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)

Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99 +/- 10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36 +/- 1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2-5.0 microliters islet tissue volume (3,300-3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited.
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PMID:An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation. 132 62

We have successfully developed a technique for culturing human islet cells obtained from the cadaveric pancreata of children. Within 24-48 h of in vitro culture, collagenase-digested human pancreatic tissue formed epithelioid monolayers. Scattered within these monolayers were insulin-positive cells, as detected by immunocytochemical and dithizone staining. Treatment of the beta cell-containing epithelioid-cell monolayers with EDTA resulted in the formation of spherical cellular clusters, i.e., pseudoislets. These pseudoislets differed from isolated islets of Langerhans in that they showed a more peripheral distribution of insulin-positive cells. Our studies have demonstrated that insulin-positive cells can be detected in monolayers obtained from human pancreata 3-4 weeks after culture. When exposed to varying concentrations of glucose, these cells secreted insulin. The development of this in vitro technique for culturing human pancreatic islet tissue could provide a model for systematically studying in vitro islet function.
Pancreas 1992
PMID:Formation of pseudoislets from human pancreatic cultures. 137 49

Type I diabetes is characterized by insulin insufficiency due to lack of functional beta cells. To replace injection therapy, schemes such as the Hybrid Artificial Pancreas (HAP) were developed. This consists of an acrylic housing enclosing a semipermeable hollow fiber membrane. Donor islets can be seeded in the annular space through a port in the housing, and thus are separated from the recipient's bloodstream or perfusate. Before scaling the HAP to human size, the dynamics of its insulin response to a perfusion glucose challenge must be better understood. In this study, the HAP's insulin response after a step increase in the lumenal glucose concentration was determined as a function of the radial thickness of the annular space (0.173-0.973 mm) and islet distribution at a flow rate of 1 ml/min. Devices containing a single, 65 mm long fiber were used. Rat islets were isolated using standard collagenase digestion techniques. In unseeded HAP perfusions, the washout time for glucose and insulin from the annular space was dependent on flow rate and radial thickness. Both solutes were removed in < 3 min from the smallest devices when perfused at 10 ml/min. Thus, solute transport within the HAP is very fast. In the seeded HAP perfusions, the devices were subjected to a step increase in the lumenal glucose concentration. Sequential samples of the HAP effluent were collected and assayed for glucose and insulin. The spatial distribution of the islets in the annular space was one of the most important factors in determining the HAP's insulin response.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro perfusion of hybrid artificial pancreas devices at low flow rates. 145 99

The binding of GRP (gastrin-releasing peptide) to mouse pancreatic islets was studied. Binding of 100 pM 125I-GRP to collagenase-prepared isolated islets at 22 degrees C was one-half maximal after 15 min and maximal at 60 min. At 60 min, total binding was 1.62% of total radioactivity per 50 islets; nonspecific binding (presence of 1 microM unlabeled GRP-1-27) was 0.05-0.61% of total radioactivity. GRP binds specifically to a high-affinity site (Kd1 = 0.81 nM; Bmax1 = 12.8 fmol/50 islets). The specific binding is saturable. Hormones with the intact C-terminus of GRP-1-27, such as N-acetyl-GRP-20-27 and neuromedin C (GRP-18-27), possess the same inhibition curve as GRP-1-27. GRP-1-16, with a cleaved C-terminus, does not inhibit binding of 125I-GRP. However, hormones that virtually are not structurally related to GRP, such as eledoisin, galanin, and VIP (vasoactive intestinal peptide) do not compete for GRP binding. The rank order of GRP analogs such as GRP-1-27, N-acetyl-GRP-20-27, and GRP-1-16 is similar though not identical with respect to inhibition of 125I-GRP binding and insulin secretory potency. We found that 1 and 10 nM GRP-1-27, at a stimulatory glucose concentration, increases the breakdown of phosphatidylinositol to Ins-1,4,5-P3, the biological relevant isomer of Ins-P3; 10 nM GRP-1-27 is effective even at a nonstimulatory glucose concentration in this respect. In a virtually Ca(2+)-free medium, 5 nM GRP-1-27 increases the 45Ca2+ efflux from 45Ca(2+)-prelabeled islets. These data indicate that (a) specific binding sites for GRP are present in mouse pancreatic islets; (b) GRP superimposes the maximal insulinotropic effect of glucose; and (c) Ins-1,4,5-P3 is probably involved as a second messenger in the biological effects of GRP-1-27, which is underlined by the efflux of Ca2+ from intracellular stores but is not a sufficient signal by itself.
Pancreas 1992
PMID:Gastrin-releasing peptide: binding and functional studies in mouse pancreatic islets. 159 56

