Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of fibrogenesis in the pancreas is not well known. We analyzed the role of prolylhydroxylase and collagenase activities in the development of fibrosis in chronic alcoholic pancreatitis (CAP). Nineteen patients with CAP and 11 controls (organ donors) with normal pancreatic histology were included in the study. Pancreatic tissue was obtained from all subjects to measure (a) area of fibrosis (histomorphometric method); (b) prolylhydroxylase activity (PHase), which reflects the intracellular synthesis of collagen (Hutton's method); and (c) collagenase activity, which reflects the degradation of collagen (collagenase assay system, 3H). The percentage of the fibrosis area in relation to the total area of pancreatic tissue was significantly higher in CAP than in the control group (70.6+/-20.2% vs. 4.6+/-1.8%; p<0.001). Mean pancreatic PHase activity was also significantly higher in CAP than in the control group (775+/-258 cpm/mg protein/h vs. 405+/-151 cpm/mg protein/h; p<0.001). The collagenase activity was significantly lower in CAP than in the control group (8.7+/-3.5 cpm/cpm added/mg protein vs. 18.0+/-3.9 cpm/cpm added/mg protein; p<0.001). A significant correlation was observed between percentage fibrosis evaluated histomorphometrically and PHase activity in all patients (r = 0.72; p<0.001), and between PHase and collagenase activities in controls (r = 0.70; p = 0.024), but not in CAP. Pancreatic tissue in CAP has an increased fibrogenic activity and an impaired collagen-degradation capacity. These findings might explain the excessive development of fibrosis in CAP.
Pancreas 1999 Jan
PMID:Synthesis and degradation of collagen in pancreatic fibrogenesis. 988 58

Expression of Galalpha(1-3)Gal on endothelium has been implicated in the rejection of porcine xenografts. The aim of this study was to determine whether expression of Galalpha(1-3)Gal on pig islets varies between pigs aged 5, 12 and 24 weeks, and to investigate whether it is expressed on islets isolated by collagenase digestion or islets maintained in tissue culture. Samples of pancreas were obtained from pigs aged 5, 12 and 24 weeks. Islets were isolated by manual collagenase digestion and density gradient separation. Samples were taken immediately after isolation or after maintenance in tissue culture. Pancreas and islet samples were processed, sectioned and stained with the lectin BS1-B4 (which binds to Galalpha(1-3)Gal residues), and anti-insulin antibody using a double staining technique. There was no significant difference in the staining patterns to sections of pancreas obtained from 5, 12 and 24 week old pigs. Vascular endothelium, connective tissue and the luminal surface of duct epithelial cells stained with BS1-B4 in all sections; endocrine and exocrine cells did not stain. Preliminary experiments showed that lectin staining to isolated islets was inconsistent between preparations, but expression did not appear to differ significantly between ages: lectin staining of some beta-cells was evident in the majority of freshly isolated preparations, but was not detectable on beta-cells following tissue culture. In conclusion, expression of Galalpha(1-3)Gal did not differ significantly in pancreata from 5, 12 and 24 week old pigs. Preliminary experiments showed that Galalpha(1-3)Gal was expressed by beta-cells immediately following isolation, but not after maintenance in culture.
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PMID:Expression of the GALalpha(1-3)GAL epitope on pig islets. 993 Sep 56

A new method has been developed for the dissociation of rat pancreas into acinar suspension by introducing an in situ flow-through perfusion with a Ca(2+)-chelating buffer before in vitro enzymatic digestion with collagenase. As a result of the perfusion step and other minor modifications, a high-quality and uniform suspension of acini and small acinar complexes is obtained that offers the possibility to work with 50 parallel samples from a single rat for protein synthesis and degradation measurements. Initial cellular viability is 95-99% and remains > 85% even after 6 h in vitro. Electron-microscopic observations show that the cells in isolated acini retain their polarity, tight junctions remain intact, and desmosomes and basement membranes disappear. Protein synthesis shows strong dependence on the extracellular supply of amino acids and 30-50% stimulation by insulin. With the help of the new system of isolated acini, data on the degradation of intracellular proteins of the exocrine pancreatic tissue is presented for the first time in the literature. After a 2-h labeling period with [14C]valine, 2-4% of the radioactive protein is degraded per hour. Of this protein breakdown, 30-40% appears to take place in lysosomes, as measured in the presence of leupeptin, an inhibitor of lysosomal degradation.
Pancreas 1999 May
PMID:Complete dissociation of rat pancreas into acini by a perfusion-incubation method: measurement of protein synthesis and the degradation of endogenous proteins. 1023 38

