Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alpha-toxin, the major cytolysin of Staphylococcus aureus, preferentially attacks human platelets and cultured monocytes, thereby promoting coagulation and the release of interleukin-1 and tumor necrosis factor. Titers of naturally occurring antibodies in human blood are not high enough to substantially inhibit these pathological reactions. In the present study, F(ab')2 fragment preparations from hyperimmune globulin obtained from immunized volunteers were tested for their capacity to inhibit the cytotoxic action of alpha-toxin in vitro and in vivo. These antibody preparations exhibited neutralizing anti-alpha-toxin titers of 80 to 120 IU/ml, whereas titers in commercial immunoglobulin preparations were 1 to 4 IU/ml. In vitro, the presence of 2 to 4 mg of hyperimmune globulin per ml protected human platelets against the action of 1 to 2 micrograms of alpha-toxin per ml. Similarly, these antibodies fully protected human monocytes against the ATP-depleting and cytokine-liberating effects of 0.1 to 1 microgram of alpha-toxin per ml. Intravenous application of 0.5 mg (85 to 120 micrograms/kg of body weight) of alpha-toxin in cynomolgus monkeys elicited acute pathophysiological reactions which were heralded by a selective drop in blood platelet counts. Toxin doses of 1 to 2 mg (170 to 425 micrograms/kg) had a rapid lethal effect, the animals presenting with signs of cardiovascular collapse and pulmonary edema. Prior intravenous application of 4 ml of hyperimmune globulins per kg inhibited the systemic toxic and lethal effects of 1 mg (200 micrograms/kg) of alpha-toxin. In contrast, normal human immunoglobulins exhibited no substantial protective efficacy in vitro and only marginal effects in vivo. It is concluded that high-titered anti-alpha-toxin antibodies effectively protect against the cytotoxic actions of alpha-toxin.
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PMID:Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo. 277 80

Tissue thromboplastin probably plays an important role in the development of post-traumatic pulmonary microembolism. Infusion of purified human tissue thromboplastin in animals resulted in an intravascular coagulation and respiratory insufficiency. This could be inhibited by previous infusion of phospholipase C (PLC) from Bacillus cereus. We have studied the effects of PLC infusion on the course of post-traumatic pulmonary microembolism, induced by a high-energy (c. 700 J) missile trauma to the hind legs of pigs. The trauma resulted in a major muscular injury and an indirect femoral fracture. Untreated pigs developed intrapulmonary microemboli. The degree of microembolism in the lungs was measured quantitatively by external detection over the right lung of radiolabeled platelets and fibrin. Infusion of 80 micrograms PLC/kg/hour resulted in an accumulation of blood PLC associated with toxic reaction leading to increasing tachycardia and circulatory collapse after 10 hours. PLC infusion of 20 micrograms/kg/hour did not inhibit the pulmonary microembolism. A PLC-dose in between, viz. 40-50 micrograms/kg/hour, proved to efficiently inhibit most of the microembolism during the infusion period. Cessation of PLC infusion after 24 hours was accompanied by a later increase in pulmonary trapping of platelets and fibrin and decreases in paO2. Concomitantly there were opacities seen on chest X-rays. The results show that tissue thromboplastin is an important etiologic factor in post-traumatic pulmonary microembolism and that inhibition with phospholipase C can be of value in the prophylaxis of the syndrome.
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PMID:Effects of phospholipase C, a tissue thromboplastin inhibitor, on pulmonary microembolism after missile injury of the limb. 333 92

Haemolytic paramyxoviruses interact with cells in the following way: a potentially leaky viral envelope fuses with the plasma membrane, creating a hydrophilic pore of approximately 1 nm in diameter; this allows ions and low molecular weight compounds, but not proteins, to leak into and out of cells. Other viruses act similarly if the pH is reduced to 5. Leakage (measured by collapse of membrane potential, by movement of monovalent cations and by loss of phosphorylated intermediates from cells) is prevented by extracellular Ca2+. Ca2+ does not affect binding or fusion of virus to cells. It inhibits leakage as well as preventing it, and it aids in the recovery (i.e. the restoration of non-leakiness) of cells. Certain 'anti-Ca2+' drugs have an opposite effect. Experiments with the bee venom protein melittin, with the alpha-toxin of Staphylococcus aureus and with activated complement, show that the lesions produced by these agents, too, are sensitive to extracellular Ca2+ and to 'anti-Ca2+' drugs. The mechanisms of these effects are discussed.
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PMID:Cell damage by viruses, toxins and complement: common features of pore-formation and its inhibition by Ca2+. 393 3

Haemolysis by Sendai virus, alpha-toxin, and activated complement is inhibited by high concentrations of divalent cations. In Daudi cells, sublytic amounts of these agents induce the following changes: collapse of surface membrane potential, uptake of Na+ and loss of K+ from cells, and leakage of phosphorylated metabolites from cells. The changes induced by Sendai virus and complement are sensitive to physiological concentrations of extracellular Ca2+. It is concluded that fluctuations in plasma Ca2+ concentration may affect the damaging action of certain pore-forming agents on susceptible cells.
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PMID:Common action of certain viruses, toxins, and activated complement: pore formation and its prevention by extracellular Ca2+. 609 41

