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)

Arterial endothelial cells were obtained from bovine aortae by mild treatment with collagenase and medium perfusion. These cells were cultured in RPMI-1640 medium containing 15mM Hepes buffer and 35% fetal calf serum at pH 7.35. Essentially all (90-95%) the effluent cells were viable and 80% of these cells attached to the substratum within 1 hour. Small patches of attached cells coalesced to form confluent monolayers in 3-5 days. Confluent monolayers of endothelial cells consisted of a homogeneous population of tightly packed, polygonal cells. Selected cultures were serially subcultured (trypsin-EDTA) for 12-14 months (30-35 passages) without any apparent change in morphology or loss of growth characteristics. Primary and three-month old (15 passages) cultures had population doubling times of 32-34 hours and 29-31 hours, respectively. These cells (primary and subcultures) did not require a minimum cell number to become established in culture. Bovine endothelial cells (primary, first, fifth and thirteenth passages) were characterized ultrastructurally by the presence of Weibel-Palade bodies, pinocytotic vesicles and microfilaments and immunologically by the presence of thrombosthenin-like contractile proteins and Factor VIII antigen. The intercellular junctions of post-confluenct cultures stained specifically with silver nitrate. From these data, we concluded that identifiable endothelial cells could be obtained from bovine aortae and cultured and maintained for prolonged periods of time.
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PMID:Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. 17 37

Islets of Langerhans were isolated from four human kidney donors, aged 16 to 21 years by the collagenase method described for isolation of rodent islets. So far the human islets have been kept in tissue culture, without attachment, in medium RPMI 1640 supplemented with 10% calf serum for more than 9 months, with preservation of the ability to release insulin in response to glucose stimulation. Replacement of calf serum with serum from normal human subjects did not affect B-cell survival, but resulted in elevated insulin values partly due to lower insulin degrading activity. Thus the described technique presents a valuable tool for studying chronic effects of metabolites and hormones on islet function, as well as for islet storage prior to transplantation into humans.
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PMID:Preservation of beta cell function in adult human pancreatic islets for several months in vitro. 36 59

For continued studies of GnRH receptor regulation in the winter flounder, we have developed an in vitro system consisting of cultured pituitary cells dissociated by collagenase. Using immunocytochemical staining methods for gonadotropin, growth hormone, and prolactin, these cell types were represented at the levels of 25, 20, and 19.5% of total pituitary cell population, respectively. Receptors for GnRH were characterized in intact monolayered attached pituitary cells, maintained in RPMI culture medium. The cell GnRH receptor characteristics were compared with those previously described using pituitary homogenates. The cells were capable of binding GnRH in a similar manner on Day 2 or Day 3 of culture, indicating the integrity of GnRH receptors. The specificity of binding was demonstrated since only high doses of cold GnRHa competed with 125I-GnRHa uptake, different peptides being without effect. The specific binding is saturable and the data suggest the presence of a single class of high-affinity (apparent Ka = 1.50 x 10(9) M-1), high-capacity sites (binding capacity = 25.03 fmol/2.5 x 10(5) cells or 242.23 x 10(3) sites/gonadotroph) which is in accordance with the characteristics of GnRH receptors present in homogenates of pooled male and female pituitary glands. All these observations suggest that such an in vitro pituitary cell system would be appropriate for studying GnRH receptor characteristics under different physiological conditions.
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PMID:Evidence of GnRH receptors in cultured pituitary cells of the winter flounder (Pseudopleuronectes americanus W.). 131 5

The expression of a novel regenerating (reg) gene has been reported previously in the regenerating islets of a surgical model of diabetes in rats. We exposed collagenase-isolated rat islets for three days to nutrient and non-nutrient growth factors in minimally supplemented RPMI medium (2.7 mmol/l glucose, 2% fetal calf serum), and investigated the relationship between reg gene expression and islet cell replication. RNA was prepared from half of the islets by homogenisation in guanidinium isothiocyanate followed by phenol/chloroform extraction. Northern/dot blot analyses were used to semi-quantify reg mRNA. Islet cell replication was estimated by culturing the remaining islets in radiolabelled thymidine to determine de novo DNA synthesis. Thymidine uptake was stimulated by the following factors: 11 mmol/l glucose (50% increase); 10% amino acids (126% increase); 10% fetal calf serum (39% increase); 100 ng/ml insulin (45% increase); 250 ng/ml growth hormone (65% increase); 1.5 nmol/l aldosterone (29% increase); 2 U/ml platelet derived growth factor (116% increase). The results are expressed as a percentage of the thymidine incorporated into control islets cultured in minimal RPMI (1118 +/- 100 (SD) cpm/microgram protein, n = 15). Increased islet cell replication was paralleled in each case by a clear rise in reg mRNA expression compared to controls. Furthermore, the rank order for reg gene expression was the same as that for thymidine uptake (r = 0.90). The present findings suggest a clear association between reg gene expression and islet cell replication in vitro, and are the first to demonstrate reg gene expression in response to individual growth factors.
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PMID:Expression of an islet regenerating (reg) gene in isolated rat islets: effects of nutrient and non-nutrient growth factors. 137 94

