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:3.1.21.1 (DNase)
7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

More efficient methods of islet isolation must be developed for islet transplantation to become clinically routine. During collagenase dispersal of human pancreas, an amorphous, viscous, gellike material often develops and entraps large numbers of islets, thereby reducing the yield. When donor human pancreas is minced and treated with collagenase, the gel forms most abundantly if the digestion temperature is less than 35 degrees C and if pH falls below 7.2 +/- 0.2. Gel formation appears to be proportional to warm- or cold-ischemia time and may be related to tissue trauma during collection. Once gel has formed, trapped islets cannot be released by filtration, dilution, DNase, incubation temperature, or pH adjustment. These characteristics suggest that the material is gelatin derived from collagen released enzymatically from pancreatic stroma. We demonstrate that gelation is greatly reduced or eliminated when 1) the incubation medium includes glycerol--a common gelatin solvent--at 5% (vol/vol), 2) the minced tissue-to-total incubation volume ratio is greater than or equal to 1:10, 3) free-islet exposure to pancreatic digestion products is minimized by frequent separation of islets, and 4) collagenase concentration is optimized by titration. Gelation is also minimized by maintaining 5) incubation temperature at 38 +/- 1 degree C and 6) pH in the range 7.7-7.9. Variations in these physical and chemical conditions were analyzed by determining islet yields (stereoscopic microscope counts of serially diluted samples) and by insulin radioimmunoassay of acid alcohol extracts of isolated islets after separation through discontinuous Ficoll gradients. When isolation conditions are optimized as stated, we typically recover 3.3 +/- 1.0 x 10(4) islets/g pancreas, corresponding to greater than 10(6) islets per donor.
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PMID:Factors influencing isolation of islets of Langerhans. 264 35

Extracellular acidosis has been shown to be protective during ischemia in renal tubule cells. However, the mechanism of protection remains unknown. Since ischemia leads to disruption and polymerization of the cortical actin cytoskeleton, we hypothesized acidosis may better preserve the actin cytoskeleton during ischemia. Therefore, the purpose of our studies was to examine the effect of pH on the integrity of the actin cytoskeleton during ATP depletion and ATP repletion. To do this, we used an in vitro model of reversible ATP depletion in LLC-PK1 cells at extracellular pH values (pHo) of 6.9, 7.4, and 7.9. Immunofluorescent studies with rhodamine-phalloidin demonstrated more marked redistribution and clumping of cortical actin at pHo 7.9 and 7.4 vs. 6.9 after 90 min of chemical anoxia. After 15 min of ATP depletion, G-actin, quantified by the deoxyribonuclease assay, decreased from 53.7 +/- 0.8 to 43.2 +/- 1.5 microgram/mg protein at pHo 6.9 vs. 37.6 +/- 1.8 microgram/mg protein at pHo 7.4 (P < 0.001). After 60 min, there still was significantly less conversion of G-actin to F-actin at pHo 6.9 vs. 7.4, with a decrease from 55.9 +/- 2.0 to 39.6 +/- 2.0 micrograms/mg protein at 6.9 vs. 35.8 +/- 2.4 at 7.4 micrograms/mg protein (P < 0.05). Furthermore, extracellular acidosis during the phase of ATP repletion resulted in more rapid normalization of cellular G-actin levels (95 +/- 3% of control vs. 82 +/- 2% for pH 6.9 vs. 7.4, respectively, P < 0.01). Together, these findings indicate the actin cytoskeleton is better preserved in an acidic environment during ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Extracellular acidosis minimizes actin cytoskeletal alterations during ATP depletion. 794 55

The activity of deoxyribonucleases was altered in the myocardium of New Zealand rabbits under normal conditions, in diabetes mellitus as well as in various experiments, where model heart perfusion was carried out using media of various ion composition and pH value. Studies of Ca2+, MG(2+)- dependent deoxyribonuclease from myocardial nuclear extracts exhibited that protective cell mechanisms appear t be induced at the first steps of tissue ischemic impairments as well as that diabetes decreased cardiac sensitivity to ischemia.
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PMID:[Nuclear endo-DNAse from rabbit myocardium in ischemia and diabetes mellitus]. 858 87

