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)

Adult rat heart was dissociated into a single cell suspension by a perfusion technique which used 0.05% collagenase and 0.1% hyaluronidase in Krebs-Ringer phosphate buffer (KRP). The non-muscle cells of the suspension were separated from the myocytes by centrifugation through 3% Ficoll solution in KRP with 0.01 mM Ca2+. An approximately 90% pure suspension of isolated single muscle cells was obtained with this method. The effects of the successive steps in the dissociation procedure on the ultrastructure of the heart were studied by scanning and transmission electron microscopy. After 30 minutes of enzyme digestion, dissociation of the inner endothelial lining of the ventricle into single cells or small groups of cells became apparent. In addition, the underlying cardiac skeleton began to disintegrate and linear arrays of cardiac muscle cells were observed. After 45 minutes of enzyme digestion the number of released single cells was higher because of the separation of intercalated discs. The majority of non-muscle cells were by now dissociated from the surfaces of muscle cells. Widening of the lateral intercellular spaces between the myocardial cells was associated with separation of desmosomes. In some regions of the heart, intact desmosomes, fasciae adherentes and gap junctions were observed even though lateral intercellular spaces had widened greatly. The majority of myocardial cells had become separated from one another after 60 minutes of enzyme digestion. Separation of gap junctional sites took place in two ways: (1) by 'unzipping' them through enzyme action; (2) by tearing them mechanically. Gap junction remnants were sometimes observed in a vesiculated state within the cell. The dissociation of the heart was ineffective when perfused with media containing 1.0 or 2 mM Ca2+. Alcian blue treatment after 60 minutes of enzyme digestion revealed that the basement membrane, and its accompanying collagen fibrils, was still present on the plasma membrane of dissociated single cells. The isolated myocardial cells retained their normal morphological characteristics. This study has enabled us to understand in detail how dismantlement of highly ordered adult cardiac tissue into a single cell suspension takes place. Cell suspensions of this type should be invaluable in the study of metabolic and synthetic activities in adult myocardial cells.
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PMID:Dissociation of adult mammalian heart into single cell suspension: an ultrastructural study. 12 Mar 52

The effect of the enzyme collagenase (40-200 units-ml-1) on the spontaneous mechanical activity in vitro and on the fine structure of the activity of the taenia was enhanced both in the isometric and isotonic recordings; after several minutes the muscles became slack or elongated to up to twice their resting lengths. The structural changes were dramatic but a number of muscle cells remained apparently unaltered even with the highest concentration and the longest incubation time (120 minutes). The large variety of structural changes were tentatively grouped into two separate sequences. One sequence involved swelling of the muscle cell, dispersion of the filaments and breaking up of the cell membrane: the thick myofilaments increased considerably in size and became heterogeneous in size and shape, but were still recognizable after disruption of the cell membrane. The other disruptive sequence involved separation of the superficial part of the muscle cell, which became electron-lucent, from the core of the cell where filaments were very densely packed. Few or no changes were observed in non-muscle cells.
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PMID:Effect of collagenase on mechanical activity and fine structure of an intestinal smooth muscle. 18 88

Experiments were performed to determine the cellular associations of the molecular forms of acetylcholinesterase (AChE) in adult rat heart. For this purpose, a cardiac muscle and a non-muscle fraction were isolated from rat heart ventricles after perfusion with collagenase and hyaluronidase, extracts of these fractions were subjected to ultracentrifugation on linear density gradients of sucrose (5-20%), and fractions of these gradients were analyzed for AChE activity. The results show that only globular AChE molecular forms were present in isolated cardiac muscle cells. Globular AChE forms were also present in the non-muscle cells fraction but in different proportions. The proportions of globular AChE forms plus the high specific activity of choline acetyltransferase in the non-muscle cell fraction suggest that this fraction contains cholinergic nerve fragments. The results of this study also show that asymmetric AChE is released during the perfusion of heart with the digestive enzymes, which suggests that asymmetric AChE is bound to the extracellular matrix of heart.
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PMID:Acetylcholinesterase molecular forms in muscle and non-muscle cells of rat heart. 258 21