Culturing of islets is essential for various purposes before transplantation. It is necessary to establish optimal culture conditions for each animal species for their preservation in culture. In this study, attempts were made to preserve the Indian bonnet monkey islets in culture. The islets were isolated from monkey pancreas by the collagenase digestion method. They were separated from acinar cells by dextran density-gradient centrifugation. They were preserved in a humidified atmosphere of 5% carbon dioxide and 95% air for 7 days. The culture medium used was RPMI-1640. Various optimal conditions such as volume of the culture medium used, number of islets in one culture dish, concentration of glucose in culture medium, and fetal calf serum percentage were tested for their better preservation in culture. After the culture period, they were tested for their insulin secretory capacity by exposing them to various secretagogues. Histologic appearance of the cultured islets also was examined. Both insulin secretory characteristics and histologic structure were found to be normal.
Pancreas 1999 Jul
PMID:Optimization of culture conditions for the preservation of monkey pancreatic islets in culture. 1041 97

A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.
Pancreas 2000 Mar
PMID:Improved quality and yield of islets isolated from human pancreata using a two-step digestion method. 1070 35

Neovascularization may be necessary for better and longer function of transplanted islets. Vascular endothelial growth factor (VEGF) is known to be one of the most important factors of angiogenesis. Recently, VEGF was reported to be expressed in islets of normal pancreas. We studied the expression of VEGF and neovascularization related peptides in transplanted islets. To determine the angiogenic microcapillary, immunochemical staining was performed for Factor VIII-related antigen (von Willebrand factor [vWF]) and platelet endothelial cell adhesion molecule-1/CD31 (PECAM-1), both of which are known as markers of the angiogenic microvessel. Transplantable islets were isolated from Lewis rats (8-10 weeks of age) by discontinuous dextran gradient after collagenase digestion. Seven to twelve hundred islets were injected into the portal vein (IPV group, n = 7) or transplanted into subnephrocapsular cavity (SNC, n = 12) of the same descent rats. In the IPV group, the liver was resected 1 hour, 1 week, or 4 weeks after transplantation (Tx). In the SNC group, the kidney was resected 1, 3, 7, or 28 days after Tx. Each tissue was fixed in formaldehyde and embedded in paraffin. Serial 4-microm slices were immunostained for insulin, VEGF, PECAM-1, or vWF using specific antibodies. In IPV group, insulin-positive cells were VEGF positive as were in the normal pancreas at all time points. Islets of 1 hour after Tx were barely PECAM-1 positive as were in normal pancreas, but islets became weakly stained at 7 and 28 days after Tx. In vWF staining, transplanted islets showed stronger staining than those in the normal pancreas. In SNC group, VEGF was also stained in insulin-positive cells at 1, 3, 7, and 28 days. In PECAM-1 staining, islets of 1 day after Tx were barely stained as were in normal pancreas. However, the staining was increasingly enhanced from 3 to 7 days and then appeared weakened at 28 days after Tx. In vWF staining, islets were always vWF positive, as was seen in IPV group. This study revealed that PECAM-1 appeared in islets after islet Tx, suggesting that neovascularization occurs within the islet grafts. On the other hand, VEGF of transplanted islet did not obviously vary with time. Enhancement of the neovascularization may lead to better results of islet Tx.
Pancreas 2000 Aug
PMID:Immunohistochemical studies on vascular endothelial growth factor and platelet endothelial cell adhesion molecule-1/CD-31 in islet transplantation. 1097 11

Islet beta cell adaptation to dexamethasone-induced insulin resistance was characterized with respect to glucose-stimulated insulin secretion and islet innervation. Male Sprague-Dawley rats were injected daily with dexamethasone (2 mg/kg for 12 days), which resulted in hyperinsulinemia and hyperglycemia compared with controls (which were injected with sodium chloride). Insulin secretion was characterized in collagenase-isolated islets. Islet innervation was examined by immunocytochemical analysis of tyrosine hydroxylase, neuropeptide Y (sympathetic nerves), and vasoactive intestinal polypeptide (cholinergic nerves). In islets isolated from the insulin-resistant animals, the insulin response to 3.3 or 8.3 mM glucose was three times greater during perifusion compared with controls (p < 0.001). Incubation of islets at 0 to 20 mM glucose revealed a marked leftward shift of the glucose dose-response relation after dexamethasone treatment (potency ratio, 1.78; p < 0.01), with no difference at 0 or 20 mM glucose. Thus, the potency but not the efficacy of glucose was increased. The number of islet nerves did not differ between dexamethasone-treated rats and controls. Dexamethasone-induced insulin resistance leads to adaptively increased glucose responsiveness of the islet beta cells, with increased potency, but not increased efficacy, of glucose to stimulate insulin secretion without any evidence of altered islet innervation.
Pancreas 2001 Mar
PMID:Beta cell adaptation to dexamethasone-induced insulin resistance in rats involves increased glucose responsiveness but not glucose effectiveness. 1124 69

