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)

Guinea pig megakaryocytes were isolated from femoral marrow and cultured in the presence of radioactive amino acids. Radioactivity was incorporated into several proteins including a 42 000 dalton polypeptide identified as actin by DNAase agarose affinity chromatography. Quantitative immunoelectrophoresis of megakaryocyte extract revealed that 3.0% of the total solubilized cellular protein was fibrinogen. Immunoabsorption studies using anti guinea pig fibrinogen beads failed to reveal the presence of newly synthesized radioactive fibrinogen in the cellular extract, however, radioactive actin was detected in the eluates obtained from the immune beads. When guinea pig fibrinogen was clotted with thrombin in the presence of radioactive megakaryocyte extract, a complex formed between a high molecular weight species of fibrin and actin. No actin fibrinogen complex was detected. The results suggest that actin synthesized by megakaryocytes complexes with fibrin formed from a relatively large pool of non-radioactive intracellular fibrinogen.
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PMID:Synthesis of actin by cultured guinea pig megakaryocytes. Complex formation with fibrin. 10 Dec 53

Occlusive vascular diseases are promoted by a "prethrombotic state" with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow-cytometric bio-assay and the related change in the actin status by using the DNase-I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F-actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein "actin" and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow-cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.
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PMID:Flow-cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets. 169 96

Vascular injury and microvascular thrombosis are prominent features of systemic lupus erythematosus, as are circulating DNA-binding antibodies (DNAb). Experimental glomerulonephritis can be induced by anti-endothelial cell antibodies, and polyreactive DNAb might be pathogenetic by binding to endothelial cells, perhaps influencing their non-thrombogenic nature. To test this hypothesis, eight monoclonal antibodies (mAb) that bind to DNA derived from (NZB x NZW)F1 or MRL/Mp-lpr/lpr mice, were tested for their ability to bind to human umbilical vein endothelial cells (HUVEC). Binding was assessed using flow cytometry, fluorescence microscopy and cellular ELISA. Three of the eight mAb, at concentrations employed in this study, bound to HUVEC and dermal fibroblasts. Of these three mAb, one bound also to platelets. Two of the three demonstrated strong binding to (1) freshly isolated, collagenase-digested HUVEC, (2) 2nd passage HUVEC in suspension after trypsinization and, (3) 2nd passage HUVEC growing on plastic plates. To determine whether DNA itself acted as a ligand in this binding, prior treatment with DNAase was studied. Treatment of the endothelial cells with DNAase had no effect on the binding of one mAb, but DNAase treatment of this monoclonal itself resulted in a 60% reduction in binding to HUVEC, suggesting that the binding might be mediated through DNA in the form of a DNA/anti-DNA immune complex. In contrast, DNAase digestion of the endothelial cells caused a 40% reduction in the binding of the other two monoclonal antibodies. Furthermore, one of the two mAb bound 30% more to HUVEC after themselves being subjected to DNAase treatment. These two monoclonals may therefore be binding directly to HUVEC, possibly to DNA associated with the membrane. Prior DNAase digestion of dermal fibroblasts had a more profound effect on the binding of all three autoantibodies compared to HUVEC after similar treatment. Therefore, DNA can bind independently to either antibody or cell, thus supporting build up of complexes and capture of preformed complexes. Functionally, the binding of mAb to HUVEC did not influence thrombin-induced prostacyclin synthesis, in contrast to a control monoclonal anti-endothelial cell antibody EN4, which did.
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PMID:A role for DNA in anti-DNA antibodies binding to endothelial cells. 191 Apr 25

The status of platelet actin has been studied analytically by the DNase-I inhibition assay. Exposure of platelets to diamide, which oxidizes sulphydryl groups, results in an increase of filamentous actin. Neutralization of the effect of diamide by addition of 2-mercaptopropionylglycine (2-MPG) or washing out the excess of diamide is associated with a normalization of the cellular actin status. These findings strongly suggest that the redox state of platelets is somehow involved in the process of actin polymerization. Alterations of the redox state in metabolically altered platelets could therefore account for changes in the G- to F-actin equilibrium. In the thrombin-induced actin polymerization the redox state of the cell seems to be not involved, since 2-MPG preincubation of platelets (1.25 mM, 30 min) does not inhibit the filament assembly.
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PMID:The role of the actin status in platelet behaviour. 246 44

Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3H]borohydride method and lysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) Ib. Approximately 70% of the platelet GP Ib was present in this skeleton. Several other minor glycoproteins, including greater than 50% of the GP Ia and small amounts of three unidentified glycoproteins of Mr greater than 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actin-binding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and co-isolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP Ib were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 Mr on the plasma membrane.
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PMID:Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib. 293 70

Colloidal gold is an electron-dense, lyophobic colloid that readily forms a stable electrostatic interaction with a variety of macromolecules. Monodispersed colloids ranging from 3-150 nm in diameter can be produced to provide the researcher with flexibility in selecting the optimally sized probe. Gold labeling of antibodies and lectins has been extensively used to study surface antigens and cell components. Recently, the use of gold labeling has been extended to study receptor-ligand binding, enzyme-substrate reactions, and transcellular pathways. Published applications include gold labeling of metabolites (low-density lipoproteins), enzymes (DNAase and RNAase, RNA polymerase, thrombin, collagenase, elastase), hormones (insulin, epidermal growth factor, glucagon), circulating plasma proteins (asialoglycoprotein, alpha 2-macroglobulin, factor VIII-von Willebrand factor), and endotoxins (tetanus toxin, cholera toxin). This broad spectrum of applications emphasizes the versatility and usefulness of colloidal gold as a probe in areas of cell biology related to receptors, endocytosis, transport, and functions of proteins.
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PMID:Colloidal gold: a pluripotent receptor probe. 635 33

