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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Leukocyte adhesion may play a central role in the pathogenesis of preservation-reperfusion injury to liver grafts. We previously showed that lymphocyte adhesion to sinusoids is dependent on the length of cold ischemia. In the present study we examined the mechanisms of lymphocyte adherence after harvesting combined with a short and a long preservation time. The effects of lymphocyte adherence on liver function were also examined. Rat livers were stored at 1 degrees C in University of Wisconsin solution for 45 min or 30 hr and then reperfused at 37 degrees C in the isolated perfused rat liver with isogeneic lymphocytes in an asanguineous perfusate. The role of reactive oxygen intermediates was investigated with allopurinol, a vitamin E analog and ascorbate or superoxide dismutase and catalase. For us to determine the role of Kupffer cells, Kupffer cell blockade was produced by gadolinium chloride. Leukotriene B4 effects were examined with the lipooxygenase inhibitor, nordihydroguaiaretic acid. We evaluated the possible presence of mechanical obstruction by studying flow rates and the circulation of red blood cells. We examined the role of adhesion molecules by pretreating lymphocytes with trypsin or neuraminidase and by exposing livers to arabinogalactan. We investigated the effects of lymphocyte adhesion on liver function by comparing perfusate liver enzymes in livers reperfused with and without lymphocytes, with trypsinized lymphocytes and with an increased number of lymphocytes. Allopurinol significantly reduced hypoxanthine degradation, and nordihydroguaiaretic acid inhibited leukotriene B4 release into the perfusate. The ability of gadolinium chloride to inhibit Kupffer cells was shown by colloid carbon uptake. In livers harvested and preserved for 45 min, lymphocytes decreased about 40% during reperfusion. In livers preserved for 30 hr, the reduction was significantly greater (about 80%). Lymphocyte adherence was lessened in livers preserved for 45 min by all three of the reactive oxygen intermediate protectants and by gadolinium chloride. In contrast, neither reactive oxygen intermediate protectants nor gadolinium chloride reduced adherence in livers preserved for 30 hr. Nordihydroguaiaretic acid had no effect in livers preserved for either 45 min or 30 hr. Portal flow in livers preserved for 45 min and 30 hr was similar, suggesting an absence of mechanical obstruction, and this finding was supported by a complete absence of red cell trapping. Trypsinization of lymphocytes and exposure of livers to arabinogalactan significantly lessened lymphocyte adherence in livers preserved for 30 hr but not in those preserved for 45 min.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Lymphocyte adherence in the reperfused rat liver: mechanisms and effects. 838 Jul 89

ATP-sensitive K+ current (IK.ATP) channels are thought to play a role in the K+ efflux observed in cardiac ischemia. Intracellular acidosis is a prominent early effect in ischemia; therefore, the effects of acidosis on IK.ATP may have certain pathophysiological implications. Increased intracellular proton concentration (pHi) is known to regulate IK.ATP in frog skeletal muscle by increasing open probability. The pHi effect on IK.ATP is not clearly understood in heart because, unlike frog skeletal muscle, low pHi causes IK.ATP run-down in inside-out patches. This would tend to mask any opening effect of low pHi if it exists. Trypsin modification of IK.ATP has recently been shown to prevent run-down in inside-out patches. We used single channel recordings in inside-out patches to study IK.ATP after exposure to trypsin. After trypsin treatment, the open probability of IK.ATP was not sensitive to pHi in the absence of ATP. In the presence of ATP, however, a decrease in pHi consistently increased the open probability of trypsin-modified IK.ATP by reducing ATP inhibition. In the absence of ATP the mean open probability was 0.43 +/- 0.07 at pHi 7.4, and 0.5 mM ATP decreased the mean open probability to 0.03 +/- 0.04 at pHi 7.4, but mean open probability was significantly increased to 0.20 +/- 0.07 at pHi 6.3 (n = 7, p < 0.01). Ca2+ did not affect the activity of trypsin-modified IK.ATP in either the absence or presence of ATP at pHi 7.4. However, Ca2+ was able to antagonize the low pHi effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intracellular H+ and Ca2+ modulation of trypsin-modified ATP-sensitive K+ channels in rabbit ventricular myocytes. 838 26

