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 patch-clamp study of Ca2+ currents and spectrofluorometric detection of the intracellular Ca2+ concentration [Ca2+]i was performed on porcine myometrial cells that had been isolated by collagenase. Isolated myometrial cells were the typical long cylinder shape. The main length and diameter of myometrial cells were 505 +/- 20 and 11 +/- 0.5 microns (n = 40), respectively, in the prepartum period and 265 +/- 22 and 7 +/- 0.4 microns (n = 40), respectively, in the luteal phase. Immunocytochemistry with an antibody against desmin stained 90% of the cells positively, and about 95% of the cells excluded Trypan blue dye. The basal [Ca2+]i of myometrial cells in the luteal phase and the prepartum period was 119 +/- 12 (n = 30) and 154 +/- 31 nmol l-1 (n = 48), respectively. In prepartum myometrial cells, oxytocin (10(-7) mol l-1) and carbachol (10(-4) mol l-1) increased [Ca2+]i in a biphasic pattern, with a sharp peak followed by a plateau. In cells in the luteal phase, adrenaline (10(-7) mol l-1) plus propranolol (10(-6) mol l-1) produced a biphasic increase in [Ca2+]i. However, in the absence of propranolol, the increase in [Ca2+]i by adrenaline was small. Prostaglandin F2 alpha (10(-6) mol l-1) induced a monophasic increase in the [Ca2+]i in cells in the luteal phase. By depolarizing the cells from -30 to +50 mV from a holding potential of -50 mV, Ca2+ currents were evoked with a threshold at -20 mV, reaching a maximum between 10 and 30 mV.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of freshly dispersed porcine myometrial cells: evidence for voltage-dependent Ca2+ channels and regulatory receptors. 779 25

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

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

Nearly homogeneous preparations of stromal cells derived from human proliferative endometrium can be obtained by treating endometrial fragments with collagenase in order to disperse stromal elements, filtering the mixture a 25 microns opening sieve to separate them from gland, and incubating the dispersed cells to culture dishes. Exposure of stromal cell cultures to db-cAMP, 8-Br-cAMP or forskolin in RPMI 1640 medium containing 2% ct-FCS and 0.1 U/ml insulin induces the expression of prolactin (PRL), evident from 1) its secretion to the culture medium, measured by radioimmunoassay and by Western blot analysis, 2) the incorporation of 35S-methionine in a protein precipitated with a PRL antibody and co-migrating with authentic PRL during electrophoresis, and 3) the synthesis of PRL mRNA determined by Northern blot analysis. The cAMP effect on PRL production is enhanced by progestins, which by themselves are weak PRL inducers under similar experimental conditions. As expected from previous findings in our laboratory, showing that addition of PRL to the culture medium induces decidualization of endometrial stromal cells, cAMP derivatives not only induce PRL but also provoke differentiation of the fibroblast-like stromal cells to the decidual phenotype, as evident from morphologic changes and by the expression of products characteristic of decidual cells, e.g. IGFBP-1, desmin, hsp 27 and laminin. These findings suggest a PRL-mediated, progesterone-enhanced decidualization mechanism initiated by physiologic agents increasing cAMP levels in stromal cells.
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PMID:Mechanisms involved in the decidualization of human endometrial stromal cells. 798 67

The ultrastructural and immunocytochemical characteristics of microvascular cells from human subcutaneous fat tissue were studied after the addition of collagenase and Percoll density gradient, respectively. Monoclonal and polyclonal antibodies directed against antigens specific for endothelial cells (factor VIII, Ulex europaeus, CD31, and CD34), pericytes (muscle-specific actin and desmin), adipocytes (S-100 protein), and monocytes-macrophages (MAC 387 and 150.95 protein) were demonstrated by alkaline phosphatase monoclonal anti-alkaline phosphatase and protein A-gold techniques. In addition, to determine whether the harvesting method interfered with microvascular cell function, DOT immunoassays of factor VIII and CD34 were conducted on solutions recovered at collagenase incubation as well as after nylon filtration and Percoll administration, respectively. After the collagenase step, the vast majority of microvascular cells had the typical ultrastructural and immunophenotypical features of endothelial cells. In sharp contrast, following the Percoll step, only 1% to 18% of microvascular cells stained with factor VIII, Ulex europeaus, and CD31, whereas 90% of them expressed the CD34 antigen. Surprisingly, DOT immunoassay revealed the presence of factor VIII in the washing buffer recovered after the Percoll step only. Consequently the decreased expression of common endothelial cell markers (factor VIII, Ulex europaeus, and CD31) observed at the end of the cell isolation procedure was related to the adverse effects of Percoll on endothelial cell function. The CD34 surface molecule, being highly resistant, is particularly well suited for unequivocal characterization of microvascular cells as true endothelium.
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PMID:Electron microscopic and immunocytochemical profiles of human subcutaneous fat tissue microvascular endothelial cells. 812 56

