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)

We have previously shown that 3F8, a murine IgG3, monoclonal antibody (MoAb) specific for the ganglioside GD2, mediates tumor cell kill in vitro and in vivo. We now describe receptor requirements of polymorphonuclear leukocytes (PMN) in 3F8-mediated cytotoxicity (ADCC) of human GD2 (+) melanoma and neuroblastoma cell lines. PMN from a child with leukocyte adhesion deficiency (LAD) were devoid of CD11/CD18 adhesion molecules and mounted no detectable ADCC. MoAb to CD11b, CD11c, and CD18 each efficiently blocked ADCC by normal PMN. In contrast, a panel of different MoAbs to CD11a had no significant inhibitory effect on ADCC, a finding consistent with the low-to-absent expression of the CD11a ligand, intercellular adhesion molecule-1, on the target cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the expression of CD11b, CD11c, and CD18 on normal PMN, decreased the expression of Fc receptors (FcR), and enhanced ADCC by normal but not by LAD PMN. MoAbs to FcRII and FcRIII each efficiently blocked ADCC; anti-FcRI MoAb had no effect. Flow cytometry using anti-FcRII MoAb versus anti-FcRIII MoAb did not show cross competition, suggesting that inhibition of ADCC was not a steric effect resulting from FcRII proximity to FcRIII. PMN deficient in FcRIII (obtained from patients with paroxysmal nocturnal hemoglobinuria) and PMN depleted of FcRIII by treatment with elastase or phosphatidylinositol (PI)-specific phospholipase C produced low ADCC, supporting a role for the PI-liked FcRIII. Thus, optimal ADCC using human PMN, human solid tumor cells, and a clinically active MoAb (conditions that contrast with the heterologous antibodies and nonhuman or nonneoplastic targets used in most models of PMN ADCC) required CD11b, CD11c, FcRII, and the PI-linked FcRIII. Furthermore, in this clinically relevant system, GM-CSF enhancement of antitumor PMN ADCC correlated with increased expression of CD11/CD18 molecules.
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PMID:Absolute requirement of CD11/CD18 adhesion molecules, FcRII and the phosphatidylinositol-linked FcRIII for monoclonal antibody-mediated neutrophil antihuman tumor cytotoxicity. 134 7

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) both stimulates hematopoietic precursor cells to grow as well as enhances the function of mature effector cells, such as neutrophils, eosinophils and macrophages. All of the biological actions of GM-CSF appear to be mediated via binding to a single class of high-affinity receptors present on all responsive cells. Affinity cross-linking experiments demonstrate that the same 98 kDa cross-linked species seen on other GM-CSF-responsive cells is also detected on a choriocarcinoma cell line, JAR. However, JAR cells express significantly increased numbers (10,000 sites/cell) of low-affinity (Kd approximately 1.5 nM) GM receptors. The GM-CSF receptor is a glycoprotein which binds to wheat germ agglutinin-sepharose. It is dramatically downregulated on neutrophils by phorbol esters and formyl-methionyl-leucine-phenylalanine (fMLP), but not by phosphatidylinositol-dependent phospholipase C. GM-CSF primes neutrophils for enhanced response to secondary stimuli, such as ionophore and chemotactic factors. Specifically, GM-CSF enhances 3H-arachidonic acid release, synthesis of leukotriene B4 and platelet activity factor in response to fMLP and the calcium ionophores.
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PMID:GM-CSF: receptor structure and transmembrane signaling. 215 79

The generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF). Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked approximately 100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production. GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)P2-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance of PdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence of ethanol. The formation of PEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism of action of GM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.
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PMID:Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM-CSF): priming of human neutrophils in vitro with GM-CSF is associated with accumulation of phosphatidic acid and diradylglycerol. 220 47

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF) enhances the release of newly synthesized PAF as measured by [3H]acetate incorporation into PAF in human neutrophils. The response was dose-dependent, rapid, transient, and inhibitable by the PAF antagonist BN-52021. The non-metabolizable bioactive PAF analogue (C-PAF) but not lyso-PAF enhances the release of newly synthesized PAF. Newly synthesized PAF was also released after stimulation of these cells with fMet-Leu-Phe. The human granulocyte-macrophage colony-stimulating factor potentiates the stimulated release of PAF. The intracellular calcium chelator BAPTA inhibits the rise of [Ca2+]i and the release of PAF but not the Na+/H+ antiport activity. PAF release, but not the rise in the intracellular concentration of free calcium, was inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The release of PAF in pertussis toxin-treated cells was also inhibited in cells stimulated with fMet-Leu-Phe or opsonized zymosan. These results suggest that functional pertussis toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for PAF release produced by physiological stimuli. It appears that PAF release requires a coordinated action of receptor-coupled G-proteins, calcium, and other parameters.
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PMID:Calcium is necessary but not sufficient for the platelet-activating factor release in human neutrophils stimulated by physiological stimuli. Role of G-proteins. 251 17

