed considerably more antiinflammatory potency than prednisolone in animals but also caused less deleterious effects on bone mineral metabolism than equivalent amounts of other glucocorticoids in man. In this study, we have compared the effects of deflazacort with those of dexamethasone on the synthesis of collagen in various rat bone cell populations and chondrocytes. Three bone cell populations were prepared by sequential time-dependent collagenase treatment of 1-day-old rat calvaria. Each cell population was further purified on a Percoll gradient (10-90%) yielding three populations of which two are different in alkaline and acid phosphatase and response to parathyroid hormone. A 3-day treatment of bone cell populations with deflazacort and dexamethasone (10(-11)-10(-5) M) revealed that both glucocorticoids, although at different concentrations, inhibited collagen synthesis. 21-desacetyl-deflazacort (5 beta, 11 beta, 16 beta)-11,21-dihydroxy-2'-methyl-5-H-pregna-1-enol [17,16-d]oxazole-3,20-dione), the presumably active form of the steroid, which is formed in vivo after administration, produced nearly identical results as its precursor. Glucocorticoid concentrations at which inhibition was initially observed were 10(-9) M and 10(-7) M for dexamethasone and deflazacort respectively. Inhibition of collagen synthesis was significantly impaired only in cells isolated from bone during early tissue digestion, and not in those obtained during extensive collagenase treatment. Chondrocytes isolated from articular cartilage of 3-month-old rabbits and grown in primary cultures did not respond to either steroid.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparative study of deflazacort, a new synthetic corticosteroid, and dexamethasone on the synthesis of collagen in different rat bone cell populations and rabbit articular chondrocytes. 643 Apr 98

Giant cell tumors of bone dissociated by collagenase digestion were found to be composed of four different cell types defined by morphology, growth in culture, and pattern of staining with monoclonal antibodies. Giant cells comprised an average of 0.8% of the cells recovered, with the remainder consisting of small stromal cells. Of the giant cells, 20-57% expressed Ia antigens, while all lacked IgG Fc receptors and five differentiation antigens associated with mature members of the monocyte-macrophage lineage (M phi S-1, M phi P-9, M phi P-15, M phi S-39, and 63d3). One antigen, M phi U-50, found on early monocytoid forms was expressed on Ia+ giant cells. 6-36% of the remaining stromal tumor cells formed a second subpopulation that assumed either a rounded or elongated shape in culture. These cells bore Ia antigens, IgG Fc receptors, and five antigens of the monocyte-macrophage lineage usually found on blood monocytes. However, these cells differed from monocytes or macrophages in that the antigen M phi R-17 generally found on tissue macrophages was absent, and the M phi U-50 antigen present on more primitive cells was well expressed. A very limited endocytic capacity was demonstrable. A third population of up to 24% of the tumor cells was defined by the presence of intense staining for Ia antigens but the absence of antigens of mature monocytes. A proportion of these cells expressed M phi U-50 and a minority had IgG Fc receptors. The two Ia(+) populations of stromal cells were not identifiable after 2 wk of culture, nor did tumor cells selected for the presence of Ia antigens proliferate in culture. A fourth population of cells lacked Ia and monocyte lineage antigens, but showed pronounced intracellular staining for acid phosphatase. These cells had a distinctive plump epitheloid to fibroblastoid morphology and were readily established in long-term culture where they gave rise to large multinuclear Ia(-) cells containing acid phosphatase. The possibility is discussed that the cell types of these tumors relate to various stages in the development of osteoclasts from precursors in the mononuclear phagocyte lineage.
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PMID:Delineation of four cell types comprising the giant cell tumor of bone. Expression of Ia and monocyte-macrophage lineage antigens. 657 16

Sequential collagenase digestion of mice calvariae provides populations of bone cells that express either osteoclasts (OC) or osteoblastic (OB) activities after growth for 6 days in similar culture conditions consisting of minimal essential medium supplemented with 10% fetal calf serum (FCS). The OC characteristics (acid phosphatase activity and hyaluronate synthesis, and their stimulation by PTH) were recovered in the cell populations released early from calvariae, but these also contained OB cells and numerous spindle-shaped alkaline phosphatase positive cells that resembled fibroblasts. We have attempted to select for growth of OC cells in these early populations by exploiting differences in growth requirements of OC, OB, and fibroblastic cells. We find that after growth for 6 days in low serum (2% FCS), OC cell populations demonstrated a threefold increase in OC activity/cell, and cell yield was reduced to one-third of that obtained in 10% FCS. Spindle-shaped cells were absent in 2% FCS and OB marker activities (alkaline phosphatase and citrate decarboxylation) were reduced threefold. In contrast to OC cells, high serum (10% FCS) favored the growth and phenotypic expression of OB cells (late populations). Cell yield and OB marker activities/cell were twofold higher in OB cells grown in 10% FCS vs 2% FCS, whereas growth but not phenotypic expression was retained at 5% FCS. These data suggest that differential serum dependence of OC and OB cells may provide a basis for further enrichment for each cell type following sequential digestion.
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PMID:Differential serum dependence of cultured osteoclastic and osteoblastic bone cells. 665 53

