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)

In an attempt to develop an improved method for preserving the pancreas prior to islet isolation, the effects of warm and cold ischemia were examined on rat pancreases, from which reproducible high yields of islets can be obtained when fresh. Both warm and cold preservation rapidly decreased islet yield. Use of Hanks' or modified Sacks' solution also led to marked decrease in islet yield. After 6 hrs of preservation, the islet yield was 1/5-1/10 of those of fresh pancreases (374 +/- 74, n = 14), and no islets were obtained after 24 hrs of preservation regardless of the preservation solution. Monitoring of ductal pressure during forced injection of Hanks' solution in 6 hrs-preserved pancreas with HBSS showed a significantly earlier and lower peak of pressure than those of fresh pancreases. On the other hand, simple hypothermic preservation after pancreatic ductal distention with collagenase Hanks' solution at the time of harvesting resulted in a significantly higher islet yield up to 6 hours (171 +/- 58, n = 14, P less than 0.01), as compared with conventional methods. The viability of the islets isolated by this method was confirmed by the ability to restore normoglycemia of STZ-induced diabetic B6 mice on transplantation of 400 islets in the renal subcapsular space. These findings indicated that loss of the integrity of the ductal system against forced collagenase injection during cold preservation led to poor distention and digestion of the pancreas, ductal collagenase injection at the time of pancreas harvesting followed by simple preservation is recommended to obtain viable islets from the preserved pancreas.
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PMID:Successful islet isolation from preserved rat pancreas following pancreatic ductal collagenase at the time of harvesting. 196 77

Previous studies have shown that the yield and viability of islets obtained from human or large mammal pancreas depends upon techniques used for islet isolation and upon factors that affect the quality of the donor pancreas. In the present studies, the efficacy of collagenase digestion by ductal perfusion or automated techniques was compared in a canine model of purified islet isolation. The ductal perfusion technique was then developed for human pancreas which was excised before (n = 8) or after (n = 8) multiple organ procurement and without in situ arterial perfusion of hypothermic preservation solution or significant cold storage. Studies with canine pancreas showed that ductal perfusion yielded 2.0 +/- 0.7 microL of islets/g of pancreas and when combined with automated digestion, yields improved to 3.6 +/- 0.8 microL/g (versus perfusion alone, not significant). The yield and viability of human islets was improved when the pancreas was excised before procurement of other donor organs. Results of islet isolation from eight consecutive human pancreases procured in this manner revealed a total yield of 355.2 +/- 44 x 10(3) islets, corresponding to 5345 +/- 600 islets/g (+/- SEM, mean islet diameter 150 microns). Six of eight Ficoll purification attempts succeeded, yielding 186.6 +/- 31 x 10(3) islets of purity ranging from 45-60%. Perifusion with glucose elicited biphasic insulin secretion with a three-fold rise. Islets from two of the isolations were utilized to initiate a clinical trial of islet transplantation in insulin-dependent diabetes mellitus.
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PMID:Isolation of purified large mammal and human islets of Langerhans. 196 80

The efficiency of Eurocollins or modified University of Wisconsin (UW) solution (MUW) in preserving rat livers was compared. After cold storage with one of the solutions, the livers were transplanted or perfused by collagenase for isolation of hepatocytes. Five of the 6 rats receiving a graft preserved with MUW versus none of the 6 rat receiving a graft preserved with Eurocollins solution survived 24 h or more. A significantly greater number of hepatocytes were isolated from livers preserved with MUW than from livers preserved with Eurocollins solution. This suggests a better reperfusion of MUW-preserved livers by collagenase resulting from less endothelial injury. LDH release by cultured hepatocytes, ketone body production and stimulation by glucagon were not significantly different between the two groups. These results confirm the superiority of MUW solution over Eurocollins in preserving liver grafts. They suggest that the advantage of MUW solution results from better protection of vascular endothelium rather than of hepatocytes.
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PMID:Comparison of rat liver preservation with Eurocollins and a modified University of Wisconsin solution: transplantation and isolation of hepatocytes for culture. 207 86

