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 use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobblestone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors, Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.
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PMID:Isolation and characterization of equine microvascular endothelial cells in vitro. 280 14

This paper presents a study on the structure and function of Kupffer cells (KC) and liver endothelial cells (LEC) isolated by a simple and rapid technique involving 1) perfusion of the liver with collagenase; 2) cell separation by means of density centrifugation in Percoll; and 3) cell culture, taking advantage of the fact that KC and LEC differ in their preferences for growth substrate. The KC, which attach and spread under serum-free conditions on surfaces of glass or plastic during the first 15 min in culture exhibit a typical macrophage-like morphology including membrane ruffling and a heterogenous content of vacuoles. Moreover, these cells express (a) Fc receptors (FcR) for binding and phagocytosis of erythrocytes covered with immune globulin G (E-IgG), and (b) complement receptors (CR) for binding and serum dependent phagocytosis of erythrocytes covered with either human C3b or mouse inactivated C3b (iC3b). The cells also bind fluid phase fluoresceinated C3b. Approximately 30% of the KC express immune response-associated (Ia)-antigens. The LEC attach and spread on fibronectin coated surfaces, but not on glass or plastic surfaces, during the first two hours in culture with or without serum, and are morphologically distinct from KC. Cultured LEC are well spread out with no membrane ruffling and with numerous large vesicles surrounding the regularly shaped nucleus. These cells bind, but do not ingest E-IgG via the FcR, but no binding of fluid phase C3b or particle fixed C3b or iC3b can be observed. Incubation of LEC with fluorescein amine conjugates of ovalbumin or formaldehyde treated serum albumin, but not with fluoresceinated native serum albumin, results in accumulation of fluorescence specifically localized in the large perinuclear vesicles. Neither KC nor any other cell types tested have the ability to accumulate fluorescence upon incubation with these compounds. Ia-antigens are not present on the LEC. Cytochemical demonstration of unspecific esterase, acid phosphatase, and peroxidase reveals different patterns and intensities of staining in KC as compared to LEC.
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PMID:Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll, and selective substrate adherence. 299 96

Serotonin (5-HT) is known to be a potent stimulator of aldosterone release in vitro and, to a lesser degree, in vivo. Since ketanserin, a specific 5-HT2-receptor antagonist, decreases aldosterone levels in some hypertensive patients, we have performed in vitro experiments to elucidate the mechanism by which ketanserin interferes with aldosterone regulation. After collagenase dispersion, rat zona glomerulosa cells were either perfused and resuspended in Biogel or incubated in Earle's medium and bovine serum albumin. The cells were stimulated with serotonin, angiotensin II, potassium (K+) and adrenocorticotrophic hormone (ACTH) in the presence or absence of ketanserin. Ketanserin did not decrease baseline aldosterone secretion although it reduced the stimulatory capacity of serotonin and K+, and had a minimal effect on ACTH. Furthermore, we demonstrated that ketanserin is able to have a significant effect on the adrenal response to angiotensin II. These data indicate that antiserotoninergic agents may act directly at the level of the adrenal glomerulosa by interfering with specific serotoninergic receptors and modifying the adrenal sensitivity to angiotensin II and K+. The importance of these findings in the clinical use of these antihypertensive drugs remains to be established.
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PMID:Effect of ketanserin on the in vitro regulation of aldosterone. 300 55

A novel gelatin-binding 21 kDa protein was identified in the culture medium of fibroblastic and sarcoma cells by affinity chromatography on gelatin-Sepharose. Its affinity for gelatin was lower than that of the other gelatin-binding proteins, fibronectin and the 70 kDa protein, as judged by stepwise elution by urea and arginine. The protein bound also to spermine and to some extent to heparin but not to staphylococcal protein A, bovine serum albumin, concanavalin A or plain Sepharose 4B. In gel filtration chromatography the protein eluted in fractions differing from those of fibronectin and the Mr 70,000 protein and retained its ability to bind to gelatin-Sepharose, indicating that the binding was not mediated by the two other gelatin-binding proteins. It contains intrachain disulfide bridges, as judged by analysis under nonreducing and reducing conditions. The protein is composed of two major subtypes with pI values of 5.85-6.10 and 6.55-6.75. It was sensitive to trypsin but not to collagenase or thrombin. Antiserum was raised in rabbits against the gelatin-binding proteins isolated from serum-free conditioned fibroblast culture medium. The antiserum reacted with fibronectin, the Mr 70,000 protein and the Mr 21,000 protein in immunoprecipitation experiments. Absorption of the antiserum with human plasma fibronectin did not decrease its reactivity with the Mr 70,000 and 21,000 proteins. However, absorption with the Mr 70,000 protein abolished also the reactivity against the Mr 21,000 protein, suggesting immunological cross-reactivity. The protein was synthesized independently from the Mr 70,000 protein, as shown by pulse-chase labeling experiments of cells. The production of the Mr 21,000 protein in cultured cells was enhanced by transforming growth factor-beta.
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PMID:Characterization of a novel gelatin-binding 21 kDa protein secreted by cultured adherent cells. 301 29

