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 lack of a technique that allows mass isolation of intact, viable human islets is part of the reason that islet transplantation has not become available to the human diabetic. This report outlines the history of islet isolation and presents two new technical modifications that have been developed in the dog. Many of the current problems in islet isolation are presented, including the difficulty in obtaining enough human pancreatic tissue with minimal warm-ischemia time; inadequate distention of the pancreas to provide sufficient disruption for maximal enzymatic reaction to release intact islets; inefficient chopping methods; the use of collagenase of variable composition; different digestion methods for obtaining isolated islets; and inefficient methods for separating and purifying the islets from the ductal, acinar, and fibrous components. The first new modification involves distention of the dog pancreas through the venous system of the gland rather than the ductal system. This results in improved intralobular disruption, which improved the yield of isolated dog islets by permitting more efficient collagenase digestion. The second new modification eliminates the concept of isolating intact islets: the dog pancreas is digested by trypsin to a single-cell preparation that is partially purified by Ficoll gradients; further purification of the endocrine cells results from selective aggregation using rotational culture. This process produces pseudoislets that contain all the islet cell types and can be kept in culture for up to 4 wk, releasing their hormones in response to appropriate stimuli. These modifications may assist in the struggle to isolate the elusive human islet for safe and effective islet transplantation in the diabetic patient.
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PMID:Isolating the elusive islet. 624 91

To assess the possibility of using hepatocytes from ischemic liver, as a bioreactor of a hybrid artificial liver, we investigated the influence of warm ischemia on the isolation and culture of hepatocytes in rats. Warm ischemia was induced by clamping the liver hilus and the animals were divided into 3 groups according to the duration of ischemia: group A (no ischemia), group B (10 minutes) and group C (20 minutes). Hepatocytes were isolated by the collagenase perfusion method and cultured for 5 days. The yield and viability of the isolated hepatocytes were lower in group C. Rate of attachment was decreased as the duration of ischemia increased. There was no significant difference observed in functions in culture. Sufficient hepatocytes, as a bioreactor, can be isolated and cultured from warm ischemic liver within 10 minutes. Though the number of available hepatocytes were diminished, hepatocytes procured from longer warm ischemic liver could be utilized as a bioreactor.
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PMID:Influence of warm ischemia on isolation and primary culture of hepatocytes from rat liver for a hybrid artificial liver. 760 54

Sinusoidal cells were isolated by collagenase perfusion and metrizamide gradient centrifugation, from liver resected for partial hepatectomy performed under warm ischemic conditions. Kupffer cells were then separated from this population by centrifugal elutriation. Isolated Kupffer cells showed good viability, with the typical features of Kupffer cells and were engaged in the endocytosis of foreign particles. They showed numerous morphological criteria of activation. However, cultured Kupffer cells were no longer in an activated state. Kupffer cells were preserved in maintenance cultures for 2 weeks. Purity of these cultures was to 93-97%. During culture, Kupffer cells retained their ultrastructural characteristics and were active in the endocytosis of latex beads and opsonized zymosan particles. It is thus possible that partial hepatectomy performed under warm ischemia could provide valuable material for the study of Kupffer cells in vitro.
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PMID:Ultrastructure of human Kupffer cells maintained in culture. 775 43

The early pathogenetic steps that finally lead to acinar cell necrosis in acute pancreatitis have been characterized only scarcely as yet. Among a lot of hypotheses, one concept favors disturbances of cellular energy metabolism as a major factor that contributes to preterm cell decline. To investigate, whether an experimental acute pancreatitis alters cell respiration, the respiratory capacities of acinar cells isolated from rats with acute pancreatitis were measured. Acute pancreatitis was induced using Popper's model, i.e., a combination of duct obstruction, secretory stimulation, and mesenteric short-term ischemia with subsequent reperfusion. Acinar cells were isolated using a collagenase digestion technique. The respiratory rates of the isolated cells in suspension were measured at 37 degrees C in 100% oxygen-saturated N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid-buffered Eagle's-minimal essential medium. Resting respiration of the acinar cells uniformly amounted to about 60 pmol of O2/s x 10(6) cells in both the control and the pancreatitis group. Cellular respiration could significantly be stimulated by stepwise uncoupling of oxidative phosphorylation by means of 2,4-dinitrophenol in all cell suspensions investigated. The maximum rate of stimulated respiration was diminished in the cells isolated from rats with acute pancreatitis as compared with the controls (79.3 +/- 5.0 vs. 160.2 +/- 15.5 pmol of O2/s x 10(6) cells, p < .05), however. This reduced respiratory load capacity of the acinar cells in acute pancreatitis reflects the restricted ability of the cells to increase respiration on enhanced cellular demand. Since mitochondrial respiration is coupled to oxidative phosphorylation, an altered energy-transforming potential of the acinar cells in acute pancreatitis becomes evident.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Acinar cell respiration in experimental acute pancreatitis. 777 97

