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)

The glycosaminoglycans (GAG), glycoproteins and collagen in bovine aorta and venous tissue have been studied. The concentration of hyaluronic acid and dermatan sulphate was significantly more in the venous tissue while chondroitin sulphates were higher in the aorta. Sequential extraction with phosphate buffered saline (PBS) collagenase, hyaluronidase and urea was also carried out with the two tissues. The GAG extractable by PBS and collagenase digestion were more in the aorta. The total aortic glycoproteins had significantly lower hexose and higher sialic acid. The PBS extractable glycoproteins of the venous tissue had more hexose and fucose. The glycoproteins released by collagenase digestion of the venous tissue had lower sialic acid and higher fucose, while glycoprotein released by hyaluronidase digestion had lower sialic acid and higher hexose and fucose. Urea extractable glycoproteins had lower fucose and sialic acid in the venous tissue. Venous tissue had higher total collagen and acid and salt soluble collagen while insoluble collagen was more in the aorta. The total GAG in the venous tissue had greater anticoagulant activity while the aortic GAG bound significantly more serum lipoproteins.
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PMID:Studies on the macromolecular components of the bovine aortic and venous tissue. 309 9

We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.
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PMID:Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate. 321 12

The present observations demonstrate that quiescent calcium-tolerant adult rabbit cardiac myocytes can be isolated by collagenase-hyaluronidase perfusion and maintained in primary culture for at least 2 wk. Culturing large numbers of myocytes requires that the freshly isolated cells be attached to a suitable substratum such as laminin, type IV collagen, or fetal bovine serum. The cultured myocytes retain their rod-like morphology for approximately 7 days before gradually spreading into a flattened conformation by 14 days. During the 1st wk of culture, contaminating interstitial cells rapidly proliferate, making cultures unsuitable for long-term study. Pure myocyte populations can be established and maintained if freshly isolated cells are cultured in the presence of cytosine arabinoside (Ara-C, 10 microM). This antimetabolite does not appear to adversely affect high-energy phosphates, since ATP and creatine phosphate (CrP) content of the myocytes is maintained at levels normally found in biopsy samples of rabbit myocardium. These results illustrate that an energetically stable population of adult cardiac myocytes can be maintained in primary culture in sufficient numbers to make them useful for future investigations of myocyte function.
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PMID:Attachment and maintenance of adult rabbit cardiac myocytes in primary cell culture. 338 98

Prior work has suggested that cryopreserved venous allografts may serve as an effective arterial substitute. To determine the relative thrombogenicity of this material, platelet uptake of cryopreserved canine jugular veins (CJV) before and after deendothelialization was compared to fresh CJV before and after deendothelialization, and to PTFE. CJV were frozen in Medium 199 (M199) with 10% dimethylsulfoxide to -70 degrees C. CJV were deendothelialized with collagenase (165 u/mg Worthington type II in phosphate-buffered saline) for 15 min at 37 degrees C. Canine platelets were harvested, labeled with indium-III-oxine, and suspended in 1 liter of M199. Labeled platelets were used to perfuse each graft in a nonpulsatile flow loop for 120 min. Deendothelialization led to a significant increase in platelet uptake (P less than 0.05). Frozen deendothelialized CJV showed the highest affinity for platelets. PTFE, cryopreserved, and fresh CJV showed similar affinity for platelets. Cryopreservation alone did not seem to inhibit the ability of endothelial cells to act as a nonthrombogenic surface and did not alter the histologic structure of the veins.
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PMID:Platelet avidity of cryopreserved veins: thrombogenicity of cryopreserved veins. 341 54

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.
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PMID:Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. 346 23

A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture.
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PMID:Isolation, long-term culture, and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells. 357 Oct 99

A method is described for the isolation of large numbers of viable disaggregated cells from human tissues. This method combined the mechanical action of a Stomacher Model 80 Lab Blender, 0.1 mg/ml trypsin or 0.5 mg/ml collagenase, and 0.1 mM [ethylene bis(oxyethylenenitrolo)]-tetraacetic acid (EGTA). Tissue (0.2 to 1.0 g) obtained from human fetal intestine, kidney, liver, lung, and skin were separately minced into approximately 1-mm3 pieces. The pieces were placed in a sterile bag containing 60 ml of calcium- magnesium-free phosphate buffered saline, the appropriate enzyme (0.1 mg/ml trypsin or 0.5 mg/ml collagenase) plus 0.1 mM EGTA, and 0.1% methylcellulose. The bag was then placed into the blender and mixed at a low speed for 3 to 20 min at room temperature. After a single cell suspension was observed by phase contrast microscopy, 10 ml of bovine calf serum was added to the cell suspension to inactivate the proteolytic enzymes. At this time 130 ml of cold Hanks' balanced salts solution containing 5% bovine calf serum was added and the entire cell suspension passed through a tissue sieve (100 mesh, 140 micron) and the cells collected by centrifugation. These cells were then resuspended into the appropriate culture medium. In comparison to other methods for establishment of cell cultures from human tissues, the method described requires shorter incubation times with relatively low concentrations of proteolytic enzymes, and yields two- to three-fold greater number of cells per tissue with 86 to 93% viability. Also, depending on the cell type, 50 to 75% of the isolated cells attached to the culture vessel within 24 h. Variation of the time and concentration of digestive enzymes can be used to select different cell types for culture.
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PMID:A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment. 375 94

