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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 simple procedure which provides a large yield of isolated ferret ventricular myocytes is described. The enzymatic dissociation was performed by perfusion of the whole heart with the "Langendorff method" at 37 degrees C, without an incubation period. Special attention was given to the period of perfusion with Ca-free or low-calcium containing solutions and to the proportion of both collagenase and elastase used. The viability and calcium tolerance of the isolated cells were tested by ultrastructural and electrophysiological studies. Photo-microscopy showed that 60 to 80% of the isolated cells had an elongated shape (18 microns in diameter, 150 microns in length) and did not beat spontaneously in normal Tyrode solution. The morphological and ultrastructural integrity of these cells was shown in SEM by their smooth surface with regularly spaced T-tubule openings and in TEM by the regular distribution of the transverse tubular system, mitochondrium and sarcomeres. Using the whole-cell patch-clamp technique, they had a resting membrane potential of -72 mV, two types ("Purkinje like" and "ventricular like") of action potentials could be elicited and they were correctly affected by well-known modulators of calcium channels. This technique was successfully applied to the rat heart and could be used for heart dissociation of small mammals. It can simultaneously provide isolated cells of different regions of the heart and can be easily and routinely used by any investigator.
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PMID:An efficient isolation procedure of Ca-tolerant ventricular myocytes from ferret heart for applications in electrophysiological studies. 210 19

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

For the improvement of the adult osteoblast culture, the osteoblasts of young adult rabbit endosteal from long bones were isolated by collagenase digesting separation. 0.1% of type-I collagen precoated culture flasks were used as substrate for isolated bone cell growth. Morphological examination of cultured cells under a phase-contrast microscope, SEM and TEM observations showed a structure similar to osteoblast in vivo. Histochemical examination of alkaline phosphatase demonstrated 97% purity of cultured osteoblasts. The presence of calcium deposit activity in cultured cells was demonstrated by Van Kossa stain. High activity of alkaline phosphatase and inorganic pyrophosphatase in cultured osteoblasts as determined by biochemical analysis. High calcium uptake in cultured osteoblasts was demonstrated by radioisotope labelled 45CaCl12. According to these methods, it was indicated that the cells isolated from young rabbit long bone endosteal were osteoblast-like and still maintained their biological function. Our system for culturing osteoblast-like cells is a successful attempt in growing bone tissue in vitro starting from isolated bone cells. Therefore, this modified method for bone cell culture on collagen precoated culture flasks could be used as the experimental model in studies concerning the osteoblasts in vitro.
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PMID:Functional evaluation of cultured rabbit osteoblast-like cells. 255 21

The skin of Rana pipiens excretes H+ and this excretion is increased by metabolic acidosis. The mitochondria-rich (MR) cells of the skin have been found to mediate this H+ transport. The purpose of this study was to determine if there is a change in the MR cells of the skin during metabolic acidosis and if the isolated split epithelia of frog skin maintains its capacity to excrete H+. Metabolic acidosis was induced by injecting 120 mM NH4Cl (0.025 ml/g body wt) into the dorsal lymph sac three times a day for 2 days. The frogs were sacrificed and collagenase-split skins from the abdomen of normal and metabolic acidotic frogs were mounted between 2-ml chambers. H+ fluxes into both the mucosal and serosal media were measured and reported in units of (nmol) (cm2)-1 (min)-1. An increase in H+ flux was seen on both the mucosal and serosal sides of the acidotic split skins. The isolated epithelia were fixed, postosmicated, and dehydrated in the chamber. They were then embedded in Spurr's resin and 1-micron sections were cut and stained with Paragon multiple stain. Coded slides were used to count various cell types. Sections were randomly selected and approximately 40,000 cells were counted. Four basic cell types were noted and confirmed by TEM photomicrographs; basal (B) cells, granular (G) cells, keratinized cells, and MR cells. The ratio of G + B cells:MR cells in the normal skins was 1.0:0.021. The ratio in acidotic skins was 1.0:0.34. The average percentage of cell population of MR cells in the normal skins was 2.08 + 0.18 and in acidotic skins 3.20 + 0.36 (P less than 0.005). We conclude that the split skin maintains the capacity to acidify the mucosal fluid. Additionally, during metabolic acidosis there is an increased number of MR cells in the skin and this increase may be an adaptive mechanism of the skin to excrete excess H+ during acidosis.
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PMID:Morphological changes in the skin of Rana pipiens in response to metabolic acidosis. 349 53