Local or systemic prostaglandin (PG) administration leads to the known softening and dilatation of the cervix uteri. Lysosomal enzymes are involved in connective tissue degradation. The question arises whether the effect of PG on the cervix uteri is mediated by lysosomes. Five pregnant women (volunteers after informed consent) in the first trimester received 500 micrograms of PGE2-derivative (Nalador) i.m. at 12 and 8 h before termination by curettage. Five pregnant women without PG-treatment served as controls. Small biopsies were obtained from the endocervical canal and were immediately immersed in cold 2.5% glutaraldehyde and after further preparations examined under a Zeiss electron microscope 9S-2. A second portion of tissue was sliced and prepared for histochemical analysis of the acid phosphatase on lysosomes. Examination of the ultrastructure of the cervix uteri showed vesicles in the extracellular matrix. These were surrounded by a single membrane and contained either fine granular material of myelin-like whorls of membranes. These vesicles lay between collagen fibers, showed the reaction product of acid phosphatase and were often surrounded by an electron-lucent halo. We conclude that these matrix vesicles were "matrix lysosomes" extruded from the cervical myo-fibrocytes into the extracellular space as a result of the PG-E2-administration. Here they are not under cellular control and can initiate the proteolytic degradation of connective tissue. This might be the crucial step in cervical dilatation which, on ultrastructural examination, can be seen as decreasing electron density of the extracellular ground substance near the matrix lysosomes. The relationship between PGE2 and collagenase production is generally accepted. If one believes that lysosomal cathepsin D and cathepsin B act synergistically with collagenase, it can be assumed that PGE2 is involved in a lysosomal degradation of the connective tissue. The morphological sign of this occurrence is the release of matrix lysosomes by PGE2 as described in the present study. Extracellular lysosomes and their physiological significance in cervical function are discussed in detail.
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PMID:The effect of prostaglandins on the lysosomal function in the cervix uteri. 666 Sep 24

Epithelial-cell-enriched primary cultures were established from canine prostate. Minced tissue was dissociated with 750 units/ml of collagenase in F12K tissue culture medium containing 1% fetal bovine serum. This treatment resulted in the gradual removal of stromal elements from the base of the epithelial cells. After 30 minutes of digestion, aggregates of epithelial cells free of stroma were dislodged from the minced pieces of prostate. These aggregates were washed and plated at high density in F12K plus 10% fetal bovine serum. After 12-16 hours in vitro the unattached cellular aggregates were removed from the culture dishes, washed, and reinoculated into new culture vessels containing fresh medium. After 48 hours in vitro, the aggregates had attached to the culture vessels and spread out to yield discrete patches of epithelial cells. By 120 hours in vitro the patches of cells had grown and coalesced to form a confluent monolayer of epithelial cells. Ultrastructural examination of these cultures indicated that adjacent cells were joined by desmosomes and tight junctions and had tonofilaments and microvilli, giving the cells an epithelial appearance. The cells contained rough endoplasmic reticulum, Golgi apparatus, and secretory granules similar to those of the epithelial cells in the intact organ. In addition, intracellular "blebs" containing acid phosphatase were observed in the monolayers and were found to increase in size and number with time in vitro. Differentiated function of the cultures was demonstrated by the presence of ornithine decarboxylase and acid phosphatase and the ability of the cultures to metabolize testosterone to primarily 5 alpha-reduced metabolites.
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PMID:Primary epithelial cell cultures derived from canine prostate: isolation, culture, and characterization. 713 51

