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)

Oval cells emerging in rat liver at the early period of 3-methyl-4-dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to alpha-fetoprotein (AFP) and cytokeratin differential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et al., Cancer Res., 45:673-681, 1985). In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer. The use of monoclonal and polyclonal antibodies directed against cytokeratins of Mr 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BDS7) and normal hepatocytes (HES6) allowed us to establish the ductular phenotype of the oval cells. A highly enriched preparation of oval cells was obtained by perfusion/digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anti-HES6 antibody, and cell separation by isopyknic centrifugation in a Percoll gradient. The procedure yielded about 8 x 10(7) cells, of which 95% expressed CK39, CK52, and BDS7, 84% gamma-glutamyl transpeptidase, and 5% albumin and AFP. The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by [3H]thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase. Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled. Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells. When the cells were cultured in the presence of an elaborate combination of supplements [minimum essential medium containing 1 mM pyruvate, 0.2 mM aspartate, 0.2 mM serine, 1 mM tyrosine, 1 mM proline, 1 mM phenylalanine and supplemented with 20% clot blood extract, 10 ng/ml oxidized bile acids, 17 microM bilirubin, 10 ng/ml cholera toxin, 1 microM dexamethasone, 2.5 micrograms/ml insulin, 50 mM beta-mercaptoethanol, and 5 micrograms/ml transferrin (medium MX)], the labeling index increased to around 30% and the level of cell fusion greatly decreased. The addition of dimethyl sulfoxide further enhanced the initiation of DNA synthesis, while sodium butyrate acted as an inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Promotion of growth and differentiation of rat ductular oval cells in primary culture. 244 46

In considering the mechanism of transformation of epithelium to mesenchyme in the embryo, it is generally assumed that the ability to give rise to fibroblast-like cells is lost as epithelia mature. We reported previously that a definitive embryonic epithelium, that of the anterior lens, gives rise to freely migrating mesenchyme-like cells when suspended in type I collagen matrices. Here, we show that a highly differentiated epithelium that expresses cytokeratin changes to a vimentin cytoskeleton and loses thyroglobulin during epithelial-mesenchymal transformation induced by suspension in collagen gel. Using dispase and collagenase, we isolated adult thyroid follicles devoid of basal lamina and mesenchyme, and we suspended the follicles in 3D collagen gels. Cells bordering the follicle lumen retain epithelial polarity and thyroid phenotype, but basal cell surface organization is soon modified as a result of tissue multilayering and elongation of basal cells into the collagenous matrix. Cytodifferentiation, determined by thyroglobulin immunoreactivity, is lost as the basal epithelial cells move into the matrix after 3-4 days in collagen. By TEM, it can be seen that the elongating cells acquire pseudopodia, filopodia and mesenchyme-like nuclei and RER. Immunofluorescence examination of intermediate filaments showed that freshly isolated follicles and follicles cultured on planar substrata react only with anticytokeratin. However, all of the mesenchyme-like cells express vimentin and they gradually lose cytokeratin. These results suggest that vimentin may be necessary for cell functions associated with migration within a 3D matrix. The mesenchymal cells do not revert to epithelium when grown on planar substrata and the transformation of epithelium to mesenchyme-like cells does not occur within basement membrane gels. The results are relevant to our understanding of the initiation of epithelial-mesenchymal transformation in the embryo and the genetic mechanisms controlling cell shape, polarity and cytoskeletal phenotype.
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PMID:Cytoskeleton and thyroglobulin expression change during transformation of thyroid epithelium to mesenchyme-like cells. 246 Mar 6

Single cardiac myocytes were isolated from hearts of 9 to 12-week-old rats by means of collagenase (100 U/ml). After assessment of their functional integrity they were processed for immunofluorescence microscopy of the cytoskeletal proteins tubulin, microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), plectin, vimentin, and vinculin. Antibodies to tubulin decorated a delicate filamentous network that apparently was unrelated to any sarcomeric organization. The distribution of MAP-1 and MAP-2 was strikingly different from that of tubulin, as both antigens were confined to Z-line structures. These structures were also prominently stained by affinity-purified antibodies to plectin and a monoclonal antibody to vimentin. Co-distribution of plectin and vimentin was also observed at the former intercalated disk region of the heart cell. Anti-vinculin antibodies decorated an intricate meshwork consisting of delicate filaments with predominantly irregular orientation and occasional assembly into whorls. These immunolocalization data indicate that the cell shape and cytoskeletal architecture characteristic of cardiac myocytes in tissues is maintained in single isolated cells. Furthermore, intermediate filaments rather than microtubules seem to be instrumental in the preservation of cell morphology.
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PMID:Morphological integrity of single adult cardiac myocytes isolated by collagenase treatment: immunolocalization of tubulin, microtubule-associated proteins 1 and 2, plectin, vimentin, and vinculin. 299 82

