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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent taxonomic studies on black-pigmented anaerobic rods, a group of bacteria found on mucosal surfaces of humans and animals, led to the subdivision of existing species and to the creation of new species. The aim of this study was to characterize all 11 currently recognized species of black-pigmented bacteria (55 strains) for their ability to hydrolyse a variety of natural and synthetic substrates and for their lectin reactivity. Although most of the strains demonstrated some activity against proteinaceous substrates, Porphyromonas gingivalis was the only species able to hydrolyse type I collagen. Most strains possessed glycylprolyl protease activity, elastase-like activity and phospholipase C activity, whereas trypsin-like activity was restricted to P. gingivalis, Porphyromonas salivosa and Bacteroides macacae. beta-Lactamase activity was demonstrated in five strains belonging to the saccharolytic group. The lectin reactivity of the bacteria was determined by a dot-blot procedure using horseradish-peroxidase-conjugated lectins. Three lectins, LOTUS A, RCA-I and ConA, failed to react with any of the bacteria tested. WGA reacted strongly with the cell surface of human biotypes of asaccharolytic black-pigmented bacteria (P. gingivalis, Porphyromonas asaccharolytica and Porphyromonas endodontalis) and Prevotella intermedia. The animal biotype strains of P. gingivalis showed a higher affinity for SBA and PNA than for WGA.
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PMID:Hydrolytic enzymes and lectin-binding activity of black-pigmented anaerobic rods. 801 4

The cell activation inhibitor CI-959 [5-methoxy-3-(1-methylethoxy)-N-1H-tetrazol-5-ylbenzo[ b]thiophene-2- carboxamide, monosodium salt] was evaluated for its effects on human neutrophil functions. CI-959 inhibited spontaneous migration and chemotaxis toward N-formyl-methionyl-L-leucyl-L-phenylalanine (fMLP) with 50% inhibition (IC50) values of 3.6 and 3.1 microM, respectively. CI-959 also inhibited superoxide anion generation in response to C5a, fMLP, serum-opsonized zymosan (SOZ), concanavalin A (Con A), and calcium ionophore A23187 with IC50 values of 2.5, 4.7, 14.5, 5.4, and 14.8 microM, respectively. In comparison, CI-959 inhibited myeloperoxidase microM, respectively. In comparison, CI-959 inhibited myeloperoxidase release in response to C5a, fMLP, SOZ, and Con A with IC50 values of 11.6, 16.1, 7.5, and < 1.0 microM, respectively, while inhibiting the response to A23187 by only 5.5% at 100 microM. At concentrations up to 100 microM, CI-959 had no effect on the respiratory burst or degranulation in response to L-alpha-1,2-dioctanoylglycerol (DiC8) or phorbol 12-myristate 13-acetate (PMA). In addition, the compound inhibited leukotriene B4 release stimulated by fMLP and SOZ (IC50 values 4.0 and 2.5 microM, respectively), while having less activity against the A23187-stimulated response (IC50 > 100 microM). These results demonstrate that CI-959 inhibits cellular responses to stimuli that mobilize intracellular calcium. For cellular responses to inophore-mediated calcium influx, only oxygen radical production was inhibited by CI-959. CI-959 was further evaluated for its effects on neutrophil stimulus-response coupling. At 100 microM, CI-959 had no effect on human neutrophil phospholipase C or protein kinase C. CI-959 inhibited fMLP-stimulated intracellular calcium mobilization and calcium influx with IC50 values of 16.7 and 3.1 microM, respectively, and exhibited less potent calmodulin antagonist activity (IC50 = 90.5 microM). These results indicate that CI-959 may exert its stimulus- and response-specific inhibitory effects on neutrophil functions, in part, through inhibition of calcium-regulated signalling mechanisms.
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PMID:Selective regulation of human neutrophil functions by the cell activation inhibitor CI-959. 814 14