The objective of this study was to examine interactions of bovine myoepithelial and epithelial cells in vitro. Mammary tissue was dissociated with collagenase into myoepithelial and epithelial cells. Myoepithelial and epithelial cells were separated by differential centrifugation. Both cell types were cultured on plastic in RPMI-1640 and Iscove's Modified Dulbecco's Modified Eagle Medium supplemented with 10% horse serum and 5% fetal bovine serum. Our data revealed that conditioned medium from epithelial cells caused a small but significant reduction in proliferation of myoepithelial cells from fetal mammary glands. Myoepithelial-epithelial cell interaction in culture was characterized by myoepithelial cells with extended filopodia that could grow on top of confluent monolayers of epithelial cells, imitating the in vivo situation. In confluent monolayers of epithelial and myoepithelial cells in coculture, small domelike structures consisting of mixtures of epithelial and myoepithelial cells were observed. These structures greatly resembled the in vivo organization of the bovine mammary gland. Furthermore, myoepithelial cells were capable of migration toward individual colonies of epithelial cells or single epithelial cells. Myoepithelial cells organized epithelial cells into well-defined colonies. Myoepithelial cells may play an important role in organizing the architectural framework of the mammary gland during growth and development.
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PMID:Bovine mammary myoepithelial cells. 2. Interactions with epithelial cells in vitro. 147 5

A primary culture method was established by comparing the different effects of four methods of enzymatic separation--trypsin, collagenase with and without trypsin pretreatment, and a trypsin-collagenase mixture--and five media: DMEM, DMEM and Ham's F 12 mixture, F 12, RPMI 1640 and Medium 199. The trypsin pretreatment/collagenase method was most preferable considering the high number of isolated cells, satisfactory adhesion, good growth and a single population at subconfluence. DMEM and the DMEM/F-12 mixture resulted in the best adhesion, cell growth and cell number at confluence. Primary cells separated by the trypsin pretreatment/collagenase method and cultured in DMEM were responsive to parathyroid hormone at the proliferating stage and had higher alkaline phosphatase activity than cells cultured from gingiva and mucosa after reaching confluence. The long-term cultured cells formed nodules that were slightly mineralized. These results indicate that the cultured pulp cells had properties characteristic of pulp cells in vivo. This enzymatic separation method may be useful in studies of the regulation of pulp metabolism and odontoblast differentiation.
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PMID:Establishment of primary cultures of pulp cells from bovine permanent incisors. 166 Feb 58

In patients undergoing aortic valve replacement, allograft valves stored at 4 degrees C in a nutrient medium have been associated with excellent immediate and long-term results. The effects of this method of prolonged storage on the antigenic, immunological and cellular characteristics of these grafts are incompletely understood. This study was designed to study these phenomena in rat aortic valves subjected to antibiotic sterilization and stored for up to 3 weeks in RPMI containing 10% fetal calf serum. Selected valves from Brown Norway rats were implanted heterotopically into the abdominal aorta of Lewis rats. Other valves were studied prior to transplantation. Antigenicity was determined by immunocytochemical staining using monoclonal mouse antibodies directed at Class I and Class II rat antigens. Immunogenicity was determined by duration of second-set skin graft survival following heterotopic aortic valve implant. Endothelial cell viability was determined by flow cytometric analysis of endothelial cells harvested from aortic valve allografts by collagenase digestion. Only fresh valves and valves stored for 1 day were positive for Class I antigens; no valves were positive for Class II antigens. Duration of skin graft survival was prolonged with greater duration of storage, but grafts remained immunogenic after 21 days of storage. Endothelial cell viability declined from 95% in the fresh valves to 64% after 21 days of storage. With prolonged duration of allograft valve storage at 4 degrees C, there is an attenuation of antigenicity, immunogenicity, and endothelial cell viability. Loss of endothelial cells may contribute to the changes in immunological responses to the valve allografts. The expression of antigens on the endothelial surface is not a reliable predictor of immunological response.
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PMID:Immunogenicity, antigenicity, and endothelial viability of aortic valves preserved at 4 degrees C in a nutrient medium. 181 69