A subpopulation of cells was derived from the Hs431 connective tissue sarcoma cell line which possessed high affinity (estimated Kd = 0.38-0.55 nM) binding sites for human recombinant [125I]-IL-1 alpha. Binding at 4 degrees C was slow approaching equilibrium by 4 hrs. Dissociation of [125I]-IL-1 alpha was also slow and unaffected by high concentrations of cold ligand. The binding site also underwent ligand-induced internalization at 37 degrees C. An Mr = 83,000 protein was identified in affinity crosslinking studies. Despite these similarities to previously reported IL-1 receptors, Hs431 cells did not exhibit biological responses to IL-1 which have been observed in other cell lines. IL-1 did not induce PGE2 or collagenase synthesis. IL-1 also failed to induce ornithine decarboxylase activity (ODC) or stimulate [3H]-thymidine incorporation. In contrast, the Hs431 cells did contain a functional epidermal growth factor (EGF) receptor as determined from binding studies, protein kinase activity, induction of ODC, and stimulation of [3H]-thymidine incorporation. Thus, the refractoriness of Hs431 cells to IL-1 was fairly specific and did not result from a generalized defect associated with cell transformation.
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PMID:Characterization of a high affinity interleukin-1 (IL-1) specific binding site in a human synovial sarcoma (Hs431) cell line. 216 29

A simple technique for the controlled collagenase digestion of the human pancreas is described. The pancreas is distended with collagenase, and a biopsy taken and divided into 5 pieces that are placed in Universals containing minimal essential medium and dithizone at 39 degrees C. The pancreas itself is incubated in MEM at 39 degrees C. Starting at 5 min and at intervals thereafter, a Universal is removed from the water bath, shaken for 30 sec, and the contents examined by microscopy. As soon as free cleaved islets are seen, the pancreas is placed into one compartment of a kidney-bowl divided in half by a 1-mm mesh. The pancreas is gently teased apart and fluid digest in the empty half of the bowl aspirated and passed through a 500-micron mesh into ice-cold MEM containing 20% newborn calf serum. This process is repeated until the digestion process has ceased. Using this technique on 20 consecutive pancreata, median wt. (range) 53.9 (45.2-72.9) g, we have counted 131,672 (43,516-400,000) islets in the digest, equivalent to 2394 (715-8000) islets/g pancreas. The volume of islet tissue in the digest was 299 (26-1341) mm3 equivalent to 5.81 (0.36-26.81) mm3/g pancreas. In conclusion, we have found this simple technique to be an effective method for the controlled collagenase digestion of the human pancreas.
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PMID:A simple method for the release of islets by controlled collagenase digestion of the human pancreas. 216 34

To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5-30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20-30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.
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PMID:Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron. 216 56

Rat livers were perfused and stored for 48 hr in cold University of Wisconsin solution before dissociation by the two-step collagenase method. At that time, glycogen content was significantly reduced, but no obvious changes in albumin, beta-actin and aldolase B mRNAs and in glutathione levels were observed. Enzymatic perfusion yielded 280 +/- 30 x 10(6) viable hepatocytes vs. 520 +/- 40 x 10(6) viable hepatocytes from unstored organs. Cell viability determined by trypan blue exclusion was 74% and 90%, respectively. Hepatocytes from University of Wisconsin-preserved livers had a 29% reduced adenosine triphosphate content, but glutathione levels did not significantly differ from those found in unstored cells. When put into culture, hepatocytes formed typical monolayers of granular epithelial cells and did not exhibit alteration of their fine structure when compared with cells from unstored organs. After 24 and 48 hr, they showed variations in cytochrome P-450 content and ethoxyresorufin O-deethylase activity similar to those observed with unstored cells. By contrast, overall protein synthesis and albumin secretion rate were 40% and 30% lower, respectively. Hepatocytes from University of Wisconsin-preserved organs could be cryopreserved and further cultured as unstored cells. The University of Wisconsin solution was also used to preserve isolated hepatocytes. Viability of freshly isolated hepatocytes was decreased by only 10% after 48 hr of hypothermic liver storage when assayed by intracellular lactate dehydrogenase content. However, after 4 hr of storage, in contrast with hepatocytes preserved in L15 Leibovitz medium, the cells attached poorly to plastic and exhibited morphological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Primary culture of adult rat hepatocytes after 48-hour preservation of the liver with cold UW solution. 225 48