A population of stem cells has been isolated from embryonic avian and neonatal rat skeletal muscle. These cells differentiate into several mesodermal phenotypes in culture upon treatment with dexamethasone. This study reports the isolation of a similar population of stem cells from another mesodermal tissue, the heart. Hearts were excised from 3- to 5-day- old rats, minced, and treated with a collagenase-dispase solution. Single cells were collected by centrifugation, washed, and plated in dishes. The cells were grown to confluence, trypsinized, and frozen at -80 degrees C in 7.5% dimethylsulfoxide. After at least 24 hr, the cells were thawed and plated in 24-well plates and treated with media containing dexamethasone at concentrations of 10(-6)-10(-10) M for 4 weeks. Control cultures contained mononucleated cells with a stellate morphology. Treatment with dexamethasone resulted in the appearance of several mesodermal phenotypes. Bone and cartilage nodules were identified with von Kossa and Alcian blue staining respectively. Adipocytes were identified using Sudan black B stain. Smooth muscle cells were identified by an anti-smooth muscle alpha-actin antibody, and skeletal myotubes were stained with anti-myosin antibody. Large binuclear cells with obvious fibers were noted and stained with anti-desmin. These binuclear cells appeared in both the control and the dexamethasone-treated cultures and were tentatively identified as cardiomyocytes. These data strongly suggest the existence of a population of mesenchymal stem cells in neonatal rat heart.
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PMID:A population of cells isolated from rat heart capable of differentiating into several mesodermal phenotypes. 863 45

After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.
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PMID:Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note. 935 Jun 55

To investigate the mechanisms responsible for the increased shortening capacity (delta Lmax) of airway smooth muscle in ragweed pollen sensitized dogs, the alterations of biophysical and biochemical properties of cytoskeleton and extracellular collagen in tracheal smooth muscle (TSM) were studied. Smooth muscle passive elastic properties were not significantly altered by removal of cytoskeleton with guanidine HCI plus 2-mercaptoethnol; collagenase digestion reduced smooth muscle force development, but did not affect its delta Lmax and passive elastic properties in both sensitized and control dogs. There were no significant differences in the amount of cytoskeletal intermediate filament proteins, desmin and vimentin between sensitized and control TSM. The content of total collagen, collagen type I, and collagen cross-linking in sensitized TSM were significantly greater than in control. Collagen fibres in sensitized TSM was more resistant to collagenase attack. We conclude that increased delta Lmax in sensitized canine TSM is not the result of alterations in passive cytoskeletal and extracellular collagen structures.
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PMID:The cytoskeleton and the extracellular matrix in sensitized canine tracheal smooth muscle. 936 Nov 52