Due to the complexity of interacting organ systems, in vivo the interpretation of results obtained from whole-animal experiments of acute pancreatitis remains difficult. To enlighten cause-and-effect-relationships, functional isolated parts of the pancreas are applied increasingly in research into the pathogenesis of the disease, therefore. By means of a collagenase digestion technique, intact acinar cells from normal as well as from pretreated rat pancreas could reproducibly be obtained in high yield. Animals were pretreated in situ by induction of either mesenteric ischemia/reperfusion, juice edema, or acute pancreatitis (AP). Pancreatic acinar cells isolated from these pretreated rats consumed oxygen at comparable rates under resting conditions throughout all experimental groups. A reduced stress capacity of cell respiration as well as a preterm decline in short-term culture was observed in the cells of the AP group, however. These results demonstrate that the induction of AP has found its reflection within the acinar cells themselves. Interestingly, these effects were not clouded by the isolation procedure. The manner of in-situ pretreatment of the respective animal proved to be decisive for later viability of isolated acinar cells in short-term culture. Uncoupling of oxidative phosphorylation by 2,4-dinitrophenol (DNP) lead to an accelerated decline of the acinar cells in all groups. The intactness of cellular energy metabolism seems to play a crucial role in maintaining cellular integrity, therefore. In additional experiments in vitro, the intracellularly mediated cell damage by DNP was compared with one induced from the extracellular environment of isolated cells by antibodies directed against cell surface antigens plus complement.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Mechanisms for cell damage in acute pancreatitis using isolated acinar cells]. 784 69

One of the major steps toward successful islet transplantation for the treatment of type diabetes is to obtain islets of sufficient number and viability. Using a standardized method of isolating islets, the goal of this study was to analyze the factors influencing the outcome of islet isolation. A total of 104 cadaveric human pancreata were processed for islets by the same team. Data from the islet-processing charts were reviewed retrospectively. The two endpoints were the recovery of islets, viable after 2 days of culture (group V = viable, group NV = nonviable) and the islet yield. Viable islets were recovered in 61% of cases (n = 63). Minimal blood glucose recorded during hospitalization was very significantly lower in group V (124 +/- 5 vs. 148 +/- 9, P = 0.01). Lack of significant medical history in the donor was associated with better viability as compared with various donor predispositions (chi-2 4.21, P = 0.04). Cold ischemia time (8.1 +/- 0.5 hr in group V vs. 9.8 +/- 0.9 hr in group NV, P = 0.07) and collagenase lot (5 lots tested, chi-2 13.1, P = 0.01) also affected the recovery of viable islets. Hospital time was shorter in group V (65.3 +/- 6.8 vs. 80.9 +/- 17.9 hr, P = 0.35). Multivariate logistic regression analyses of viable islet recovery identified minimal blood glucose (P = 0.03) and collagenase lot (P = 0.06) as the most significant risk factors. However, the best multivariate predictive model--which includes blood glucose, collagenase lot, donor age and surgical procurement team--correctly predicted 66.2% of cases only. Multivariate analysis of final islet yield designed hospitalization length, cardiorespiratory arrest, surgical procurement team, and collagenase lot as the best predictors. These data obtained in a large series of pancreata emphasized several donor and technical factors that should target the attention of islet transplant researchers in order to improve islet yield and viability.
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PMID:Human islet isolation in 104 consecutive cases. Factors affecting isolation success. 801 87

Inflammatory reactions induce the production of reactive oxygen species (ROS): the reverse sequence of these events is also true. Moreover, many components of these reactions interact with a synergistic effect. In this short comprehensive review we analyze some of these interactions which may have pathological effects. Inflammatory reactions are triggered off by exogenous or endogenous aggressions and are characterized by cellular and vascular events. The activated leucocytes leave the circulating blood and reach the site of the aggression where they release a large amount of ROS as well as the content of their granules. The granular content is made in a large part by molecules with killing and degradative activities such as myeloperoxidase, defensins, elastase, collagenase, cathepsins and lysozyme. The inflammatory reaction is beneficial for humans when its effects are limited to the pathogens. The insufficiency of a component of the inflammatory reaction such as the production of ROS which is seen, for example in chronic granulomatous disease, leads to severe and recurrent bacterial infections. In other situations inflammatory reactions are deleterious because they are directed against normal tissues instead or in addition to pathogens. In some cases the behaviour of the phagocytes is modified because they have been primed by inflammatory molecules such tumor necrosis factor, LPS, interleukins or interferons. Priming often leads to a decreased speed of locomotion of the leucocytes with an increased susceptibility to their stimuli. The combination of these effects leads to a premature release by the phagocytes of their killing and degradative factors. Production of ROS such as that seen during irradiation, drug metabolism, or ischemia followed by reperfusion for example, induces inflammatory reactions with a secondary amplification of ROS production. Acute ROS production can also lead to thrombosis, whereas chronic ROS production can induce a chronic inflammatory reaction of the endothelium with atherosclerosis as a possible consequence. Some examples are also given to show that ROS might control positively or negatively the activity of inflammatory molecules. The multiplicity of the cross reactions between ROS and inflammation allows to suggest that drugs that disconnect these two events might be therapeutically used.
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PMID:[Reactive oxygen species and inflammation]. 801 8