Leukocyte chemoattractants markedly alter the morphology and membrane functions of leukocytes. Bacterial collagenase causes a change in cell shape similar to that seen with the leukocyte chemoattractant, f-Met-Leu-Phe, and also promotes capping of concanavalin A. Human neutrophils in suspension or adherent to cover glasses were exposed to clostridial collagenase (10-250 units/ml) for up to 30 min at 37 degrees C and then fixed. Collagenase (125 units/ml) caused more than 85% of PMNs to assume an asymmetric or motile morphology even in the presence of 1% gelatin or 10 mg/ml bovine serum albumin. Trypsin alone (0.01-1%) did not induce a shape change. A similar morphology was seen in some untreated PMNs (less than 5% of all cells) and is characteristic of f-Met-Leu-Phe-treated cells (more than 90%). Collagenase inhibitors (i.e., reduced glutathione, cysteine, and acid-soluble collagen), however, prevented the shape change induced by collagenase but not by f-Met-Leu-Phe. At 4 degrees C, fluorescein-Con A (20 micrograms/ml) bound uniformly to both untreated and collagenase-treated cells. Upon further incubation at 37 degrees C, Con A was internalized over the entire cell periphery of the rounded, untreated cells but on collagenase-treated PMNs was rapidly gathered into a cap overlying the uropod or protuberant region of cytoplasm where it was subsequently internalized. Checkerboard Boyden chamber assays showed clostridial collagenase to be chemokinetic and chemotactic for human PMNs. In receptor binding experiments, the clostridial collagenase preparation competed poorly with [125I]formylhexapeptide for binding to PMN formylpeptide receptors (less than 15% reduction in binding at 200 units/ml collagenase). Thus, collagenase does not seem to interact strongly with the neutrophil formylpeptide receptor and may stimulate PMN motility by interacting at an altogether different site.
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PMID:Clostridial collagenase. A chemoattractant for human neutrophils. 302 90

Prepuberal (P) gilts were induced to ovulate with pregnant mare serum gonadotropin followed 72 h later by human chorionic gonadotropin (hCG). Three P gilts and three mature (M) gilts each were ovariectomized on d 10, 14, 18, 22 and 26 (d 0 = day of hCG for P gilts and onset of estrus for M gilts). Gilts ovariectomized on d 14, 18, 22 and 26 were hysterectomized on d 6 to ensure maintenance of the corpora lutea (CL). Two to five grams of minced luteal tissue were dispersed using collagenase and hyaluronidase in HEPES buffered salt solution supplemented with glucose and bovine serum albumin. Dispersed cells were rinsed in Dulbecco's Modified Eagle Medium (DMEM), counted (ratio of large to total number of luteal cells determined) and then incubated for 1 h in DMEM. With aliquots standardized to 2.5 X 10(4) viable, large cells (greater than 25 micron diameter) were incubated in 1 ml DMEM for 2 h in the presence of either 10, 50, 100 or 1,000 ng luteinizing hormone (LH); .1, 1, 10 or 100 ng hCG; 10, 100 or 1,000 ng norepinephrine (NE) or either .75, or 1.5 mM dibutyrl cyclic adenosine monophosphate (dbcAMP). Progesterone (P4) in the medium was quantified by radioimmunoassay. Basal P4 production (no P4 stimulator added to the medium) on d 10, 14, 18, 22 and 26 for P gilts was 246 +/- 9, 66 +/- 4, 64 +/- 6, 41 +/- 3 and 69 +/- 6 ng/ml medium, respectively, and for M gilts was 281 +/- 12, 128 +/- 8, 53 +/- 4, 82 +/- 6, 101 +/- 5 ng/ml medium, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of induced corpora lutea from prepuberal gilts and spontaneous corpora lutea from mature gilts: in vitro progesterone production. 303 Sep 95

The use of a bovine serum albumin (BSA) density gradient for isolation of rat pancreatic Islets of Langerhans after collagenase digestion has been compared with the standard Ficoll separation technique. The criteria studied were islet yield (insulin extraction of the islet interfaces and pellet), purity of preparation (amylase content of the islet preparation), insulin release characteristics, and the result of isologous transplantation in diabetic rats. The islet interface of the BSA gradient contained 62.2% of the total insulin, whereas the corresponding interface of the Ficoll gradient contained only 36.6% (P less than 0.001). The amylase content of the Ficoll-separated islet preparation was 6133 U/L, as opposed to 1230 U/L with BSA (P less than 0.001). BSA-isolated islets gave similar insulin release characteristics to non-density-gradient-isolated islets, whereas Ficoll-separated islets showed suboptimal insulin release. Single-donor-single-recipient transplantation was successfully performed with BSA-isolated islets whereas multiple donors were required with Ficoll-separated islets. Thus significantly improved results were found with the bovine serum albumin density gradient separation in all criteria and consequently the use of this gradient represents an advance in islet isolation techniques.
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PMID:Bovine serum albumin density gradient isolation of rat pancreatic islets. 310 87