Utilizing an intrasplenic canine islet autotransplant model, the effects of cold storage preservation on pancreatic tissue prior to and after collagenase dispersion were examined. A control series, in which freshly retrieved and prepared tissue was transplanted, yielded a 75% success rate (6/8). In contrast, when the pancreas was stored in modified silica gel filtered plasma (SGF I) for 24 hr, no autotransplant was successful (0/6). However, when the islet tissue was prepared following pancreatectomy and then stored in a mannitol-containing modification of SGF (SGF III), autotransplantation was successful in 83% (5/6) after 24 hr of preservation and in 60% (3/5) after 48 hr of preservation. Similarly, the islet tissue was stored in a hyperkalemic hydroxyethyl starch solution (HES) and this was successful in 20% (1/5) after 24 hr of preservation and in 50% (1/2) after 48 hr of preservation. Cold storage preservation techniques for the pancreas prior to islet isolation need to be refined, but dispersed islet-enriched pancreatic tissue can be successfully maintained at 4 degrees C for up to 48 hr prior to transplantation in dogs using established pancreas preservation solutions.
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PMID:Cold storage preservation of pancreatic tissue prior to and after islet preparation in a dog autotransplantation model. 247 12

Cold-storage preservation of the canine pancreas prior to islet isolation has previously been noted to reduce the intrasplenic islet autograft success rate; but the mechanism of this deleterious effect has not been determined. We undertook a study in both outbred dogs and Lewis (RT1-1) rats to determine the influence of cold-storage preservation interval, preservation solution, and flushing technique on islet yield and islet viability. The preservation solutions used were those that had proved most efficacious in preserving segmental canine pancreases--namely, the modifications of silica gel fractionated plasma (SGF-III and SGF-IV) and an hydroxyethylstarch/lactobionate solution (UW-1). In the first set of experiments, the traditional vascular flush was used; this was followed by storage at 4 degrees C. After brief periods of preservation (3 hr in the rat, 12 hr in the dog) there was a significant (P less than 0.006) reduction in islet yield. The reduced yields were similar with each solution tested, were made worse with increasing intervals of storage, and resulted in a significant reduction in autograft success rate. The second set of experiments examined the effect of using an intraductal flush prior to preservation, along with the effect of adding collagenase to the preservation fluid. Islet yields were maintained at control values in both animal models using preservation intervals of up to 24 hr. These islet yields produced auto- or isograft success rates similar to those obtained by transplanting freshly obtained tissue; verifying adequate islet viability. We recommend that a pre-storage ductal flush technique be used for cold-storage preservation of the pancreas prior to islet isolation and transplantation.
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PMID:Cold-storage preservation of the canine and rat pancreas prior to islet isolation. 249 30

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4 degrees C for 0, 3, 12, 24, and 48 hr (n = 16-21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (+/- standard errors of the means) were controls = 819 +/- 58 (n = 21), 3 hr = 867 +/- 51 (n = 20), 12 hr = 770 +/- 71 (n = 16), 24 hr = 805 +/- 62 (n = 18), and 48 hr = 722 +/- 55 (n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 +/- 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose greater than 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose less than 200 mg/dl) were 100% after storage times of 0 hr (n = 10), 3 hr (n = 8), and 12 hr (n = 8); 86% with a storage time of 24 hr (n = 7); and 0% after 48 hr (n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Experimental pancreatic preservation prior to islet isolation and transplantation. 249 69


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