Endothelial cells have ectonucleotidases that rapidly catabolize extracellular nucleotides. Our aim was to study whether the metabolism of extracellular nucleotides and adenosine are influenced by exposure of endothelial cells to reactive oxygen metabolites at concentrations relevant to human pathology. Human umbilical vein endothelial cells were incubated with hypoxanthine (100 microM) and xanthine oxidase (80 mU/ml), to generate superoxide, or with hydrogen peroxide (100 microM). The cells were then washed, and the metabolism of radioactive substrates was followed. After exposure to hypoxanthine-xanthine oxidase the half time of disappearance of [14C]ATP (5 microM) was prolonged from 9.9 +/- 5 to 28.3 +/- 15.6 min and that of [14C]AMP from 9.5 +/- 2.5 to 25.0 +/- 9.9 min. The conversion of extra- into intracellular nucleotides via adenosine was also decreased (mean for [14C]ATP 0.25 vs. 0.90 and for [14C]AMP, 0.075 vs. 0.75 nmol/10(6) cells in 30 min compared with parallel controls, respectively). Hydrogen peroxide or trypsin had no significant effect on the metabolism of extracellular adenine nucleotides and neither did a short (up to 15 min) exposure to the superoxide-generating system. The conversion of [14C]adenosine into intracellular nucleotides and hypoxanthine was not influenced by either hypoxanthine-xanthine oxidase or by hydrogen peroxide. We conclude that superoxide radicals inhibit the catabolism of extracellular adenine nucleotides by the ectonucleotidases of endothelial cells and may thus modify the pathophysiology of ischemia-reperfusion injury.
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PMID:Metabolism of extracellular adenine nucleotides by human endothelial cells exposed to reactive oxygen metabolites. 844 61

We have characterized the distribution of immunoreactive epidermal growth factor (irEGF) in control and ischemia-injured rat kidneys. Kidneys that had undergone ischemic injury contained levels of soluble irEGF that were six times those of uninjured kidneys. The predominant forms of soluble irEGF were native and des-Arg-epidermal growth factor (EGF), both of which are biologically active. Crude membrane fractions from whole kidneys were solubilized in Triton X-100 and tested for irEGF. Amounts of irEGF were slightly decreased in the ischemia-injured kidney membranes. However, when solubilized membrane fractions were digested with trypsin, which generates a single immunoreactive species which appears identical to native EGF, the amount of irEGF in control fractions increased 13-fold and the amount in injured fractions increased only 4-fold as measured by radioimmunoassay. To better characterize the membrane-associated irEGF, Triton X-100-solubilized membrane fractions from control animals were affinity purified and subjected to high-performance liquid molecular sieve chromatography. Three major peaks of material exhibited immunoreactivity to EGF antibodies, bound the EGF receptor, and stimulated [3H]thymidine incorporation in growth-arrested fibroblasts. Trypsin digestion of the two high-molecular-mass peaks enhanced these activities. The third peak eluted with native EGF and showed no change in activity with trypsin addition. We propose that EGF is released from membrane-associated EGF precursors and can then act in an autocrine or paracrine fashion to promote cell growth after ischemia-induced acute renal failure.
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PMID:Increased soluble EGF after ischemia is accompanied by a decrease in membrane-associated precursors. 845 64

A porcine pancreatic transplantation model was used to investigate possible protease activation in the pancreatic graft during preservation. After perfusion with Perfadex and cold ischemia for 24 h, but prior to reperfusion, activated carboxypeptidase B was demonstrated in tissue samples from the graft parenchyma with a Western blot technique, indicating that graft pancreatitis may already be initiated during the preservation phase. A higher degree of carboxypeptidase B activation was observed in grafts perfused at a pressure of 130 cm H20 than after perfusion at 70 cm H20. During reperfusion, the fraction of activated carboxypeptidase B gradually declined but was still detectable after 2 h. One group of pigs received aprotinin intravenously during reperfusion, but the protease inhibitor did not influence the degree of carboxypeptidase B activation in the biopsy specimen. Immunoblotting against cationic trypsinogen/trypsin was also performed. When activated trypsin was detectable, it never presented more than a few percent of the total amount of uncomplexed immunoreactive trypsinogen/trypsin.
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PMID:Protease activation in the porcine pancreatic allograft during preservation. 857 79

Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100-treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca(2+)-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pre-treated with 1 mmo1/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 mumol/L per 30 mIU/mL) or H2O2. pMgATP-rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the absence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03 +/- 0.17 (n = 21) to 4.22 +/- 0.22 (n = 4) after 25 minutes of incubation in the presence one of 30 mIU/mL. XO and 100 mumol/L xanthine or to 4.04 +/- 0.1 (n = 11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced on increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.
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PMID:Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils. 863 32

Very recent experimental data, obtained by using the permeabilized cell technique or tissue homogenates for investigation of the mechanisms of regulation of respiration in the cells in vivo, are shortly summarized. In these studies, surprisingly high values of apparent Km for ADP, exceeding that for isolated mitochondria in vitro by more than order of magnitude, were recorded for heart, slow twitch skeletal muscle, hepatocytes, brain tissue homogenates but not for fast twitch skeletal muscle. Mitochondrial swelling in the hypo-osmotic medium resulted in the sharp decrease of the value of Km for ADP in correlation with the degree of rupture of mitochondrial outer membrane, as determined by the cytochrome c test. Very similar effect was observed when trypsin was used for treatment of skinned fibers, permeabilized cells or homogenates. It is concluded that, in many but not all types of cells, the permeability of the mitochondria outer membrane for ADP is controlled by some cytoplasmic protein factor(s). Since colchicine and taxol were not found to change high values of the apparent Km for ADP, the participation of microtubular system seems to be excluded in this kind of control or respiration but studies of the roles of other cytoskeletal structures seem to be of high interest. In acute ischemia we observed rapid increase of the permeability of the mitochondrial outer membrane for ADP due to mitochondrial swelling and concomitant loss of creatine control of respiration as a result of dissociation of creatine kinase from the inner mitochondrial membrane. The extent of these damages was decreased by use of proper procedures of myocardial protection showing that outer mitochondrial membrane permeability and creatine control of respiration are valuable indices of myocardial preservation. In contrast to acute ischemia, chronic hypoxia seems to improve the cardiac cell energetics as seen from better postischemic recovery of phosphocreatine, and phosphocreatine overshoot after inotropic stimulation. In general, adaptational possibilities and pathophysiological changes in the mitochondrial outer membrane system point to the central role such a system may play in regulation of cellular energetics in vivo.
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PMID:On the regulation of cellular energetics in health and disease. 890 74