Staphylococcus aureus produces a variety of proteins, including alpha-toxin and protein A, that could contribute to corneal tissue damage during keratitis. We examined corneal infections produced by intrastromal injection of four S. aureus strains--three isogenic mutants, one lacking alpha-toxin (Hly- Spa+), one lacking protein A (Hly+ Spa-), and one lacking both alpha-toxin and protein A (Hly- Spa-), and the wild type (Hly+ Spa+)--in a rabbit model of experimental keratitis. Rabbit corneas were injected intrastromally with 100 CFU of one of the four strains, and the eyes were examined by slit lamp biomicroscopy over a 25-h period. Corneal homogenates were used for determination of CFU and neutrophil myeloperoxidase activity at 5-h intervals. All strains had the same logarithmic growth curve from 0 to 10 h postinfection, after which CFU remained constant at 10(7) CFU per cornea. By 15 h postinfection, slit lamp examination scores were significantly higher for eyes infected with Hly+ strains than for Hly(-)-infected eyes. At this time, distinct epithelial erosions were seen in Hly(+)-infected eyes but not in Hly(-)-infected eyes. Myeloperoxidase activity was significantly greater for Hly(+)-infected corneas than for Hly(-)-infected corneas at both 20 and 25 h postinfection. Spa(+)- and Spa(-)-infected eyes showed no differences in slit lamp examination scores or myeloperoxidase activities. These results suggest that alpha-toxin, but not protein A, is a major virulence factor in staphylococcal keratitis, mediating the destruction of corneal tissue in eyes infected with this bacterial pathogen.
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PMID:Corneal virulence of Staphylococcus aureus: roles of alpha-toxin and protein A in pathogenesis. 818 73

Staphylococcal alpha-toxin, the prototype of an oligomerizing, pore-forming cytotoxin, is sensitive to biochemical modifications and cannot be labeled with biotin or fluorescein under preservation of its biological activity. In this study, we have used site-directed mutagenesis to introduce cysteine residues at positions 69, 130, and 186. Each mutant was fully and rapidly reactive with several sulfhydryl-specific reagents, indicating superficial location. Coupling of SH-groups with fluorescein-maleimide or biotin-maleimide was tolerated without loss of hemolytic activity at position 130, and the formed hexamers were visible on target cells by fluorescence microscopy and could be detected on electroblots by reaction with streptavidin-peroxidase. At the two other positions, modification caused significant loss of activity. However, the labeled proteins still bound to red cells, as shown by fluorescence microscopy and electroblotting. Intrinsically labeled alpha-toxin represents a novel tool to study the interaction of this pore-former with target membranes.
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PMID:Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186. 850 20

We present a new method to analyze the hydrolysis of phosphatidylcholine by either phospholipase C (PLC) or phospholipase D. The method is nonradioactive, rapid, and very sensitive and is based on chemiluminescence. It relies on the peroxidase-catalyzed chemiluminescent oxidation of luminol by the H2O2 derived from choline oxidation. The enzyme activity can be quantified by calculation of produced choline in a standard curve. Data are accurate and reproducible in a large range of choline concentration. In fact, the response of PLC to GTP in plasma membranes was similar to the activity measured with radioactive methods of diacylglycerol or phosphatidylinositol triphosphate determination. The method can be applied to study the hydrolysis of phosphatidylcholine in plasma membrane preparations and in intact cells.
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PMID:A chemiluminescence method to analyze phosphatidylcholine-phospholipase activity in plasma membrane preparations and in intact cells. 859 73