Abnormalities of tubular membrane structure and composition have been proposed as the primary defect in nephronophthisis (NEF). In order to characterize the protein composition of tubular cells in NEF, in vitro methods were developed to culture and propagate tubular cells obtained from biopsy fragments. Accordingly, microdissected cortical slices (1 x 3 mm) were first digested with collagenase and DNAse and then grown in RPMI medium supplemented with 10% NU serum and conditioned serum deriving from 3T3 cultures. At confluence, cultured cells from NEF showed characteristics which were typical of normal tubules, i.e. presence of cytokeratin and positivity for succinic dehydrogenase and alkaline phosphatase stainings, and presented no morphological alterations compared to cultured cells from normal tubular epithelium. Moreover, no difference was observed for fibronectin, collagen IV and laminin stains. Analysis by two-dimensional electrophoresis of cellular extracts revealed several changes in protein composition of NEF, the main one being the decrease in NEF cells of a polypeptide with a molecular weight of 120 kD and a pI of 4.8; this polypeptide was a constant finding in normal kidneys. These observations demonstrated that human tubular epithelial cells can be successfully cultured from very small biopsy fragments, which represents a new approach to the study of molecular disorders involving tubular cells in inherited disease. Cultured cells from NEF maintain the same morphological, immunological and cytochemical characteristics as normal tubular cells, but present a few alterations in polypeptide composition which may have pathogenetic relevance. A more careful analysis of these alterations is needed to define the molecular disorder(s) involving the tubule in NEF.
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PMID:Tubular epithelium culture from nephronophthisis-affected kidneys: a new approach to molecular disorders of tubular cells. 207 4

Endocrine-rich monolayers of pig fetal pancreas that are free of fibroblasts have been established with the ultimate aim of providing guidelines for the culture of the human equivalent. The immunogenic potential of the monolayers--hence their capacity to be grafted--has also been analyzed. Fetuses ranging from 50 to 90 days were used, and, following digestion with collagenase (4 mg/ml, 15-20 min), the pancreatic suspension was plated onto tissue culture vessels containing RPMI 1640. The fetal calf serum concentration was kept low (5%) initially to inhibit fibroblast proliferation, but subsequently increased to 7%. Monolayers from a typical litter of 8-10 fetal pigs produced 6-8 x 10(8) viable epithelial cells by day 10 of culture, of which 75% were endocrine cells. This represents an 8-fold increase in a two-week period. The ratio of beta:alpha:delta:pancreatic polypeptide cells was 19:33:18:5. These monolayers synthesized both DNA, (pro)insulin and protein, and displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+ and 1.3 microM 12-0-tetradecanoyl-phorbol-13-acetate. Static stimulation with 20 mM glucose however, did not elicit a response in insulin secretion. These cells displayed no reaction to allogeneic lymphocytes in a mixed lymphocyte culture, whereas freshly obtained porcine epithelial cells did. Methods may need to be found to increase the proportion of B cells in this enriched endocrine cell population. In general however, guidelines have been established that may be useful in developing a monolayer of human fetal pancreatic cells with the eventual aim of transplantation. The reduction in immunogenicity of the pig fetal pancreatic cells suggests that they too might be a potential source for transplantation.
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PMID:Pig fetal pancreatic monolayers. A model of potential use in transplantation. 219 44

Cell suspensions prepared by collagenase digestion of pancreata obtained from 21.5-d-old rat fetuses were preincubated in RPMI medium containing 10% fetal bovine serum (FBS), to ensure cell adhesion. Twenty hours later, this medium was replaced by a chemically defined medium. Dulbecco's modified Eagle's (DME)-F12 was used alone or supplemented with various combinations of transferrin, sodium selenite, or Ultroser G. The evolution of the culture and the islet ultrastructure were similar in defined and serum-containing media. However, in the defined medium, the neoformed islets seemed less numerous, and the fibroblast layer less dense, when compared to the RPMI + 10% FBS control medium. At Day 7, in defined media, the total insulin content per dish was half that of control cultures. None of the tested additives improved the yield of the cultures. The fractional insulin release per day was elevated in defined media. In subsequent incubations, glucose and leucine stimulated insulin release in a way characteristic of these cells of fetal origin. The labeling index of islet cells cultured in DME-F12 reached 10.7%, which is not far from that observed in RPMI + 10% FBS. Such a defined medium is useful to study B cell physiology, avoiding the possible interaction of serum components with substances to be tested.
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PMID:Culture of endocrine pancreatic cells in protein-free, chemically defined media. 224 53


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