Gut-origin sepsis is a serious medical complication of military injuries following hemorrhage. Splanchnic ischemia induces intestinal necrosis leading to systemic bacteremia. Rat and mouse models of hemorrhagic shock were used to investigate bacterial translocation from the gut. Orally administered ameliorative treatments using the cytokine interleukin-6 (IL-6) were able to reduce or eliminate sepsis following hemorrhage. To mimic battlefield wounds and hemorrhage, anesthetized mice were bled from the femoral artery, held at a mean arterial blood pressure of 35 mm Hg for 1 hour, and then resuscitated with shed blood and 2-fold volume lactated Ringer's solution. Anesthetized rats were bled from the carotid artery at a rate of 15 ml/kg at 1 ml/minute. Bacteriological cultures of livers and mesenteric lymph nodes from hemorrhaged animals given recombinant IL-6 had significantly fewer colonies per gram of tissue than saline-fed controls. 125I-labeled IL-6 remained in the gut for up to 6 hours giving regional protection, whereas labeled interleukin-2 was disseminated throughout the body in the same time. In vivo and vitro studies of IL-6 showed that long incubations with high doses of trypsin, chymotrypsin, or intestinal contents were necessary to inactivate the bioactivity of this cytokine. Electron microscopy showed that epithelial cells from hemorrhaged mice fed saline had sparse or missing villi and vacuolated cytoplasm. Epithelial cells from control mice or mice hemorrhaged and fed cytokine appeared completely normal. Oral administration of IL-6 on the battlefield may be an important treatment for the prevention of sepsis following hemorrhage.
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PMID:Systemic sepsis following hemorrhagic shock: alleviation with oral interleukin-6. 915 11

Activated mast cells are present in human coronary atheromas, as well as in the adventitia of patients with variant angina, and may play an important role in plaque rupture and coronary vasomotion. To assess whether or not activation of mast cells is a primary event, we measured serum levels of tryptase, a specific marker of mast cell activation, in 8 patients with unstable angina during a spontaneous ischemic episode (Group 1) and in 5 patients with variant angina (Group 2) during ergonovine-induced coronary spasm. Blood samples were collected as soon as possible after the onset of pain and ECG changes (0 min), and after 5, 15 and 60 min. Tryptase levels in Group 1 were 0.13 U/l (range 0.017-0.44) at the onset of pain and significantly raised to 0.75 U/l (range 0.05-2.49) at 5 min, decreasing to 0.076 U/l (range 0.018-0.16) at 15 min and to 0.085 U/l (range 0.01-0.25) at 60 min (p = 0.035). Conversely, tryptase levels in Group 2 were 0.09 U/l (range 0.07-0.13) at 0 min, 0.11 U/l (range 0.07-0.22) at 5 min, 0.10 U/l (range 0.07-0.18) at 15 min, 0.11 U/l (range 0.07-0.17) at 60 min (NS). In conclusion, tryptase levels raise during spontaneous ischemic episodes in unstable angina, but not after ergonovine-provoked ischemia in variant angina, suggesting that a primary, yet unknown stimulus, may activate mast cells during some ischemic episodes in unstable angina.
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PMID:[Tryptase levels are elevated during spontaneous ischemic episodes in unstable angina but not after the ergonovine test in variant angina]. 955 75

We report the average insulin response to acute glucose measured by in vitro perifusion of pancreatic islets isolated from 80 consecutive human organs. Different perifusion parameters were considered [basal release, stimulation index (SI), time to peak, incremental area under the curve delta-AUC alpha)], and the correlation among them was determined. SI positively correlated with delta-AUC alpha (p < 0.001, r = 0.80) while negatively with time to peak (p < 0.05, r = -0.23). We also evaluated several variables of the isolation procedure that might affect responsiveness to glucose by human islets. Sex and age of pancreas donors, cold ischemia time, duration of the digestion, collagenase concentration, and lot characteristics (collagenase, trypsin, clostripain, and proteases activity), and final islet yield were considered. Multivariate regression analysis showed only an independent association between SI and the concentration of collagenase (p = 0.01).
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PMID:Lessons from in vitro perifusion of pancreatic islets isolated from 80 human pancreases. 1070 99


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