The small heat-shock proteins appear to have a regulatory role in actin dynamics. Since cytoskeletal disruption is integral to ischemic renal injury, we evaluated expression and intracellular distribution of heat-shock protein 25 (HSP-25) in rat renal cortex after 45 min of renal ischemia. HSP-25 was constitutively expressed and induced by ischemia with peak levels reached by 6 h reflow. Ischemia caused a shift of HSP-25 from the detergent-soluble into the insoluble cytoskeletal fraction. By 2 h reflow, the majority of HSP-25 had redistributed into the soluble fraction. HSP-25 was predominantly localized in a subapical distribution in control proximal tubules, a pattern intermediate between deoxyribonuclease (DNase)-reactive and filamentous actin. After ischemia, HSP-25 dispersed through the cytoplasm with small punctate accumulations similar to DNase-reactive actin. During later reflow, all three proteins were found in coarse intracytoplasmic accumulations; however, HSP-25 and DNase-reactive actin were in separate accumulations. HSP-25 and microfilamentous actin staining returned to the subapical domain. Thus the temporal and spatial patterns of HSP-25 induction and distribution suggest specific interactions between HSP-25 and actin during the early postischemic reorganization of the cytoskeleton. HSP-25 may have additional roles distinct from actin dynamics later in the course of postischemic recovery.
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PMID:Heat-shock protein 25 induction and redistribution during actin reorganization after renal ischemia. 945 42

Diadenosine polyphosphates (ApnA) have been recently discovered in the heart, and their levels found to be regulated by ischemia. These signaling molecules are believed to regulate cellular processes that alarm a cell to metabolic stress. In particular, changes in cardiac diadenosine polyphosphates (ApnA) levels may contribute to the regulation of ATP-sensitive K+ (K(ATP)) channel activity, an ion channel that couples the cellular metabolic state with membrane excitability. A feature of myocardial ischemia is the disruption of the actin cytoskeleton which critically regulates the behavior of K(ATP) channels. Whether the integrity of actin microfilaments regulates the interaction of ApnA with K(ATP) channels is not known. The inside-out configuration of the patch-clamp technique was applied to cardiomyocytes isolated from guinea-pig heart. Following patch excision, the prototype dinucleotide, diadenosine tetraphosphate (Ap4A), inhibited K(ATP) channel opening. Treatment of the internal side of membrane patches with either cytochalasin B or DNase I, disrupters of the actin cytoskeleton, prevented Ap4A-induced inhibition of K(ATP) channel opening. Application of purified actin to DNase-treated membrane patches restored the ability of Ap4A to close K(ATP) channels. This study shows that inhibition of cardiac K(ATP) channel by Ap4A, a putative alarmone, requires intact subsarcolemmal actin network. Such interaction between K(ATP) channels, the cardiomyocyte cytoskeleton and intracellular Ap4A could affect different channel-dependent functions.
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PMID:Diadenosine tetraphosphate-gating of cardiac K(ATP) channels requires intact actin cytoskeleton. 1152 Nov 71

Deoxyribonucleic acid fragmentation at nucleosomal junctions is a hallmark of neuronal apoptosis in ischemic brain injury, for which the mechanism is not fully understood. Using the in vitro cell-free apoptosis assay, the authors found that caspase-3-dependent deoxyribonuclease activity caused internucleosomal DNA fragmentation in brain-cell extracts in a rat model of transient focal ischemia. This in vitro deoxyribonuclease activity was completely inhibited by purified inhibitor of caspase-activated deoxyribonuclease protein, the specific endogenous inhibitor of caspase-activated deoxyribonuclease, or by caspase-activated deoxyribonuclease immunodepletion. The induction of the deoxyribonuclease activity was correlated with caspase-3 activation and caspase-3-mediated degradation of inhibitor of caspase-activated deoxyribonuclease. Furthermore, inhibiting caspase-3-like protease activity prevented the endogenous induction of internucleosomal DNA fragmentation in the ischemic brain. These results suggest that caspase-3-dependent caspase-activated deoxyribonuclease activity plays an important role in mediating DNA fragmentation after focal ischemia.
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PMID:Induction of caspase-activated deoxyribonuclease activity after focal cerebral ischemia and reperfusion. 1180 89

Ischemia/reperfusion is known to result in DNA fragmentation and cell death in kidney tubular epithelium, but the endonucleases responsible for this DNA damage have not been identified. DNA substrate gel analysis of extracts from normal rat kidney cortex revealed the presence of a DNase with an apparent molecular mass of 30 to 34 kD. This enzyme is not a dimer of the previously described nuclear 15-kD endonuclease in kidney cells. Partially purified DNase exhibited characteristics similar to those of rat DNase I. The DNase was able to digest circular DNA (endonuclease), required both Ca(2+) and Mg(2+) ions, and was inhibited by Zn(2+) and by aurintricarboxylic acid; it was not inhibited by G-actin. Rat kidneys were subjected to 40 min of ischemia, followed by 0, 1, 4, 16, or 48 h of reperfusion. The activity of the DNase in cytosolic and nuclear extracts, the 200-bp ladder-generating activity, and 3'OH strand breaks in nuclear DNA were simultaneously increased after ischemia, during the first hours of reperfusion. Oxidative DNA damage, measured as 8-hydroxydeoxyguanosine content, did not coincide with endonuclease-generated DNA breaks. Oxidative DNA damage was increased during ischemia and gradually decreased during reperfusion. Phosphorothioated DNase I antisense oligodeoxynucleotide introduced into cultured NRK-52E rat kidney epithelial cells inhibited DNA fragmentation and attenuated cell death induced by hypoxia/reoxygenation in vitro. The data indicate that the DNase I-like endonuclease may contribute to DNA fragmentation in reperfused rat kidneys.
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PMID:DNase I-like endonuclease in rat kidney cortex that is activated during ischemia/reperfusion injury. 1191 59