Subendothelial cells (SEC) were obtained from the inner intimal layer of adult human aorta by collagenase treatment. SEC were identified in primary culture either as smooth muscle cells by staining with FITC-labeled antisera against human smooth muscle myosin or as macrophages, foam cells and contaminating endothelial cells by their uptake of malondialdehyde treated low density lipoproteins labeled with fluorescent dye 3,3'-dioctadecylindocarbocyanine. Between 1 and 5 days in culture, along with smooth muscle cells (SMC, 38-82%), endothelial cells (0-9%), macrophages and foam cells (2-32%), one more type of cell was found. This cell type resembled SMC in size and shape, but was not stained by antisera to SMC myosin. By ultrastructural criteria these cells were characterized as modulated SMC for they contained prominent rough endoplastic reticulum and Golgi complex together with basement membrane and a large number of plasmalemmal vesicles. Like SMC they reacted with phalloidin and were stained by anti-vimentin but not by anti-desmin monoclonal antibodies. The proportion of such cells varied from 5 to 33% of total cell number and increased in parallel to macrophages and foam cells in vessels with well developed atherosclerotic lesions. We conclude that the applied technique may be used for identification of cultured vascular cells including modulated SMC.
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PMID:Identification of intimal subendothelial cells from human aorta in primary culture. 313 80

A 140 000 D glycoprotein (140 kD gp), labelled radioactively with surface-specific techniques, remained as the major cell surface glycoprotein in the detergent-resistant cytoskeletal preparations of cultured human fibroblasts. The 140 kD gp was present also in trypsinized cells and was not affected by treatment of the cells either with collagenase, chymotrypsin or thrombin. In density gradient fractionation of whole cells the 140 kD gp was recovered in the plasma membrane fraction together with small amounts of cytoskeletal components. In fractionation of cytoskeletal preparations, on the other hand, the 140 kD gp could not be dissociated from cytoskeletal proteins and together with vimentin it formed the major component of the oligomeric polypeptide complex generated by treating the surface-labelled cytoskeletal preparations with bifunctional cross-linking reagent, dithiobis succinimidyl propionate (DTPS). Moreover, the 140 kD gp seemed to copurify with vimentin upon reconstitution of intermediate filaments from urea-solubilized cytoskeletal preparations. On the other hand, low ionic-induced degradation of vimentin led to a decrease in the amount of the detergent-resistant 140 kD gp on the cell surface. In electron microscopy, a close apposition between bilayer-like plasma membrane remnants of the adherent cytoskeletons and cytoskeletal elements could be seen. The results indicate that the 140 kD gp is a plasma membrane glycoprotein which closely interacts with the detergent-resistant cytoskeleton of cultured human fibroblast. Possible mechanisms of the association are discussed.
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PMID:140 000 Dalton surface glycoprotein. A plasma membrane component of the detergent-resistant cytoskeletal preparations of cultured human fibroblasts. 633 55

In vitro studies on dispersed human endometrial cells are still difficult to perform as the techniques employed do not allow an optimal separation of the cells. In particular, epithelial glands tend to maintain their tubular structure even after enzymatic dispersion. This could be a disadvantage when monolayers of single well dispersed cells are needed. In this paper we describe a new technique to establish monolayer cultures of isolated endometrial stromal and epithelial cell populations. After a first collagenase digestion, stromal and epithelial cells were separated by differential sedimentation at unity gravity. The epithelial glands obtained were further dispersed in single cells using a short incubation with low amount of trypsin. Morphologic characterization was performed using immunohistochemistry for vimentin and cytokeratins. Compared to previous described methods, this procedure is shorter and could better preserve cell surface structures. Thus, it could be successfully employed for in vitro studies of human endometrial pathophysiology.
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PMID:Culture of human endometrial cells: a new simple technique to completely separate epithelial glands. 768 May 17

The ovarian mesothelium (OM) represents the tissue of origin of ovarian epithelial cancer. To gain insight into the regulation of this tissue, OM organoids and submesothelial ovarian stromal cells (SC) were isolated from New Zealand White rabbits by a stepwise tissue dispersal technique, while granulosa cells (GC) were aspirated from mature follicles (14 +/- 4 groups/animal). OM and SC dispersal were sequentially accomplished by: a) 1-h incubation in collagenase type I (300 U/ml), gentle scraping of the ovarian surface, and 1 g sedimentation of OM organoids (equivalent to 0.93 +/- 0.40 x 10(6) cells/animal) on 5% bovine serum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%-300 U/ml) under periodical resuspension and gentle scraping of SC (1.40 +/- 0.25 x 10(6)/animal) from OM-denuded ovaries. After a week-long in vitro expansion, OM cells (OMC) were cultured alone and with SC or GC within monocameral vessels or bicameral transfilter vessels in serumless, fibronectinrich (4 micrograms/ml) HL-1 medium. After 7 d of contact cell-cell interaction, cytokeratin-positive OMC became surrounded by fibroblastoid, vimentin-positive SC or by cytokeratin and vimentin-weakly positive GC. Filter-bound OMC humorally interacting with underlying SC or GC displayed a biphasic, epithelioid and spindle, morphology with universal cytokeratin expression. Bromo-2'-deoxyuridine (BrdU) immunoperoxidase revealed mean cell proliferation indices of 14.88% for OMC cultured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monocameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vessels over GC or SC, respectively. This model provides an experimental tool for investigating the unexplored role of stromal-mesothelial interaction in OM pathobiology.
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PMID:Ovarian mesothelial and extramesothelial cells in interactive culture. 779 49