Lysophosphatidic acid (LPA) is a mitogenic phospholipid produced by certain activated cells and present in serum. LPA stimulates phospholipase C and inhibits adenylate cyclase in its target cells, apparently by activating a specific G-protein-coupled receptor. Here, we demonstrate that LPA causes transient rounding of N1E-115 and NG108-15 neuronal cells accompanied by growth cone collapse and retraction of neurites. The effect of LPA is concentration dependent, being half-maximal at 10-20 nM, and reversibly blocked by suramin, an LPA receptor antagonist. The morphological response to LPA is indistinguishable from that evoked by thrombin or a thrombin receptor-activating peptide (TRP) (K. Jalink and W. H. Moolenaar, J. Cell Biol., 118: 411-419, 1992); yet, LPA and thrombin appear to act through distinct receptors. LPA-induced shape changes, like those induced by thrombin and TRP, are driven by contraction of the cortical actin cytoskeleton and not attributable to prior phospholipid hydrolysis and Ca2+ mobilization nor to other classic second messenger systems. Instead, LPA- and TRP-induced shape changes are accompanied by a small but significant increase in p60src protein tyrosine kinase activity. Treatment of cells with pervanadate selectively inhibits LPA- and TRP-induced shape changes as well as p60src activation. These results indicate that, in N1E-115 and NG108-15 cells, LPA and TRP trigger neurite retraction and cell rounding through a novel, receptor-mediated signaling pathway, and they suggest that p60src may play a role in this pathway.
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PMID:Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action. 768 47

Clostridium perfringens type A strains which differed in alpha-toxin (phospholipase C [PLC]) productivity were inoculated intraperitoneally or intravenously into mice, and then their 50% mouse lethal doses (LD50) were determined. Strain NCTC 8237 produced ninefold higher PLC activity than strain 13. The mean LD50 for the former was 1 log unit lower than that for the latter. Two isogenic strains were constructed from strain 13: strain 13(pJIR418 alpha) (pJIR418 alpha contains the plc gene), which produced ninefold higher PLC activity than strain 13; and strain 13 PLC-, which showed no PLC productivity at all because of transformation-mediated gene disruption. The mean LD50 for strain 13(pJIR418 alpha) was 1 log unit lower than those for strain 13 PLC- and strain 13. These results indicate that PLC functions as a virulence-determining factor when it is produced in a sufficient amount. Such a difference in LD50 was also observed between Bacillus subtilis with and without the cloned plc gene. Inoculation of B. subtilis PLC+ intravenously into mice caused marked thrombocytopenia and leukocytosis. Mice inoculated with B. subtilis at 2 LD50 died because of circulatory collapse. Histological examination revealed that intravascular coagulation and vascular congestion occurred most prominently in the lungs. These results suggest that PLC plays a key role in the systemic intoxication of clostridial myonecrosis, probably by affecting the functions of platelets and phagocytes.
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PMID:Role of alpha-toxin in Clostridium perfringens infection determined by using recombinants of C. perfringens and Bacillus subtilis. 792 85

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate- and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate-sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+-ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+-ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.
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PMID:Dynamic properties of an inositol 1,4,5-trisphosphate- and thapsigargin-insensitive calcium pool in mammalian cell lines. 901 6

For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme-substrate-water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AMI-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AMI-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure.
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PMID:Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus: a molecular mechanics and molecular dynamics study. 927 25

The studies presented here explore intracellular signals resulting from the action of repellents on growth cones. Growth cone challenge with thrombin or thrombin receptor-activating peptide (TRAP) triggers collapse via a receptor-mediated process. The results indicate that this involves activation of cytosolic phospholipase A(2) (PLA(2)) and eicosanoid synthesis. The collapse response to repellents targets at least two functional units of the growth cone, the actin cytoskeleton and substratum adhesion sites. We show in a cell-free assay that thrombin and TRAP cause the detachment of isolated growth cones from laminin. Biochemical analyses of isolated growth cones reveal that thrombin and TRAP stimulate cytosolic PLA(2) but not phospholipase C. In addition, thrombin stimulates synthesis of 12- and 15-hydroxyeicosatetraenoic acid (HETE) from the released arachidonic acid via a lipoxygenase (LO) pathway. A selective LO inhibitor blocks 12/15-HETE synthesis in growth cones and inhibits thrombin-induced growth cone collapse. Exogenously applied 12(S)-HETE mimics the thrombin effect and induces growth cone collapse in culture. These observations indicate that thrombin-induced growth cone collapse occurs by a mechanism that involves the activation of cytosolic PLA(2) and the generation of 12/15-HETE.
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PMID:Thrombin-induced growth cone collapse: involvement of phospholipase A(2) and eicosanoid generation. 1059 66

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.
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PMID:Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis. 1107 55


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