Staphylococcus aureus corneal infection results in extensive inflammation and tissue damage. Our previous studies of bacterial mutants have demonstrated a role for alpha-toxin in corneal virulence. This study analyzes, by genetic rescue experiments, the virulence of mutants affecting alpha-toxin and beta-toxin activity and demonstrates the ocular toxicity of these purified staphylococcal proteins. Three types of isogenic mutants were analyzed: (i) mutants specifically deficient in alpha-toxin (Hla) or beta-toxin (Hlb), (ii) a mutant deficient in both Hla and Hlb, and (iii) a regulatory mutant, deficient in the accessory gene regulator (agr), that produces reduced quantities of multiple exoproteins, including alpha- and beta-toxins. Plasmids coding for Hla and Hlb (pDU1212 and pCU1hlb, respectively) were used to restore toxin activity to mutants specifically deficient in each of these toxins. Either corneas were injected intrastromally with logarithmic-phase S. aureus or purified alpha- or beta-toxins were administered to normal eyes. Ocular pathology was evaluated by slit lamp examination and myeloperoxidase activity of infiltrating polymorphonuclear leukocytes. Corneal homogenates were cultured to determine the CFU per cornea. Eyes infected with the wild-type strain developed significantly greater corneal damage than eyes infected with Agr-, Hlb-, or Hla- strains. Epithelial erosions produced by parent strains were not produced by Agr- or Hla- strains. Hlb+ strains, unlike Hlb- strains, caused scleral edema. Plasmid pDU1212 restored corneal virulence to strain DU1090 (Hla-), and plasmid pCU1hlb restored corneal virulence to strain DU5719 (Hlb-). Application of purified alpha-toxin produced corneal epithelial erosions and iritis, while application of beta-toxin caused scleral inflammation. These studies confirm the role of alpha-toxin as a major virulence factor during S. aureus keratitis and implicate beta-toxin, a mediator of edema, as a lesser contributor to ocular damage.
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PMID:Specific roles of alpha-toxin and beta-toxin during Staphylococcus aureus corneal infection. 912 32

The role of polymorphonuclear leukocytes (PMN) in stemming systemic infection is executed mainly by the utilization of molecular O2 leading to the production of reactive oxygen intermediates (ROI). PMN-derived ROI also serve as intra- and extracellular second messengers providing both positive and negative feedback on cellular autoregulation. We investigated the effect of endogenous ROI on two signal transducing pathways: the receptor (R)-G-protein-phospholipase D (PLD) and receptor (R)-G-protein-phospholipase C pathways responsible for the subsequent interleukin-8 (IL-8)-induced PMN respiratory burst. Purified human PMN were primed with LPS adhered to plastic surfaces and stimulated with IL-8 with or without the presence of each of five different selective ROI scavengers/antioxidants: DMSO, N(a)N3, L-alanine, catalase, or superoxide dismutase. Total IL-8 surface receptor expression was assessed by 125I-IL-8 and 125I-labeled mAbs against IL-8R type A and B binding assays; PLD activation was assessed by measuring formation of phosphatidyl ethanol (PEt) in the presence of ethanol; PLC activation was measured by quantitative conversion of [32P]ATP-labeled phosphatidic acid (PA) into diacylglycerol (DAG); expression of G alpha-inhibitory subunit was assessed by SDS-PAGE and immunoblotting with polyclonal Abs against this subunit. Production of O2-, H2O2, HClO, and myeloperoxidase (MPO) in the experimental model was confirmed in a separate set of experiments. The overall impact of antioxidants on each component of the transducing tripartite complex was stimulatory; however, N(a)N3 and SOD exhibited the most ubiquitous effect with consistent up-regulation by N(a)N3 of IL-8R expression, whereas even trace amounts of externally added authentic MPO significantly down-regulated the functional activity of both effector enzymes. These results demonstrate a multiple site-specific targeting of the signal-transducing complex by endogenous PMN-derived ROI and an overall protective effect of ROI scavengers/antioxidants.
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PMID:Endogenous PMN-derived reactive oxygen intermediates provide feedback regulation on respiratory burst signal transduction. 926 41