Muscle satellite cells are quiescent myogenic stem cells situated between the basal lamina and plasmalemma of mature skeletal muscle fibers. Injury to the fiber triggers the activation and proliferation of satellite cells whose progeny subsequently fuse to form new myotubes during regeneration. In this paper we report the proliferation of satellite cells on single muscle fibers isolated from adult rats and placed in culture. Viable fibers were liberated from muscle with collagenase and purified from non-muscle cells. The fibers were covered with a basal lamina and retained normal morphological characteristics. Each fiber contained two to three satellite cells per 100 myonuclei. Satellite cells showed little proliferative activity in medium with 10% serum but could be induced to enter the cell cycle by chick embryo extract or fibroblast growth factor. Other polypeptide mitogens such as epidermal growth factor, multiplication stimulating activity, and platelet-derived growth factor were ineffective. Mitogen-stimulated satellite cells fused to form new myotubes after 4-5 days in culture. These results imply that satellite cells are under positive growth control since they proliferate in contact with viable mature fibers when stimulated with mitogen. The mature fibers remained viable in culture but did not give rise to mononucleated cells. After several days, however, the fibers began to extend sarcoplasmic sprouts and underwent dedifferentiative changes that led to the formation of multinucleated cells resembling myotubes. These cells reexpressed embryonic isozymes of creatine kinase not made by the mature fibers.
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PMID:Proliferation of muscle satellite cells on intact myofibers in culture. 351 58

Isolated, mononucleated, cross-striated muscle of a medusa can be activated by collagenase treatment to transdifferentiate completely to various new cell types and to regenerate autonomously the sexual (without gametes) and feeding organ of the animal. Under these circumstances all isolated muscle fragments produce smooth muscle cells and a glandular cell type (y-cells). When culture conditions are appropriate, endoderm is also formed, followed by regeneration of a complex organ of seven or eight new non-muscle cell types, including nematocytes, digestive, secretory, gland, interstitial, and presumably nerve cells.
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PMID:Isolated, mononucleated, striated muscle can undergo pluripotent transdifferentiation and form a complex regenerate. 614 22

Actomyosin was partially purified from rat parotid cells dispersed by collagenase digestion and found to possess different solubility characteristics from that from (undispersed) rat parotid tissue. This is attributed to the decrease in vascular contamination effected by the isolation of parotid cells, yielding a non-muscle actomyosin [Adelstein, Conti, Johnson, Pastan & Pollard (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3693-3697]. Myosin light-chain kinase was partially purified from dispersed rat parotid cells by calmodulin affinity chromatography and shown to be activated by Ca2+-calmodulin. The calmodulin content of dispersed rat parotid cells was shown to be 6.50 +/- 0.59 ng of calmodulin/micrograms of rat parotid-cell protein (mean +/- S.E.M.), as determined by the activation of purified bovine brain phosphodiesterase by heat-treated extracts of dispersed rat parotid cells.
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PMID:Studies on rat parotid-cell actomyosin. 633 11

To analyze direct effects of steroids on the rates of synthesis (and/or degradation) of newly synthesized proteins of the rat heart, we have used high resolution two-dimensional gel electrophoresis and autoradiography. A collective steroid domain of nineteen proteins, comprising fifteen with an increased rate of synthesis and four with a decreased rate of synthesis, was consistently seen in cultures of cardiac muscle and non-muscle cells from neonatal rats following 24 h incubation with 10(-7) dexamethasone. Similarly, incubation with 10(-7) M sex steroids, mineralocorticoids, and other glucocorticoids including the highly selective compound RU26988, established the glucocorticoid-specificity of the response. Different subsets of this glucocorticoid domain were seen for collagenase- or trypsin-dispersed primary cultures of cardiac muscle and non-muscle cells or for passaged cultures of cardiac non-muscle cells. Six polypeptides were consistently induced in all cardiac cultures, regardless of cell morphology. Two polypeptides were consistently induced only in those cultures containing cardiac non-muscle cells, whereas protein l, of identical Mr(approximately 52K) and pI (approximately 5.3) to desmin, was induced only in cultures of spontaneously contractile cardiac muscle cells. The glucocorticoid domain proteins described herein represent direct steroid effects on cardiac cells and are therefore candidate mediators of physiological glucocorticoid effects on, for example, differentiation and contractility.
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PMID:Glucocorticoid effects on newly synthesized proteins in muscle and non-muscle cells cultured from neonatal rat hearts. 651 49

