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)

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

Spectrometry has been employed to assess the levels of collagenase, catepsin D, trypsin-like proteinases and their inhibitors as well as bone acid and alkaline phosphatase both in the center and along the periphery of giant cell tumor of bone (GCTB) and chondrosarcoma. The levels of collagenase, trypsin-like proteinases and their inhibitors in the center of chondrosarcoma were much higher while those of alkaline phosphatase--lower than along tumor periphery. The catepsin D and acid phosphatase concentrations of the center and periphery of chondrosarcoma were similar. It was suggested that an extremely low concentration of trypsin-like inhibitors may contribute to degradation of the matrix in tissues adjacent to chondrosarcoma and, consequently, to tumor invasion development.
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PMID:[The comparative biochemical and morphological characteristics of chondrosarcoma and giant-cell tumor of the bone]. 1097 75

The procedures recently developed in our laboratory to observe three-dimensional structures of cell organelles in thick biological specimens by means of high voltage electron microscopy are reviewed. Thick biological specimens such as whole mount cultured cells seeded and grown on grid meshes in culture vessels or thick sections cut from embedded tissues and stained by histochemical reactions can be readily observed three-dimensionally by high voltage transmission electron microscopy at 400-1000kV. Cultured cells used were both primary cultures from animal tissues and established cell lines maintained in our laboratory. The livers of adult Wistar rats were isolated by collagenase perfusion, and hepatocytes were suspended in a Leibovitz medium and seeded on formval coated gold grid meshes in Petri dishes, incubated in a CO(2) incubator in a humidified atmosphere containing 5% CO(2) in air at 37 degrees C for a few days. Established cell lines, CHO-K1 cells, were cultured in Ham's F12 medium, while HeLa cells were cultured in Eagle's MEM under the same condition. Some of the cells were cultured under experimental conditions such as hepatocyte culture in the medium containing peroxisome proliferating agents such as clofibrate or bezafibrate and some of them were labeled with (3)H-thymidine, (3)H-uridine, (3)H-labeled precursors and (14)C-bezafibrate. Also some cells were incubated in medium containing HRP to induce pinocytosis. All the whole mount cultured cells on grid meshes were prefixed in buffered 2.5% glutaraldehyde, stained with various histochemical reactions and postfixed in 1% osmium tetroxide. The histochemical reactions used were glucose-6-phosphatase (G-6-Pase), thiamine pyrophosphatase (TPPase), cytochrome oxidase, acid phosphatase (AcPase), DAB, ZIO, PA-TCH-SP reactions and radioautography was performed after labeling with radiolabeled compounds. The whole mount cultured cells were dried in a critical point dryer and were observed with JEOL JEM-4000EX or Hitachi H-1250M high voltage electron microscopes at 400-1000kV. By tilting the specimens' stereo-pair micrographs were recorded and they were observed with stereoscopes. Rat liver, mouse intestine and pancreas tissues, fixed and stained as above, were embedded in Epoxy resin, thick sectioned at 1-2 microm and were observed as for the whole mount cultured cells at 1000kV. Stereo-pairs were further analyzed with an image analyzer JEOL JIM-5000 (JEOL, Tokyo, Japan), producing two contour lines plotted from the micrographs at a thickness of 0.2 microm and were observed with anaglyph type glasses, demonstrating the depth or heights of respective cell organelles. The results show that whole mount cultured cells and thick sections stained with histochemical reactions reveal cell organelles corresponding to marker enzymes, such as G-6-Pase in endoplasmic reticulum, TPPase and ZIO in Golgi apparatus, cytochrome oxidase in mitochondria, AcPase in lysosomes, DAB in peroxisomes and pinocytotic vesicles, PA-TCH-SP in secretory granules, (3)H-thymidine and (3)H-uridine in nuclei, (3)H-animo acids in endoplasmic reticulum and secretory granules, (14)C-bezafibrate around ER and peroxisomes. The ultrastructure of these cell organelles as well as the structural relationship between them can be demonstrated three-dimensionally with stereo-pair images. Overall, these procedures are useful for analyzing stereologically the ultrastructure of cell organelles in cells and tissues.
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PMID:Three-dimensional high voltage electron microscopy of thick biological specimens. 1107 Mar 59


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