The development of techniques for separating islets from acinar cells on a mass scale is a prerequisite to successful clinical attempts at islet transplantation. We have identified and partially characterized two blood group-reactive monoclonal antibodies (McAb), CAC1 and CAC2, with specific binding activity to acinar cells but not islets. These McAb are of the IgM subclass, and were found on immunocytochemical analysis to possess broad interspecies cross-reactivity, binding to antigens expressed in the acinar tissue of rats, dogs, and man. The antigens that these McAb recognize are glycolipid in nature and thus were not denatured by collagenase digestion of the pancreas. These properties, together with their ability to effect complement-mediated lysis, may make the McAb generally useful reagents in islet isolation and purification.
Pancreas 1991 May
PMID:Immunocytochemical identification of monoclonal antibodies with binding activity to acinar cells but not islets. 171 75

We wished to determine whether the stimulation of protein synthesis by CCK8, carbachol, and insulin in isolated rat pancreatic acini resulted from translational or transcriptional induction of protein synthesis, and whether these hormones had similar or different effects on the rates of synthesis of individual enzymes. Isolated pancreatic acini were prepared from streptozocin-treated rats by collagenase digestion, mechanical dissociation, and centrifugation through a bovine serum albumin (BSA) cushion. Sixty-minute incubations, with maximally effective doses of CCK8, carbachol, and insulin, produced a 50, 90, and 100% increase, respectively, in the rate of protein synthesis. After inhibition of transcription with actinomycin D, the hormones still produced a 23, 50, and 61% increase, respectively, in the rate of protein synthesis. The study of the effect of the three hormones and the combination of CCK8 and insulin on the rate of synthesis of trypsinogen, chymotrypsinogen, lipase, and amylase, purified by isoelectric focusing, demonstrated that the hormones induced similar effects on the pattern of enzyme synthesis, and that they all induced the rate of synthesis of chymotrypsinogen slightly more than that of the other enzymes studied. We conclude that the hormones studied exert similar posttranscriptional influences in the regulation of protein synthesis in the pancreatic acinar cell.
Pancreas 1986
PMID:Translational control of protein synthesis in isolated pancreatic acini: role of CCK8, carbachol, and insulin. 243 15

The neuropeptide galanin is present in intrapancreatic nerve fibers and is known to affect the secretion of the islet hormones. Its most potent effect is thereby the inhibition of insulin secretion. In the present study, we investigated whether galanin influences amylase secretion from isolated rat pancreatic acini. Acini were isolated by the collagenase digestion technique and incubated for 45 min in a Krebs-Henseleit medium with or without addition of the cholinergic agonist carbachol or the C-terminal octapeptide of cholecystokinin (CCK-8) in the presence or absence of galanin. Carbachol, at its optimal concentration (10(-5) M), stimulated amylase secretion to 11.8 +/- 0.5% of total amylase content compared to 4.3 +/- 0.3% in controls (p less than 0.001). Galanin, (10(-8)-10(-9) M), reduced the carbachol-induced amylase secretion to 10.4 +/- 0.3% (p less than 0.01). Galanin at concentration levels below 10(-10) M had no significant effect. At 10(-8) M, CCK-8 stimulated amylase secretion to 9.7 +/- 0.6% compared with 5.2 +/- 0.3% in controls (p less than 0.01). Galanin (10(-7) M) reduced this stimulation to 8.0 +/- 0.4% (p less than 0.05). Galanin did not affect basal amylase secretion. It is concluded that the intrapancreatic neuropeptide galanin weakly inhibits carbachol- and CCK-8-induced amylase secretion from isolated rat pancreatic acini. Thus, galanin has the capability to directly affect not only endocrine but also exocrine pancreatic secretion although its effect of inhibiting amylase secretion seems weak.
Pancreas 1988
PMID:Galanin inhibits amylase secretion from isolated rat pancreatic acini. 246 Aug 54