In order to establish a model for the in vivo assessment of islet function we have used the Rowett nude rat with transplantation of allogeneic and xenogeneic (mouse) islets into the renal subcapsular space following a minimal period of diabetic induction. Thirty-one nude rats were given streptozotocin and 30 became diabetic with blood glucose levels of greater than 20 mmol/l at 48 h. Rat and mouse islets were prepared by intraductal collagenase and bovine serum albumin density gradient isolation. Eight rats received transplants of freshly prepared allogeneic islets and 8 rats received transplants of 48 h cultured allogeneic islets. Seven rats received transplants of 48 h cultured mouse islets. Diabetes was reversed in all animals and all remained normoglycaemic for 21 days. Graft removal by nephrectomy resulted in hyperglycaemia in 22 out of 23 animals. Histological examination of the grafts showed a band of endocrine tissue beneath the renal capsule which stained strongly positive for insulin and there was no evidence of lymphocytic infiltration/rejection. One rat remained normoglycaemic after graft removal, which may represent recovery of the animal's own islets from the streptozotocin-induced diabetes. Control rats remained diabetic until death. In conclusion, the athymic nude rat can be used for the assessment of allogeneic and xenogeneic islet function when a short (48 h) period of streptozotocin-induced diabetes is used. This model offers a potential method for assessing in vivo function of isolated human islets.
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PMID:In vivo assessment of isolated pancreatic islet viability using the streptozotocin-induced diabetic nude rat. 313 50

We have tested whether mannose- and galactose-specific lectins on liver cells are able to bind antibody-antigen complexes and thus function as Fc-receptors. Rat hepatocytes and liver sinusoidal cells were isolated by collagenase perfusion and differential centrifugation. Rat erythrocytes were coated with purified IgM or IgG from rabbits immunized with rat erythrocytes. Both IgM and IgG coated erythrocytes bound to liver macrophages but not to hepatocytes. The binding of IgM and IgG coated red blood cells to liver macrophages could not be blocked by potent inhibitors for mannose- and galactose-specific macrophage lectins such as mannan, D-mannose-bovine serum albumin, N-acetyl-D-galactosamine, D-galactose-bovine serum albumin, or asialofetuin. Although lectin activity is calcium dependent and trypsin sensitive neither condition blocked rosette formation between liver macrophages and opsonized erythrocytes. Thus mannose- and galactose-specific lectins are not involved in the sequestration of IgM- or IgG-antibody-erythrocyte complexes in the liver.
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PMID:Lack of evidence for liver lectins functioning as IgM or IgG-Fc-receptors. 316 7

Hens in their sixth month of lay were injected with a series of doses of ovine LH, either following the oviposition of a mid-sequence egg (which is closely associated with an ovulation), or following the oviposition of the last egg of a sequence. The concentrations of steroid hormones were subsequently measured by radioimmunoassay in blood withdrawn at intervals after injection. No changes were observed in the concentrations of either testosterone or 17 beta-oestradiol, irrespective of the dose of LH or the stage of the ovulatory cycle. An increase in delta-4-androstenedione was observed in all cases. This increase was minimal and unrelated to the dose of LH at the mid-sequence stage, but a dose-response relationship was observed in hens injected following the terminal oviposition of a series. Consistent, dose-related changes in the concentrations of progesterone in plasma were found at both stages of the ovulatory cycle, and the magnitude of these changes was 3.5-5 times greater than those observed for androstenedione. Granulosa cells from the first, second and third ovarian follicles were dispersed by collagenase and stimulated with ovine LH either immediately or following a 24 h pre-incubation in medium 199. The amount of progesterone released into the medium after 3 h was assessed by radioimmunoassay. A progressive decrease in this response was observed in cells derived from the first to the third follicles in all cases. Cells challenged after 24 h always showed increased responses, cells from the second follicle secreted similar amounts of progesterone as cells from the first follicle that were challenged immediately, without pre-incubation. The responses obtained after 24 h were attenuated if no bovine serum albumin was present in the medium, or if ovine LH had been present in the medium continuously. These results are interpreted as evidence for an increase in the secretion of progesterone by the granulosa cells of the hen which is linked to the maturation of the follicle. The final stages of this maturation may proceed in the absence of LH.
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PMID:Plasma levels of progesterone in the domestic hen related to the maturation of ovarian follicles, and changes in the secretion of progesterone by granulosa cells cultured for 24 hours. 324 2


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