The methods reviewed here include: scanning electron microscopy (SEM) of vascular corrosion casts, SEM of intact tissue, SEM of HCl-collagenase treated tissues, light microscopy (LM) of India ink or silicon rubber injected tissues, with stereomicrography, LM of tissue stained by perfusion with hematoxylin, and a correlative study of intravital microscopy with SEM of vascular corrosion casts or LM of India ink-injected tissue. The last technique allowed for both the examination of the microvascular architecture and blood flow in a particular tissue area. This paper shows that an adequate understanding of the microvasculature of the pancreas can only be gained when a variety of SEM techniques, together with other LM and TEM techniques are employed in a coordinated fashion. The intralobular arterioles of the pancreas supply the islets of Langerhans, exocrine acini, and duct system. Blood leaving the islets flows into the capillaries in the exocrine region through the insulo-acinar portal system and insulo-ductular portal system, although some fraction of the blood drains through venules into nearby veins. Thus, these studies in the pancreas indicate that the islets of Langerhans are situated in the center of the pancreatic microcirculatory bed so that the insular secretions are transferred in high concentrations through short vascular routes to the exocrine region of both the duct system and acini, presumably to act upon these structures.
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PMID:Review of scanning electron and light microscopic methods in microcirculation research and their application in pancreatic studies. 638 19

Fluorescent conjugates of hydroxyethyl (OEt) starch of Ficoll are selectively ingested and retained in vivo by spleen marginal-zone (MZ) and lymph node marginal-sinus macrophages of mice, whereas similar conjugates of type 3 pneumococcal capsular polysaccharide (SIII) are generally retained by macrophages (Kupffer cells, histiocytes, macrophages of spleen, lymph nodes, bone marrow and peritoneal cavity). MZ and other macrophages are readily identifiable by fluorescence after injection in vivo of OEt starch and SIII labeled with tetraethylrhodamine isothiocyanate and fluorescein isothiocyanate. Collagenase digestion was required for recovery of intact MZ macrophages from spleen in single cell suspensions and for maximum yields of other macrophages. MZ macrophages are larger and morphologically distinct from other macrophages, but resemble them in respect of EA, EAC receptors and acid phosphates and nonspecific esterase content and are equally radio-resistant. The appear normal in CBA/N nude and in beige mice. Freshly isolated MZ macrophages in suspension have adherent lymphocytes, dispersible by EDTA treatment, with B but not T cell markers. It is suggested that selective adherence to MZ macrophages is a factor in determining B cell traffic. MZ macrophages did not have demonstrable surface I-A or I-EC antigens. Only 4--8% of other spleen macrophages freshly isolated by collagenase treatment expressed I-A in the same preparation, whereas 35% of other cells (lymphocytes and blasts) reacted with monoclonal anti-I-A and anti-I-EC. After adherence to glass or plastic, 40% or more red-pulp, but not MZ macrophages, became I-A-positive. When taken from mice recently restimulated with sheep erythrocytes, half the red-pulp macrophages expressed I-A even before adherence. The relation of MZ to other macrophages is not known. However, their properties are consistent with the demonstrated ability of thymus-independent antigens selectively taken up by these cells to elicit long-lasting IgM antibody responses by direct interaction with B cells. The unexpected observation that only a small proportion of spleen macrophages freshly isolated from unstimulated mice had detectable surface I-A, but that this proportion was much increased after attachment to plastic, is discussed in relation to the possibility that macrophages do not express surface Ia antigens unless they have been stimulated.
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PMID:Different macrophage populations distinguished by means of fluorescent polysaccharides. Recognition and properties of marginal-zone macrophages. 694 Jul 55

The present study details a new method for the exposure and viewing of individual microvessels located within the small intestine of rats. This procedure will selectively and consistently remove the outer muscle layers and underlying submucosa of the intestinal wall and thereby expose a variety of arterioles in their normal location within the tissue, with their normal relationship to each other undisturbed. The small intestine of the rat was initially fixed by vascular perfusion with 2.5% glutaraldehyde in 0.1 M cacodylate/HCL buffer, reinfused with heparinized whole blood, removed from the animal, and secured to a dissecting petri dish for further fixation. Subsequently, the external muscularis was dissected from the sample which exposed the submucosa. In order to remove the connective tissue elements from this layer and uncover the submucosal vasculature, the samples were first transferred to a solution of 30% potassium hydroxide for 2-5 minutes and then to a final digesting solution containing collagenase. Thereafter, the samples were routinely processed for light microscopy and for scanning (SEM) or transmission (TEM) electron microscopy. Examination of the samples revealed excellent preservation of the three-dimensional organization of the arteriolar wall with minimal membrane damage. This new technique now makes it possible to visualize the shape and position of individual smooth muscle cells along arterioles of differing size and branching order.
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PMID:A new morphological procedure for viewing microvessels: a scanning electron microscopic study of the vasculature of small intestine. 713 4