Apoptosis is an evolutionarily conserved process critical to tissue development and tissue homeostasis in eukaryotic organisms and, when dysregulated, causes inappropriate cell death. Global ischemia is a neuronal insult that induces delayed cell death with many features of apoptosis. Ischemic preconditioning affords robust protection of CA1 neurons against a subsequent severe ischemic challenge. The molecular mechanisms underlying ischemic tolerance are unclear. Here we show that ischemia induces pronounced caspase-3 activity in naive neurons that die and in preconditioned neurons that survive. Preconditioning intervenes downstream of proteolytic processing and activation of caspase-3 (a protease implicated in the execution of apoptosis) and upstream of the caspase-3 target caspase-activated DNase (CAD, a deoxyribonuclease that catalyzes DNA fragmentation) to arrest neuronal death. We further show that global ischemia promotes expression of the pro-survival inhibitor-of-apoptosis (IAP) family member cIAP, but unleashes Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI), a factor that neutralizes the protective actions of IAPs and promotes neuronal death. Preconditioning blocks the mitochondrial release of Smac/DIABLO, but not the ischemia-induced upregulation of IAPs. In the absence of Smac/DIABLO, cIAP halts the caspase death cascade and arrests neuronal death. These findings suggest that preconditioning preserves the integrity of the mitochondrial membrane, enabling neurons to survive in the face of caspase activation.
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PMID:Ischemic preconditioning: neuronal survival in the face of caspase-3 activation. 1502 68

We have previously demonstrated that ischemia caused by acute myocardial infarction induces an abrupt increase of serum deoxyribonuclease I (DNase I) activity. In this study, we examined whether hypoxia can affect the levels of DNase I activity and/or its transcripts in vitro. We first exposed the human pancreatic cancer cell line QGP-1, which is the first documented DNase-I-producing cell line, to hypoxia (2% O2), and found that this induced a significant increase in both the activity and transcripts of DNase I. This response was mediated by increased transcription only from exon 1a of the two alternative transcription-initiating exons utilized simultaneously in the human DNase I gene (DNASE1); exposure of QGP-1 cells to hypoxia for 24 h resulted in a 15-fold increase of DNASE1 transcripts starting from exon 1a compared with the expression level under normoxic conditions. Promoter, electrophoretic mobility shift, and chromatin immunoprecipitation assays with QGP-1 cells exposed to hypoxia or normoxia showed that the region just upstream from exon 1a was involved in this response in a hypoxia-induced factor-1-independent, but at least in a Sp1 transcription factor-dependent manner possibly through enhanced binding of Sp1 protein to the promoter. These results indicate that DNASE1 expression is upregulated by hypoxia in the cells.
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PMID:Hypoxia induces upregulation of the deoxyribonuclease I gene in the human pancreatic cancer cell line QGP-1. 1791 Sep 90

Ischemia and seizure cause excessive neuronal excitation that is associated with brain acidosis and neuronal cell death. However, the molecular mechanism of acidification-triggered neuronal injury is incompletely understood. Here, we show that asparagine endopeptidase (AEP) is activated under acidic condition, cuts SET, an inhibitor of DNase, and triggers DNA damage in brain, which is inhibited by PIKE-L. SET, a substrate of caspases, was cleaved by acidic cytosolic extract independent of caspase activation. Fractionation of the acidic cellular extract yielded AEP that is required for SET cleavage. We found that kainate provoked AEP activation and SET cleavage at N175, triggering DNA nicking in wild-type, but not AEP null, mice. PIKE-L strongly bound SET and prevented its degradation by AEP, leading to resistance of neuronal cell death. Moreover, AEP also mediated stroke-provoked SET cleavage and cell death in brain. Thus, AEP might be one of the proteinases activated by acidosis triggering neuronal injury during neuroexcitotoxicity or ischemia.
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PMID:Neuroprotective actions of PIKE-L by inhibition of SET proteolytic degradation by asparagine endopeptidase. 1837 43


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