Pancreas obtained from 34 adult human cadaver organ donors was divided into proximal and distal segments, and the duct to each segment was cannulated. Collagenase was injected into the proximal duct of 7 glands and into the distal duct of 7 others; the duct of the opposite segment was perfused with collagenase. The pancreas was then dispersed by teasing, trituration, and passage through filters. Perfused proximal and distal segments released 1461 +/- 287 and 2728 +/- 797 islets/g (+/- SEM) versus 710 +/- 149 (P less than 0.05) and 1950 +/- 636 after injection. Twenty other pancreases were perfused with collagenase warmed rapidly to 39 degrees C (n = 4) or warmed slowly to 37 degrees C (n = 6) or 39 degrees C (n = 10): the yield was 1625 +/- 632, 1320 +/- 116, and 2009 +/- 277 islets/g respectively. Total yields from the latter were 76 X 10(3) large (greater than 100 microns) and 85 X 10(3) small (less than 100 microns) islets with recoveries of 61% and 42%, respectively, after Ficoll density gradient purification. Histology showed highly purified islets. Perifusion with glucose elicited a biphasic release of insulin with the mean response (microU/islet/min) rising to a first peak of 0.5 and constant second phase secretion of 0.25, followed by a return to baseline. Reduced response was observed for islets from pancreas stored greater than 6 hr and tissue obtained from multiple centers. Less insulin was produced by freshly isolated islets, islets less than 100 microns, and after Ficoll separation. Secretion was similar for islets derived from proximal or distal segments. Perfusion of collagenase via the ducts of human pancreas improves islet isolation and Ficoll gradient separation yields highly purified islets. Important factors influencing insulin secretion are the source of donor tissue, cold storage of pancreas, Ficoll purification, islet size, and tissue culture.
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PMID:Studies of the isolation and viability of human islets of Langerhans. 283 43

Nine of 38 islet isolation experiments, using the duct collagenase technique, were selected for quality checks on isolated islet tissue. Pancreas was harvested, following aortic multi-organ perfusion. The total number of islets isolated amounted to 112,461 +/- 11,828 in 13.7 ml of suspension on average. In vitro secretion of beta cells was increased by a factor of 3.8 in response to glucose stimulation. Isolated islets in morphologically intact condition were detected by histological investigations. A new viability test (MTT, Sigma) for isolated pancreas islets worked well, in that it provided very soon information on islet survival in the wake of collagenase preparation. These results produced evidence to an improvement of technical conditions for clinical use of adult islet transplantation in cases of type I diabetes.
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PMID:[Isolation of islands of Langerhans from the human adult pancreas]. 314 48

Epidermal growth factor (EGF) regulates pancreatic acinar enzyme secretion. The mechanism of action of EGF in pancreatic acinar cells is not clear. In the present study we investigated the role of heterotrimeric GTP-binding proteins (G proteins) in EGF receptor signal transduction. Pancreatic acini were isolated from rat pancreas by collagenase digestion and permeabilized by digitonin. Activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) was assessed using a radioreceptor assay specific for inositol 1,4,5-trisphosphate [IP3(1,4,5)]. For measurement of amylase secretion isolated pancreatic acini were incubated with secretagogues for 30 min at 37 degrees C. Amylase released into the medium was assessed by monitoring the hydrolysis rate of p-nitrophenyl-alpha,D-maltohepatoside. The weakly hydrolyzable GTP analogue guanosine 5'-[3-O-thio]triphosphate (GTP gamma S) and guanosine 5'-diphosphate (GDP) were used to activate and inhibit G protein-mediated signal transduction, respectively. EGF (90 nM) stimulated amylase release in isolated pancreatic acini. This effect was enhanced by guanosine 5'-[3-O-thio]triphosphate (0.1 mM), which stimulates G proteins. Guanosine 5'-diphosphate (1 mM), which inhibits the activity of heterotrimeric G proteins, had no effect on basal and EGF-induced amylase release. Lower EGF concentrations (20 nM) inhibited COOH-terminal cholecystokinin octapeptide (CCK8)-induced IP3(1,4,5) production and amylase release in pancreatic acini). However, in the presence of GDP, EGF had no significant effect on CCK8-stimulated amylase release. Furthermore, coincubation of the acini with CCK8, EGF, and GDP revealed that GDP reduces the inhibitory effect of EGF on CCK8-induced IP3(1,4,5) production.(ABSTRACT TRUNCATED AT 250 WORDS)
Pancreas 1995 Apr
PMID:Epidermal growth factor receptor signaling in rat pancreatic acinar cells. 754 69


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