An attempt to determine the ideal temperature and duration of storage of human foetal chondrocytes yielded highly cellular preparations with no alteration in morphology or loss of viability. Initial digestion with activated papain was followed by incubation in 0.5 per cent collagenase. Trypan blue exclusion test revealed a viability count of 95-99 per cent and radioactive thymidine uptake a corresponding labelling index. On TEM no subcellular damage was evident. The isolated viable chondrocytes were further banked at varying temperatures of +4 degrees, -4 degrees, -30 degrees, -79 degrees and -196 degrees C, in Eagles MEM with 10 per cent dimethyl sulfoxide. Post storage morphology and viability of these cells, thawed after durations of 20 h, 1 wk, 1, 2, 3, 4 and 6 months, were compared with prestorage readings in an attempt to define the ideal temperature for banking. Storage in liquid nitrogen at -196 degrees C demonstrated excellent preservation even at the end of six months with minimal subcellular change. Electron microscopy and labelling index were found to be superior to Trypan blue exclusion test in assessing the stored chondrocytes for retention of their functions.
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PMID:Isolation & cryo-preservation of human foetal articular chondrocytes. 813 36

Phosphoprotein appears to play an important role in the mineralization of dentin during tooth development and remineralization after demineralization by dental caries. To better understand this role, we describe the extraction and characterization of phosphoprotein from immature, human root apex dentin during and after EDTA demineralization. The extraction procedure included dissociation of the demineralized dentin matrix by guanidine hydrochloride (Gdn.HCl) followed by subsequent digestion with cyanogen bromide (CNBr) and collagenase. Characterization of these extracts included 'Stains-All' staining of SDS polyacrylamide gels (SDS-PAGE) and amino acid, protein and phosphorus analyses. The ability of these matrices to remineralize was determined by TEM and measuring calcium levels in the remineralized tissue by atomic absorption spectroscopy. The staining of SDS-PAGE gels and amino acid analysis showed that an intact phosphophoryn was extracted from the dentin of the immature apices during EDTA demineralization and that it had an apparent Mr approximately 140,000. In the subsequent extracts and digests, the phosphoprotein has a range of molecular weights, some of which may have been degraded products of the intact phosphoprotein. A greater quantity of phosphoprotein was found in the EDTA-demineralized dentin matrices than in dentin after Gdn.HCl, CNBr and collagenase digests. These EDTA-demineralized matrices also remineralized to a greater extent than those dissociated with Gdn.HCl. The differences in both the quantity and the quality, as defined by the amino acid residue profile, of the phosphoprotein in the sequential extracts of the root apex dentin may be important in affecting the ability of this tissue to remineralize.
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PMID:Phosphoprotein analysis of sequential extracts of human dentin and the determination of the subsequent remineralization potential of these dentin matrices. 970 61

Osteoblasts from 21 day-old fetal rats calvaria were isolated using a collagenase digestion procedure. Cells were cultured in the presence of Laddec (a highly purified bovine xenograft) and Bio-Oss (natural bone mineral). Optical microscopic observations showed that osteoblasts attached on the plastic culture dishes and formed close contact with biomaterial particles. By day 5, the osteoblasts formed a confluent monolayer. A cytozymatic method showed intense alkaline phosphatase (ALP) staining around and between the substrate granules of the two materials. By day 14, inverted phase microscopic observations showed that osteoblasts formed bone nodules around and between substrate particles. In addition, at this time, the Von Kossa staining was positive. Using a conventional assay, ALP specific activity was higher in the presence of Laddec than in the presence of Bio-Oss. A quantitative morphological method using image analysis showed that the proportion of mineralized bone formed around biomaterial particles in relation to total bone was increased with Laddec (15% more than with Bio-Oss). Ultrastructural observations by TEM showed the presence of an electron dense collagen-free layer at the biomaterial/bone interface and a collagenous matrix deposited at the periphery, indicating the bioactivity of the biomaterials. These results indicated that Laddec increased the expression of ALP in osteoblast cultures and facilitated the formation of multiple cell layers, providing a culture environment suitable for mineralization.
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PMID:Effects of Laddec on the formation of calcified bone matrix in rat calvariae cells culture. 1039 84


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