The digestion of pancreatic tissue with collagenase is an essential part of the islet isolation procedure. However, the process exposes islets to various types of harmful factors, including collagenase contaminants, enzymes released from the acinar cells, warm ischemia, and mechanical agitation. Nitrogen oxide production and cytokine release may also contribute to islet cell damage. Protection of islets from such damage would improve the islet yield, survival, and function. Beraprost sodium (BPS) is a prostaglandin I2 analogue, is stable in aqueous solution, and has a cytoprotective effect on various types of cells. BPS has been shown to improve the yield and function of cryopreserved and/or cultured islets. These findings prompted us to examine its cytoprotective effect on islets during the islet isolation process. Canine islets were isolated by means of a two-step digestion method and purified on Euro-Ficoll density gradient solutions (the procedure used for human islets). BPS at a concentration of 100 nM was added to the collagenase solution. After purification, the islet yield was 434,561 +/- 35.691 islet number expressed as 150 microm equivalent size (IEQ)/pancreas or 8,799 +/- 345 IEQ/g of pancreas in the BPS group and 349,987 +/- 52,887 IEQ/pancreas or 7,998 +/-1610 IEQ/g of pancreas in the control group (n = 8, each). The percent viability was 88.5 +/- 0.7% in the BPS group and 82.0 +/-0.9% in the control group (P < 0.01). Therefore, the recovery of viable islets (calculated by islet number x % viability) was 384,586 +/- 46,804 IEQ/pancreas (7,743 IEQ/g) in the BPS group and 286,989 +/- 43,367 IEQ/pancreas (6,558 IEQ/g) in the control group (P < 0.02). After culture, significantly higher numbers of islets were also recovered in the BPS group than in the control group. The islet insulin content was significantly higher in the BPS group than controls (237.8 +/- 38.5 versus 92.3 +/- 25.6 microU/IEQ; P < 0.02), although islets of both groups responded with high stimulation indices (>6). These results indicate that the addition of BPS to the collagenase solution increases the recovery of viable islets, and improves beta cell function.
Pancreas 2001 Jul
PMID:Increased islet viability by addition of beraprost sodium to collagenase solution. 1145 Nov 49

Yields and function of isolated islets vary considerably in spite of the introduction of new or improved methods for isolation. In most studies, these variations have been attributed to inadequacies of the applied collagenase preparations. However, when we retrospectively analyzed our rat islet isolations, we found large variations in yield and function in spite of application of identical collagenase sources. Therefore, in the present study, we determined the effect of rat donor strain, the source of inhibition of proteolytic activity (by bovine serum albumin), and the culture conditions on yield and function. AO rats showed a twofold higher islet yield than Wistar and Lewis rats. However, a higher yield was not associated with a higher response on glucose load since this response was more pronounced with Lewis islets than with Wistar and AO islets. Rats with a higher weight donate more islets but have a lower insulin secretory capacity. Islet yield and function also vary with application of different sources of bovine serum albumin during digestion. Moreover, the culture conditions influence the functional survival of isolated rat islets. CMRL1066 preserves the insulin secretory capacity of rat islets better than RPMI1640. Finally, the number of islets surviving the culture is higher when 4 instead of 12 and 24 islets were applied per square centimeter. Our observations indicate that strain and weight of the rat donor, the source of bovine serum albumin, and the culture conditions of islets are pertinent factors in efficacious isolation of islets.
Pancreas 2004 Jul
PMID:Factors influencing isolation of functional pancreatic rat islets. 1521 Nov 19

Commercially available purified collagenases, derived from Clostridium histolyticum, contain two different classes of collagenase: class I collagenase (CI) and class II collagenase (CII) at a predetermined ratio. In this study, using purified CI and CII in separate vials, we had a unique opportunity to investigate the effect of the proportion between two collagenase classes on clinical human islet isolation. Pancreas organs derived from deceased donors were prospectively assigned to one of three different enzyme protocols: group A--CII:CI = 1:1 vial; group B--1:2; group C--1.5:1. As a result, their total collagenase activities were 2116, 2230, and 3117 Wunsch units/pancreas in groups A, B, and C, respectively but thermolysin dosage was adjusted to 624-988 Units/g pancreas. The pancreas was not efficiently digested in group C in spite of a relatively longer digestion time and significantly higher Wunsch activity, resulting in the poorest islet isolation outcome among the three groups. Additional retrospective analysis revealed that this suboptimal outcome in group C was not because of the absolute excessive amount of collagenase activity but as a result of the relatively high proportion of CII (i.e., unbalanced CII/CI ratio). Our study suggests that an excessive CII is ineffective in releasing islets from human pancreas, and rather a balanced CII/CI ratio is of paramount importance.
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PMID:Detrimental effect of excessive collagenase class II on human islet isolation outcome. 1868 Apr 82


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