Differentiated mammalian cell lines can be established by introducing viral oncogenes into primary cells. Such lines can retain their original specialised functions while being adapted to prolonged life in culture; but most transformed cell lines obtained in this way characteristically show altered properties compared with the primary cells. The result of these changes is that transformed cell lines no longer provide a good model of the original tissue, and indeed often resemble other transformed lines more than the initial cell type. In our laboratory three murine peritoneal macrophage-like cell lines have been isolated by transforming primary cells with SV40 origin-deleted DNA. These lines have been in continuous culture for approximately 1 year and have been shown to express many macrophage-specific properties throughout this time, including Fc receptors and staining for non-specific esterase. The cell lines phagocytosed IgG-coated particles, they were positive for the murine macrophage-specific marker F4/80 and they showed antigen-presentation function. Lysozyme, acid phosphatase, plasminogen activator, collagenase, prostaglandin E2 and 5'-nucleotidase activities have also been detected in these lines. In this paper the method of DNA transformation will be described as well as some of the assays used for the characterization of the three immortalized cell lines.
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PMID:Establishment and characterization of murine macrophage-like cell lines following transformation with simian virus 40 DNA deleted at the origin of replication. 808 36

The temporo-mandibular joint of aged mice develops osteoarthritic (OA) degenerative lesions. Adult chondrocytes have a low rate of cell replication, and cartilage repair potential is very limited. One of the major problems in OA is the low rate of matrix synthesis and the inability of the chondrocytes to exceed the rate of matrix degradation. These combined factors lead to the overall destruction of the cartilage as seen in OA. Cartilage degradation is mediated by elevated proteolytic activity of enzymes. Among the enzymes degrading cartilage are the metalloproteinases, stromelysin and collagenase. Other proteinases that may potentially participate in matrix degradation are the lysosomal enzymes cathepsin B, D, and L, and acid phosphatase. On the other hand, alkaline phosphatase (ALP) is an enzyme that has been shown to be a marker for anabolic activity in skeletal tissues such as bone and cartilage. The cartilage of the mandibular condyle in the T-M-J from aged mice reveals OA lesions. An overall reduction of cell proliferation and sulfated proteoglycan synthesis has been also shown in this joint. In the present study the effects of hTGF-beta on the stimulation of DNA and sulfated GAG synthesis and ALP activity were studied. Mandibular condyle cartilage obtained from 12-month-old ICR male mice were cultured in BGJb serum-free medium for 24-72 hours, supplemented with 0.1-10 ng/ml hTGF-beta 1. 3H-thymidine and 35S-sulfate were added for the last 24 hours of the culture and their incorporation into DNA and sulfated GAGs respectively, as well as the activity of ALP, were determined. Results indicated that hTGF-beta 1 enhanced the incorporation of both 3H-thymidine and of 35S-sulfate into cartilage cultures of aged mice, and also induced ALP activity. It thus appeared that in OA degenerating articular cartilage, the chondrocytes could be stimulated in vitro to proliferate and to synthesize new matrix, thus indicating induced anabolic activity in the tissue.
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PMID:Osteoarthritis in the temporo-mandibular joint (TMJ) of aged mice and the in vitro effect of TGF-beta 1 on cell proliferation, matrix synthesis, and alkaline phosphatase activity. 918 53

Pycnodysostosis (Pycno) is an autosomal recessive osteosclerotic skeletal dysplasia that is caused by the markedly deficient activity of cathepsin K. This lysosomal cysteine protease has substantial collagenase activity, is present at high levels in osteoclasts, and is secreted into the subosteoclastic space where bone matrix is degraded. In vitro studies revealed that mutant cathepsin K proteins causing Pycno did not degrade type I collagen, the protein that constitutes 95% of organic bone matrix. To determine the in vivo effects of cathepsin K mutations on bone metabolism in general and osteoclast-mediated bone resorption specifically, several bone metabolism markers were assayed in serum and urine from seven Pycno patients. Two markers of bone synthesis, type I collagen carboxy-terminal propeptide and osteocalcin, were normal in all Pycno patients. Tartrate-resistent acid phosphatase, an osteoclast marker, was also normal in these patients. Two markers that detect type I collagen telopeptide cross-links from the N and C termini, NTX and CTX, respectively, were low in Pycno. A third marker which detects a more proximal portion of the C terminus of type I collagen in serum, ICTP, was elevated in Pycno, a seemingly paradoxical result. The finding of decreased osteoclast-mediated type I collagen degradation as well as the use of alternative collagen cleavage sites by other proteases, and the accumulation of larger C-terminal fragments containing the ICTP epitope, established a unique biochemical phenotype for Pycno.
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PMID:Determination of bone markers in pycnodysostosis: effects of cathepsin K deficiency on bone matrix degradation. 1057 90

Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon epithelium in toxicological studies in vitro.
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PMID:Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes. 1096 60


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