Human umbilical vein endothelial cells (HUVEC) in primary confluent cultures lost their normal polygonal shape and assumed a 'contracted' appearance as judged by phase contrast microscopy when exposed to highly purified bovine thrombin (2 N.I.H. u/ml). Total actin in thrombin-exposed cells did not differ from that of control cells, as measured by the deoxyribonuclease I inhibition assay. However, the monomeric actin pool (unpolymerized actin) in thrombin-treated HUVEC was c. 15% smaller (P less than 0.01) than in control HUVEC (in which it represented approximately 50% of total actin). Transmission electron microscopy showed that thrombin-stimulated HUVEC contained more and thicker bundles of filamentous actin than control cells. Polymerization of actin and reorganization of actin microfilaments may contribute to the shape changes of HUVEC induced by thrombin.
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PMID:Actin pools and actin microfilament organization in cultured human endothelial cells after exposure to thrombin. 654

The temporal changes in human platelet actin polymerization, cytoskeletal morphology, and protein content that occur during thrombin-induced platelet activation were investigated by analysis of Triton-extracted platelets. Measurement of the DNase inhibitory activity of control platelets immediately after adding an equal volume of 2% Triton X-100, 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 0.1 M Tris, pH 7.4, showed that approximately 50% of the actin in unstimulated platelets was filamentous and unable to inhibit DNase-catalyzed hydrolysis of DNA. Activation of platelets with thrombin for 15 s caused the amount of actin in the filamentous form to increase to approximately 65%. Examination of the morphology and protein composition of these filamentous structures showed that the cytoskeletal structures from control platelets consisted of a random array of filaments which contained 14% of the total platelet myosin and 6% of the total actin-binding protein. In contrast, the cytoskeletal structures of thrombin-activated platelets appeared as cytoskeletal structures of individual platelets. The composition of these cytoskeletons varied depending on the time of thrombin activation. Those from platelets activated with thrombin for 15 to 30 s contained 90% of the platelet myosin and 20% of the platelets with thrombin before Triton addition resulted in a decreased association of myosin to 60% with no change in either the actin or actin-binding protein content of the cytoskeletal structures. Since these changes are rapid and precede serotonin secretion, it is suggested that they are involved in the physiological response of the platelet.
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PMID:Changes in the cytoskeletal structure of human platelets following thrombin activation. 689 99

Cytochalasin E (CE) was used in this study to evaluate relationships between platelet shape change, pseudopod extension, internal transformation, actin filament assembly, fibrinogen receptor expression, aggregation, clot tension and secretion. Various ratios of G- to F-actin, measured by the DNase inhibition assay, were achieved by using several concentrations of CE in stirred and unstirred thrombin-stimulated platelets. An increased rate of actin polymerization was found in "aggregating" conditions as compared to "activating" conditions, the latter achieved by either absence of stirring or stirring in the presence of the tetrapeptide Arg-Gly-Asp-Ser (RGDS). Larger concentrations of CE were required in aggregated than in activated platelets to obtain G-actin levels analogous to those in resting platelets. Concentrations of polymerizing actin above 20% and 55% for activated and aggregated platelets, respectively, trigger platelet shape change and the extension of pseudopods. Prevention of de novo actin polymerization by CE, added either before or after thrombin stimulation, inhibits fibrinogen binding to platelets. This suggests an involvement of the new actin polymers in fibrinogen receptor exposure. As expected, platelet aggregation and clot tension, which are dependent on fibrinogen receptor exposure, were inhibited by CE. Granular secretion was not dependent on polymerizing actin.
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PMID:Role of actin in platelet function. 792 78

The three prominently phosphorylated 29-kD proteins in thrombin-activated human platelets are forms of the mammalian 27-kD heat-shock protein (HSP27). Though the function of HSP27 is not yet known, its phosphorylation is highly correlated with platelet secretion, and recent evidence in nonhematopoietic cells suggests that HSP27 regulates cortical actin filament assembly. Therefore, the subcellular location and phosphorylation state of HSP27 in resting and thrombin-activated platelets was studied. Platelets were fractionated by established Triton X-100 lysis methods followed by differential centrifugation to obtain the 14,000g fraction (low-speed cytoskeleton), 100,000g fraction (membrane skeleton), and the 100,000g supernatant fraction containing soluble cytosolic proteins. In resting platelets, HSP27 was present principally in the 100,000g supernatant fraction. Platelet activation with thrombin led to translocation of the majority of HSP27 to the low-speed cytoskeleton. This association was reversible by DNase, supporting the idea that HSP27 is a specific component of the actin cytoskeleton. Immunofluorescence studies similarly showed HSP27 is cytoplasmic in resting platelets but colocalizes with actin in fully spread, glass-activated platelets. Immunoprecipitation studies showed a small amount of constitutively phosphorylated HSP27 in resting platelets, but phosphorylation of the majority of HSP27 after thrombin activation. After activation, virtually all phosphorylated HSP27 was found in the low-speed cytoskeletal fraction, and each of the three phosphorylated forms of HSP27 were present by two-dimensional autoradiography. Furthermore, in time-course studies, the phosphorylation of HSP27 occurred just before localization of HSP27 to the low-speed pellet. These results show that, after platelet activation, HSP27 is first phosphorylated and then translocated from the cytoplasm to the assembling cytoskeleton, and suggest that HSP27 phosphorylation may be important to the binding of HSP27 to cytoskeletal components and the cytoskeletal rearrangements characteristic of platelet activation.
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PMID:Phosphorylated HSP27 associates with the activation-dependent cytoskeleton in human platelets. 794 27


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