The use of human omentum as an alternative to veins as a source of cells for seeding onto small-caliber vascular prostheses has awakened controversy as to the identification of the predominant cell type derived from this source. Mesothelial cells from omentum were extracted by collagenase digestion, and cultured until a monolayer was formed. These cells showed positivity for monoclonal antibodies specific for endothelial cells (anti-CD34 QBEND10), antibodies to intermediate filaments (anti-vimentin and anti-desmin) and anti-smooth muscle cell antibodies (anti-actin and anti-total actin). The mesothelial cells behaved like endothelial cells derived from vein when seeded onto polytetrafluoroethylene prostheses, showing high levels of prostacyclin production. This report provides additional evidence of the non-endothelial origin of the cells derived from human omentum.
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PMID:Coatings for vascular prostheses: mesothelial cells express specific markers for muscle cells and have biological activity similar to that of endothelial cells. 755 50

The administration of 150 nM etoposide, an inhibitor of DNA topoisomerase II activity, decreased the proliferation and induced the differentiation of U937 human promonocytic cells, as determined by nitroblue tetrazolium reduction, surface accumulation of CD11b/CD18 and CD11c/CD18 integrins, and c-fms protooncogene expression. The expression of these differentiation markers started to be detected at 24 h of treatment. Etoposide caused little cell damage, as determined by trypan blue exclusion and by apoptotic-like DNA degradation, which was slightly initiated at 48 h. The treatment induced a transient increase in c-fos, c-jun, and jun B mRNA levels, with maximum values at 12 h, a transient increase in collagenase mRNA level, with maximum value at 48 h, and a progressive increase in vimentin and lamin A and C mRNAs. These changes were qualitatively similar to those produced by 12-O-tetradecanoylphorbol-13-acetate. Etoposide also caused a transient increase of total AP-1 binding activity, with maximum value at 12 h of treatment, as determined by gel retardation assays. The drug produced an early transient activation (3-6 h) of membrane-bound protein kinase C, followed by the later activation (48 h) of both the membrane and cytosolic enzyme. The protein kinase C inhibitors, sphinganine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), attenuated the induction of differentiation markers by etoposide. These results suggest that protein kinase C and AP-1-dependent gene expression could be involved in myeloid cell differentiation by DNA topoisomerase II inhibitors.
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PMID:Etoposide-induced differentiation of U937 promonocytic cells: AP-1-dependent gene expression and protein kinase C activation. 781 32

Proximal tubular cells (PTC) were isolated from porcine kidney by collagenase treatment, subsequently purified on a discontinuous density gradient and finally cultured. Porcine PTC (PPTC) in primary culture expressed keratin, characteristics of epithelia and brush border specific glycoproteins (FX1A). In addition, vimentin was present. All cells were negative for the endothelial marker pal-E. Less than 0.1% expressed the Tamm-Horsfall protein, characteristic of the distal tubule, while less than 0.3% of all cells in culture expressed desmin, characteristic of connective tissue (i.e. fibroblasts) and mesangial cells. Ultrastructural analysis revealed microvilli, tight junctions and abundant mitochondrial and lysosomes, all characteristics of proximal tubular cells. Freshly isolated PPTC were validated as in vitro model to detect nephrotoxicity by studying the effect of mercuric chloride, cis-platin, p-aminophenol and the halogenated alkenes 1,2 dichlorovinyl-l-cysteine, S-(1,1-difluoro-2,2-dichloroethyl)-L-cysteine (DCDFE-cys) and the glutathione conjugate of DCDFE on viability and mitochondrial membrane potential. The cells responded, time- and dose-dependently, to the nephrotoxic compounds with a decrease in mitochondrial membrane potential and loss of viability. The sensitivity of the porcine cells in detecting toxic effects corresponded favorably with in vitro systems derived from other animals.
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PMID:Evaluation of nephrotoxicity in vitro using a suspension of highly purified porcine proximal tubular cells and characterization of the cells in primary culture. 785 34


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