To define further the structural specificity of the taurocholate uptake site, we studied the ability of a variety of taurine-conjugated bile acids with differing hydroxyl substituents on the sterol moiety to inhibit [14C]taurocholate uptake. Rat hepatocytes isolated by collagenase perfusion were incubated in a tris(hydroxymethyl)aminomethane-phosphate buffer containing [14C]taurocholate (2.5-100 microM) in the presence or absence of inhibitor bile acid. Stronger inhibitors were studied at a fixed concentration of 5 microM, weaker ones at 25 microM. Initial uptake velocity was measured by sedimenting an aliquot of cells through silicone oil into 3 N KOH every 15 s for 1 min. Uptake velocity (nmol X mg protein-1 X min-1) could then be related to taurocholate concentration and a Vmax and Km could be determined by applying a nonlinear least squares fit to the data obtained with or without inhibitor. The kinetic parameters allowed the determination of the type of inhibition and of inhibition constants (Ki) of the various test bile acids. The data indicate that bile acids containing a 6- or 7-OH group exhibit competitive inhibition, whereas bile acids with no 6- or 7-OH group exhibit noncompetitive inhibition. Of the compounds exhibiting competitive inhibition, Ki varied with the number of hydroxyl groups on the sterol moiety. We conclude that the presence or absence of a 6- or 7-OH group dictates the mechanism of inhibition; the number of hydroxyl substituents determines the potency of competitive inhibition.
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PMID:Bile acid inhibition of taurocholate uptake by rat hepatocytes: role of OH groups. 382 74

Glycoproteins were extracted from human atherosclerotic lesions by a phosphate buffered heparin solution and by 0.15 M NaCl, followed by sequential digestion of the tissue by collagenase and elastase. Proteins obtained from the extracts by fractional precipitation by (NH4)2SO4 at 40%, 60% and 100% saturation of the salt at pH 4.0 were analyzed for total protein and for fibronectin and laminin by immunodiffusion. Greater amounts of proteins were extracted from atherosclerotic lesions than from uninvolved tissue. The buffered heparin solution extracted several-fold more protein than 0.15 M NaCl. Fibronectin was found in most protein fractions from every extract, but the presence of laminin was noted only in the protein fractions precipitated at 40% saturation of (NH4)2SO4 in the extracts of the tissue by heparin.
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PMID:Identification of fibronectin and laminin in glycoprotein extracts of human aorta. 393 60

The C-terminal telopeptide of the alpha 1 chain of type I collagen from bovine skin was isolated from a bacterial collagenase digest. Two forms of the telopeptide were obtained, one with two and the other with three residues of tyrosine. In both of these, the single lysyl residue had been oxidized to alpha-aminoadipic delta-semialdehyde. Circular dichroism spectra of the telopeptide in aqueous solution at neutral pH were interpreted as indicating the presence of little regular secondary structure. However, sodium dodecyl sulfate at a concentration of 40 mM induced some alpha helix, as predicted from the sequence, and trifluoroethanol also induced secondary structure, probably a mixture of alpha helix and beta sheet. A major feature of the circular dichroism spectra of the telopeptide in sodium dodecyl sulfate, in denaturing agents, and in sodium phosphate buffer at low temperature was a positive band at 227 nm due to tyrosine side-chain chromophores. The disappearance of this band on heating and at high pH was ascribed to the adoption by the telopeptide of a specific tertiary structure. Poly(ethylene glycol) 1000 used as a perturbant in UV difference spectroscopy caused conformational changes resulting in decreased accessibility of tyrosine side chains and transfer of these to a less polar environment. A structural model in which the four aromatic side chains of the telopeptide are arranged in two pairs with the rings antiparallel is proposed to account for these results.
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PMID:Spectroscopic study of environment-dependent changes in the conformation of the isolated carboxy-terminal telopeptide of type I collagen. 396 71


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