Cells from the muscular layer of neonatal (3-day-old) rabbit urinary bladders were dissociated with collagenase, and cultured in M199 supplemented with 10% fetal bovine serum and antibiotic-antimycotic. Cells in culture were of two types: long and short. The short cells were thick and spindle-shaped, and the long cells were flat and elongated. The long cells can be about 15 times longer than the short cells. The short cells do not divide, but the long cells divide readily. Expressions of smooth muscle and non-muscle myosins, alpha-smooth muscle actin, vimentin, and h-caldesmon were determined by immuno-fluorescence microscopy using specific antibodies. Both types of cells react strongly with antibodies against smooth and non-muscle myosins. Unlike the short cells, the long cells also contain alpha-actin and vimentin. The expression of h-caldesmon was very weak in both cell types. Also, cells dissociated from the smooth muscle layers of adult (6-month-old) rabbit bladder were cultured under the same conditions as the cells from the neonatal bladders to see if the heterogeneity of smooth muscle cells, exhibited by cells from neonatal rabbits, is also shown by cells from adult bladder. Two types of cells were also identified. The cells were then fixed and examined with the same panel of antibodies that we used for the neonatal cells. The long cells from adult bladder muscle express similar proteins to those in the neonatal long cells, and the short cells were stained positively with smooth muscle myosin, non-muscle myosin, alpha-smooth muscle actin, and lightly with caldesmon. Although the absence of vimentin in the short cells from adults is similar to that from neonatal, the strong expression of alpha-actin in the adult short cells is unlike the short cells from neonatal rabbits, in which their expression is barely detectable.
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PMID:Identification of two types of smooth muscle cells from rabbit urinary bladder. 870 36

Single smooth muscle cells (SMCs) isolated from guinea pig ileum using collagenase and papain were cultured on laminin-coated dishes in MEM containing fetal calf serum. Temporal changes in intracellular calcium ion concentration in response to carbachol and to ATP were investigated using fluo-3/AM and fluorescence microscopy. It was observed that carbachol caused an increased intracellular calcium ion in freshly isolated single SMCs but a reduced or negative response of cultured SMCs before confluence. On the other hand, ATP was observed to cause an increase in the calcium ion content of SMCs throughout the culture. SDS-PAGE and Western blot analyses revealed changes in the expression of contractile proteins as follows. l-Caldesmon and non-muscle type myosin heavy chain (NMHC) (considered to be marker molecules for dedifferentiation in smooth muscle cells) and non-muscle type tropomyosin were not observed in freshly isolated single SMCs. l-Caldesmon and NMHC appeared in the cultured SMCs within 2 days and the tropomyosin isoform was observed 6 days following seeding. Simultaneously, smooth muscle type myosin heavy chain (SMHC) decreased strikingly and the 41 kDa tropomyosin monomer was lost. The content of alpha-actin decreased gradually to a minimum on day 6 when non-muscle type tropomyosin appeared, and the cells began to proliferate rapidly. These results suggest that the loss of contractility in cultured smooth muscle cells is more closely related to changes in contractile protein profiles than to receptor-mediated signal transduction and that in addition to NMHC and l-caldesmon, non-muscle type tropomyosin may be useful as a marker molecule for de-differentiation of smooth muscle cells.
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PMID:Changes in Ca2+ signaling and contractile protein isoforms in smooth muscle cells from guinea pig ileum during culture. 1159 84