Fibrosis, a consequence of tissue repair, can become a final common pathway to organ failure, if progressive. Prevention and regression of organ fibrosis represent targets of considerable interest. The natural fate of fibrosis differs among various tissues being either persistent, progressive or regressive. Cellular and molecular responses involving myofibroblasts (myoFb), a phenotypically transformed fibroblast-like cell of considerable functional diversity, is involved in collagen turnover at sites of repair, where they govern the fate of fibrosis. Insights gained from the natural regression of established fibrous tissue may offer strategies to remove unwanted fibrosis in failing organs. In the present study, we addressed the temporal sequence to various components of collagen synthesis and degradation involved in the appearance and subsequent regression of pouch tissue induced in the rat by subcutaneous injection of air followed by instillation of the phorbol ester croton oil. Pouch tissue was collected on day 2, 4, 10, 14, 21, 28 and 35 (n=6 at each time point). Activities of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP-1 (TIMP-1) were determined by zymography and reverse zymography, respectively; collagen accumulation by hydroxyproline concentration; gene expression of TIMP-1 or tissue inhibitor of MMP-1, type I collagen and transforming growth factor-beta1 (TGF-beta1) by in situ hybridization; TGF-beta1 concentration by sandwich enzyme-linked immunosorbant assay (ELISA); and myoFb and its phenotypes by immunohistochemistry using antibodies to alpha-smooth muscle actin (alpha-SMA), vimentin or desmin. During pouch tissue formation, we found: (1) pouch weight increased progressively from day 2 to day 14 and then declined progressively thereafter; (2) type I collagen mRNA expression, barely detectable at day 2, increased at day 4, together with tissue hydroxyproline concentration (P<0.05) reaching a peak on day 10, and gradually decreased thereafter in association with declining tissue hydroxyproline concentration; (3) mRNA expression and concentration of TGF-beta1, detectable at day 2, significantly (P<0.05) increased at day 4, reached a peak at day 10, and gradually declined thereafter; (4) MMP-1 activity, low at day 2, increased continually over the course of 35 days; (5) TIMP-1 mRNA, detectable at day 2 and significantly (P<0.05) increased at day 4, gradually decreased thereafter; (6) activity of TIMP-1 increased continuously from day 2 to day 14 and then was markedly reduced thereafter; and (7) myoFb were first observed in pouch tissue at day 4 and became more extensive thereafter with their phenotype changing over time. Early appearing myoFb (day 4, 10, 14, and 21) expressed alpha -SMA and vimentin (VA phenotype), while later appearing cells (day 28 and 35) additionally expressed desmin (VAD phenotype). Thus, in croton oil-induced rat pouch model, the subcutaneous accumulation of pouch tissue hydroxyproline over the course of 10 days is initially associated with a VA-positive myoFb phenotype and its transcription of TGF-beta1, type I collagen and TIMP-1. Beyond day 10, a regression of pouch tissue collagen begins in association with the appearance of a VAD-positive myoFb phenotype and progressive increase in MMP-1 activity as the expression of TIMP-1 and TGF-beta1 are withdrawn. Regression of established fibrosis in failing organs may, therefore, be attainable through manipulation of myoFb phenotype and/or enhanced collagen degradation relative to collagen synthesis.
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PMID:Appearance and regression of rat pouch tissue. 1033 40

Coronary microvascular endothelial cells exert (patho)physiological effects on the function of cardiac myocytes, which may be studied experimentally using pure cell populations. As an essential pre-requisite to the investigation of cells from gene-modified mice, we studied the phenotypic properties of coronary microvascular endothelial cells isolated from normal mice, and biochemically characterized the superoxide production by these cells. Microvascular endothelial cells were isolated from devitalized mouse ventricular tissue after sequential digestion with collagenase, trypsin and DNase. Coronary microvascular endothelial cells were separated from cardiac myocytes and other cells by differential centrifugation, plating and culture. Mouse coronary microvascular endothelial cells showed an irregular "cobblestone" morphology at confluence, were >98% positive for CD31 by FACS analysis, and were also positive for VE-cadherin and endothelial-type nitric oxide synthase (eNOS) by confocal microscopy. The cells took up fluorescently labelled, acetylated low-density lipoprotein, but were negative for a alpha -smooth muscle actin, desmin and cytokeratin. Unlike human endothelial cells, mouse coronary microvascular endothelial cells only weakly expressed von Willebrand factor. Immunoblotting showed that the mouse cells expressed components of a phagocyte-type NADPH oxidase. They exhibited NADPH-dependent O(2)(-)-generating activity, which was increased by angiotensin II but completely inhibited by diphenyleneiodonium. Thus, mouse coronary microvascular endothelial cells express both eNOS and NADPH oxidase, interactions between which may play a role in endothelial cell pathophysiology.
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PMID:Phenotypic properties and characteristics of superoxide production by mouse coronary microvascular endothelial cells. 1144 17


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