Using rat pancreatic acini, we have recently shown that apical endocytosis is inhibited at pH 6.0 and progressively activated as the pH is increased to 8.3. Endocytotic activity correlated with the release of GP2, a GPI-linked protein, from the apical plasma membrane. To determine whether the cleavage of GPI-anchored proteins from the membrane of rat acinar cells was responsible for activation of endocytosis, cells at pH 6.0 were incubated with PI-specific phospholipase C (PI-PLC). PI-PLC treatment reversed the inhibition of endocytosis observed at pH 6.0. Reactivation of endocytosis correlated with PI-PLC-induced release of GP2 but not cleavage of phospholipids in cellular membranes. Furthermore, administration of diacylglycerol or phorbol esters had no effect on reactivation of endocytosis. PI-PLC did not alter intracellular pH or calcium levels. Two proteins were identified as GPI-linked proteins on the cell surface. One was GP2, whose release from the apical plasma membrane correlated with apical endocytosis of horseradish peroxidase (HRP). The other protein, identified by Western blotting using an antibody directed against a cryptic determinant exposed on GPI-linked proteins after cleavage with PI-PLC, has a molecular weight of 98000 in nonreducing SDS gels and 54000 in reducing SDS gels. By nondenaturing gel electrophoresis and staining with naphthylphosphate, this protein was found to be alkaline phosphatase. In contrast to GP2, alkaline phosphatase was not endogenously released at pH values of 7.4 or 8.3, conditions that activate endocytosis of HRP under physiological conditions. By electron microscopic evaluation, incubation of cells at pH 6.0 with PI-PLC led to induction of HRP uptake into vesicles at the apical pole of the cell, a reduction in apical plasma membranes, and a concomitant contraction of the acinar lumen space. Internalized HRP accumulated in the Golgi region of the cell. These results suggest that the cleavage of GPI-anchored proteins from the apical plasma membrane activates apical endocytosis.
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PMID:Cleavage of GPI-anchored proteins from the plasma membrane activates apical endocytosis in pancreatic acinar cells. 954 73

Stable transformation of Rat-1 fibroblasts by the v-Src oncoprotein results into the constitutive formation of macropinosomes. In the present report, we found that macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer horseradish peroxidase at intervals >5 minutes in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate or the phosphatidylinositol 3-kinase inhibitor wortmannin, dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of tracer entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited peroxidase accumulation only after intervals >5 minutes, due to accelerated peroxidase regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom.
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PMID:Regulation of macropinocytosis in v-Src-transformed fibroblasts: cyclic AMP selectively promotes regurgitation of macropinosomes. 968 28

The role of the inflammatory cytokine interleukin 1beta (IL-1beta) as potent agonist of the PMN respiratory burst signal transduction cascade has been described. We hypothesized that this phenomenon is self-limiting and that polymorphonuclear leukocyte (PMN)-derived reactive oxygen intermediates (ROI) might provide feedback regulation on the IL-1beta surface receptor (IL-1betaR)-G-protein-effector enzyme transducing tripartite complex that ultimately leads to NADPH oxidase activation. Therefore, we separately assessed either baseline or IL-1beta-induced activation of each member of the IL-1betaR-G-protein-phospholipase D (PLD) or IL-1betaR-G-protein-phospholipase C (PLC) signaling systems in the presence or absence of one of several specific ROI scavengers/antioxidants. Purified human PMN were lipopolysaccharide primed, adhered for 2 h, and stimulated with 100 ng/mL IL-1beta with or without 1% v/v dimethyl sulfoxide, 10 mM NaN3, 30 mM L-alanine, 200 U catalase, or 300 U superoxide dismutase (SOD). To validate the use of these antioxidants, the production of O2-, H2O2, hypochlorous acid, or myeloperoxidase (MPO) in the employed experimental model was confirmed in a separate set of experiments. The expression of IL-1betaR type I or II was assessed by binding with corresponding 125I-labeled monoclonal antibodies and corrected for nonspecific binding. PLD activation was assessed by measuring phosphatidyl ethanol formation in the presence of ethanol. PLC activation was determined by quantitative measurement of diacylglycerol. The level of Galpha stimulatory and inhibitory subunits was assessed by Western blotting. IL-1betaR type I expression was significantly up-regulated in the presence of catalase and SOD. PLD activation was increased by dimethyl sulfoxide and NaN3, and PLC activation was up-regulated by NaN3, L-alanine, SOD, and catalase. After 5 min of stimulation with IL-1beta, Gialpha expression was significantly down-regulated by NaN3 and SOD, whereas SOD had an up-regulating effect on the expression of Gs alpha. Increasing concentrations of externally added authentic MPO progressively down-regulated both PLD and PLC activity. Thus, PMN-derived ROI, in addition to their role as antibacterial/fungal agents, serve as second messengers in IL-1beta signal transduction, with MPO having the most ubiquitous role as a modulator of PMN second messenger pathways.
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PMID:The role of neutrophil-derived oxidants as second messengers in interleukin 1beta-stimulated cells. 968 92


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