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)

Dithizone, a zinc chelating agent, is known to selectively stain the islets of Langerhans in the pancreas. In the present study, we have used this stain to aid the identification of islets in material obtained by collagenase digestion of human pancreas. Islets were shown to rapidly and reversibly stain red on incubation with dithizone solution. Tissue selected on the basis of dithizone staining was shown to contain insulin-positive cells and to accumulate insulin in the medium during a subsequent period in tissue culture. Experiments with rat islets indicated that the dithizone treatment had no effect on insulin release in tissue culture, on acute responses to stimulatory glucose concentrations or on the insulin content of cells. These results suggest that dithizone staining can assist in the identification of islets from the human pancreas and may prove to be a useful tool in developing techniques for the large scale isolation of functionally intact human islets.
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PMID:Supravital dithizone staining in the isolation of human and rat pancreatic islets. 254 5

To establish a large-scale isolation procedure for adult porcine islets usable as a donor source for xenotransplantation and as a model of human islet isolation, we improved several characteristics of the conventional isolation procedure. At a slaughterhouse we first selected a breeder pig over 1.5 years old (and over 200 kg in weight) with warm ischemic time (WIT) of 15 +/- 2 minutes as nonheart-beating donors. Then, we made a special enzymic mixture that consisted of collagenase S-1 (260 U/mg, NittaZelatin, Japan), collagenase P (1.86 U/ml Lyo Boehringer-Mannheim, USA), DNase (Sigma, St. Louis, Mo), Disparse (NittaZelatin, Japan), and protease inhibitor (Sigma). Third, this mixture was injected very gently into the pancreatic duct at the time of pancreatic harvesting. To prevent overdigestion of the pancreas, the mixture was first cooled to less than 10 degrees C. Fourth, during the warm digestion of pancreas, the pancreas with the enzymic mixture was quietly put in a water bath at 37 degrees C without mechanical shaking. Fifth, we purified the islets with a COBE 2991 cell processor by the Dextran 70 gradient method, because Dextran 70 is very cheap and has the same purification effect as the Ficoll gradient. The results of 10 consecutive breeder porcine islet isolations are reported. The total yield of isolations of islets over 50 microm in the longest diameter after staining with Dithizone (DTZ) was 85,900 +/- 19,954 islets, 291,667 +/- 240,452 IEQ (2,900 +/- 2,324 IEQ/g). The purity of the isolated islets was very high: 90.2 +/- 3.8%. Glucose stimulation during in vitro incubation induced significant insulin release from isolated breeder porcine islets. In two of the diabetic rats receiving encapsulated islets grafts using a mesh-reinforced polyvinyl alcohol hydrogel bag (MRPB), a prominent reduction in serum glucose levels (less than 200 mg/dL) persisted for 13 and 19 days, respectively, after intraperitoneal xenotransplantation islets without immunosuppression. In conclusion, we succeeded in a more efficient and less-expensive isolation of a large amount of adult porcine islets from a nonheart-beating donor.
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PMID:Improved large-scale isolation of breeder porcine islets: possibility of harvesting from nonheart-beating donor. 971 Mar 9

Two types of islets of Langerhans are present in the avian endocrine pancreas: glucagon islets (A-islets) and insulin islets (B-islets). Islets from the chicken pancreas were isolated by ductal injection of collagenase, enzymatic digestion, atraumatic dispersion of the digests at an appropriate time, and nylon mesh filtrations. A- and B-islets were identified by immunohistochemistry and radioimmunological quantification of insulin and glucagon. Dithizone-positive islets proved to be mostly of the A-type by immunostaining, and radioimmunological measurements: islets from the cranial half of the body of the pancreas were almost pure (95%) glucagon islets (0.454 +/- 0.027 pmol glucagon and 0.023 +/- 0.005 pmol insulin per islet). Increasing the glucose concentration in the incubation medium from 14 to 42 mM decreased glucagon release, demonstrating that the alpha-cells maintained their glucose sensitivity after isolation.
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PMID:Isolation of functional glucagon islets of Langerhans from the chicken pancreas. 978 98

Transplantation of pancreatic islets represents a promising way of curing type I diabetes (insulin-dependent diabetes mellitus). Culture enables the survival of endocrine tissue awaiting islet transplantation and reduces islet immunogenicity prior to xenografting. In this study, attempts were made to preserve the monkey islets in culture for 7 days and to study the ultrastructure by electron microscopy. The islets were isolated from monkey pancreas by the collagenase digestion method and were separated from acinar cells by dextran density gradient centrifugation. These islets were preserved in a humidified atmosphere of 5% carbon dioxide and 95% air for 7 days. The culture medium used was CMRL-1066. After 7 days of culture the islets were processed for light and electron microscopic studies, which revealed that the cultured islets were intact and maintained their structural integrity. Semi-thin sections of the cultured islets showed morphology with occasional structural alterations at the periphery. Dithizone staining of the cultured islets showed crimson red colour, proving that the islets were pure and without any exocrine contamination. Electron microscopy showed that the cultured islets had well-preserved alpha-, beta- and delta-cells. Different cell types of the monkey pancreatic islets were identified by the presence of their characteristic secretory granules. The ultrastructural characteristics present in hormone-synthesizing cells, i.e. rough-endoplasmic reticulum, Golgi apparatus, mitochondria and secretory granules, were observed as in native islets.
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PMID:Culture prior to transplantation preserves the ultrastructural integrity of monkey pancreatic islets. 1459 2

Conceptually, pancreas islet transplantation (PIT) associated with renal transplantation (RT) should resolve not only chronic renal failure but also diabetes. Although the most frequently used site for PIT is the portal vein, genitourinary locations could be technically feasible during RT. Seventeen pigs (age 3 to 4 months; mean weight 34.5 kg) underwent the following experimental steps: On day 1 a left nephrectomy was performed and the kidney was perfused with cold Wisconsin solution. This was followed by a caudal pancreatectomy and islet isolation by means of digestion with intraductal collagenase. Islets were stained with Dithizone and cultured overnight al 37 degrees C and 5% CO(2). On day 2 a right nephrectomy and orthotopic RT of the preserved left kidney were performed. The islets were transplanted into four different sites: subcapsular in the kidney graft, in the bladder submucosa, in the testis by puncture, and in the testis by infusion through the vas deferens. On day 7 the animals were sacrificed. Islet viability was determined by histological examination with insulin immunostaining and determination of insulin in the blood of the veins draining the implantation sites. The mean weight of the pancreatic specimens was 27.8 g (13 to 46). The mean number of islets was 536,000 (16,600 to 1,5000,000). Islets were shown in the bladder submucosa and the testes after vas deferens infusion. The number of viable islets in the other implantation sites was very scarce. The insulin levels of the venous effluents were: 15.1 microU/mL for bladder submucosa, 10.2 microU/mL for intradeferential injection in the testis, 7.3 microU/mL for intratesticular injection by puncture, and 2.6 microU/mL for subcapsular implantation in the graft. In conclusion, the bladder submucosa and testis via the vas deferens might represent alternative sites for PIT. The latter route may benefit from the immunoprivileged and special trophic conditions of the testis. For the first time, the feasibility of the bladder submucosa as an implantation site for pancreas islets was demonstrated.
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PMID:Pancreas islet transplantation in the genitourinary tract associated with renal transplantation: an experimental study. 1709 10