University of Wisconsin solution has become the most commonly used vascular perfusate during multiorgan donation world-wide. In the UK however, hyperosmolar citrate remains in common use. The purpose of this prospective randomized study was to compare the effect of systemic perfusion with UW or HOC on subsequent islet yield and purification for pancreata with short cold ischemic times. Seven pancreata were randomized to each group, with the donor age, pancreas weight, and period of cold ischemia being similar in both. Perfusion with UW was shown to inhibit collagenase digestion, and a higher concentration of this enzyme was needed to achieve comparable numbers of islets with good separation of exocrine and islet tissue after a similar period of digestion. There were no differences in the number, size, purity, or viability of islets between the two groups. In conclusion, UW solution offers no benefits over HOC for pancreata with short cold ischemic times, and because of its expense and need to use greater amounts of collagenase enzyme, we continue to use HOC.
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PMID:Human islet isolation--a prospective randomized comparison of pancreatic vascular perfusion with hyperosmolar citrate or University of Wisconsin solution. 821 48

To use cultured human hepatocytes as a hybrid artificial liver, effective methods for isolating and culturing the hepatocytes from resected surgical specimens were investigated. Two different procedures for isolating hepatocytes, perfusion and agitation with a collagenase solution (Method 1) and perfusion with a mixed solution of collagenase and dispase (Method 2), were examined. The yield of isolated hepatocytes obtained by Method 2 (13.31 x 10(6) cells/g of liver) was significantly higher than that by Method 1 (0.94 x 10(6)). The warm ischemia time (0-90 min) of the liver fragments obtained did not disturb the viability and yield of the isolated hepatocytes. The gluconeogenesis and urea synthesis of the cultured human hepatocytes were well preserved for 10 days. These results show that for prolonged human hepatocyte culture (10 days), isolation from resected human liver tissues by a combination of the proteolytic enzymes collagenase and dispase was effective and warm ischemia was tolerated for up to 90 min, which indicates the possibility of using cultured human hepatocytes as a hybrid artificial liver.
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PMID:Isolation and culture of human hepatocytes from resected liver tissue as a bioreactor for a hybrid artificial liver. 833 42

Lysophosphatidylcholine (LPC) increases extracellularly during ischemia in vivo in both animals and man as judged by measurements from venous effluents, but more recent studies have shown little or no increase in buffer-perfused, isolated heart preparations. The appearance of LPC in blood and lymph in animals and in venous effluents in man in response to ischemia suggests a vascular site for the production of LPC. The present study was performed to assess whether thrombin could stimulate phospholipase A2 in endothelial cells and whether this would evoke an increase in and release of LPC. Endothelial cells were disassociated from canine aortas by incubating with 0.1% collagenase for 20 min. Cells were plated and allowed to grow to confluence. Measurement of LPC was performed using Bligh and Dyer extraction of lipids, high performance liquid chromatography separation, and quantification of LPC using a recently developed radiometric assay employing [3H]acetic anhydride. Incubation of endothelial cells with thrombin (0.05 unit/ml) resulted in a 2.5-fold increase in LPC to 2.3 +/- 0.1 nmol/mg of protein at 2 min (p < 0.01) and returned to control levels within 20 min. The increase in LPC induced by thrombin exhibited a concentration-dependent response with an ED50 = 0.04 unit/ml. A concentration-dependent increase in LPC was also elicited by stimulation with the peptide portion of the thrombin receptor's tethered ligand SFLLRNPNDKYEPF with an ED50 = 8 microM. The LPC produced was rapidly and completely released into the surrounding media. Hirudin completely blocked the thrombin-induced increase in LPC. Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (0.1 microM), which rapidly inactivates thrombin's proteolytic activity in situ without impairing binding, or phenyl-prolyl-arginyl-chloromethyl ketone (PPACK, 5 nM), which inactivates thrombin due to chemical alteration of the proteolytic site, each prevented the increase in LPC in response to thrombin. Stimulation of protein kinase C with phorbol 12-myristate-13-acetate (PMA, 1 microM) enhanced the response to thrombin. In contrast, staurosporine (100 nM), H7 (15 microM), or chronic treatment with PMA for 20 h to down-regulate protein kinase C completely prevented the increase in LPC in response to thrombin. Thus, thrombin stimulation of endothelial cells in vivo during ischemia may be a primary mechanism contributing to the marked increase in LPC extracellularly during ischemia.
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PMID:Thrombin-induced release of lysophosphatidylcholine from endothelial cells. 839 49


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