Deoxycytidine kinase (dC kinase) is the rate-limiting enzyme in the anabolism of important anticancer and retroviral nucleoside derivatives. Its activity is often decreased in resistance to these drugs. To analyze the structure, function, and control of this clinically important enzyme we isolated 15 cDNA clones for human deoxycytidine kinase from lambda gt11 thymus and Molt 4 libraries. Four clones were sequenced. The largest clone is 2.9 kilobases and codes for a 626-amino acid open reading frame. The DNA and deduced amino acid sequence of the human dC kinase clones are homologous with a previously unidentified murine cDNA clone p3.4J (EMBL:MM34j) reported to be related to granulocyte-macrophage colony-stimulating factor. Deoxycytidine kinase also has cysteine-rich regions that are homologous with thioredoxin, the beta subunit of prolyl 4-hydroxylase, phosphoinositide-specific phospholipase C, thyroid hormone-binding protein, and protein disulfide isomerase. No differences were seen in the amount and size of deoxycytidine kinase protein and mRNA between CCRF/CEM and L1210 leukemic cell lines that express and do not express enzyme activity. Genomic restriction fragments were similar between the active and inactive CCRF/CEM cell lines. These data suggest that the cells deficient in dC kinase activity have a small defect in the structural gene.
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PMID:Human deoxycytidine kinase. Sequence of cDNA clones and analysis of expression in cell lines with and without enzyme activity. 200 68

Murine peritoneal exudate macrophages (PEM) co-express granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) receptors, among others. Treatment of PEM with lipopolysaccharide (LPS) or tumor-promoting phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) induces a rapid but transient loss of M-CSF receptors in PEM. GM-CSF receptors are not affected by this treatment. The loss of M-CSF receptors induced by LPS can be inhibited by neomycin and compound 48/80, two potent phospholipase C (PLC) inhibitors, but not by phospholipase A2, calpain, protein kinase C (PKC) or protease inhibitors. On the other hand, the loss of M-CSF receptors induced by TPA has been prevented by PKC inhibitors but not by PLC inhibitors. PLC inhibitors also prevent LPS-suppressed receptor-mediated internalization of radiolabeled recombinant human (rh) M-CSF by macrophages. Similar prevention of LPS-induced M-CSF receptor downregulation was observed in human monocytes that had been pretreated with PLC inhibitors. Our results show that 1) TPA-induced M-CSF receptor loss is strictly dependent on PKC activation; 2) PLC activation alone also leads to downregulation of M-CSF receptors; and 3) LPS-induced M-CSF receptor downregulation in PEM is mediated primarily through a PLC-dependent pathway. Our data also imply that the expression of M-CSF but not GM-CSF receptors is linked to an important, yet unknown, PLC-sensitive component(s) whose hydrolysis may lead to downregulation of M-CSF receptors.
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PMID:Downregulation of M-CSF receptors by lipopolysaccharide in murine peritoneal exudate macrophages is mediated through a phospholipase C dependent pathway. 851 62

Nyk/Mer is a recently identified receptor tyrosine kinase with neural cell adhesion molecule-like structure (two immunoglobulin G-like domains and two fibronectin III-like domains) in its extracellular region and belongs to the Ufo/Axl family of receptors. The ligand for Nyk/Mer is presently unknown, as are the signal transduction pathways mediated by this receptor. We constructed and expressed a chimeric receptor (Fms-Nyk) composed of the extracellular domain of the human colony-stimulating factor 1 receptor (Fms) and the transmembrane and cytoplasmic domains of human Nyk/Mer in NIH 3T3 fibroblasts in order to investigate the mitogenic signaling and biochemical properties of Nyk/Mer. Colony-stimulating factor 1 stimulation of the Fms-Nyk chimeric receptor in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a proliferative response in the absence of other growth factors. We show that phospholipase C gamma, phosphatidylinositol 3-kinase/p70 S6 kinase, Shc, Grb2, Raf-1, and mitogen-activated protein kinase are downstream components of the Nyk/Mer signal transduction pathways. In addition, Nyk/Mer weakly activates p90rsk, while stress-activated protein kinase, Ras GTPase-activating protein (GAP), and GAP-associated p62 and p190 proteins are not activated or tyrosine phosphorylated by Nyk/Mer. An analysis comparing the Nyk/Mer signal cascade with that of the epidermal growth factor receptor indicates substrate preferences by these two receptors. Our results provide a detailed description of the Nyk/Mer signaling pathways. Given the structural similarity between the Ufo/Axl family receptors, some of the information may also be applied to other members of this receptor tyrosine kinase family.
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PMID:Mitogenic signals and transforming potential of Nyk, a newly identified neural cell adhesion molecule-related receptor tyrosine kinase. 852 23

Eosinophilic and neutrophilic granulocytes represent major effector cells in the inflammatory response. Whereas neutrophils are predominantly involved in bacterial infections, eosinophils are of essential importance in the allergic inflammation. Surface markers have been used to distinguish neutrophils (CD16+) from eosinophils (CD16-) and might indicate different functional properties of these cells. In this study, expression and functional activity of CD52 on human eosinophils and neutrophils was investigated in nonatopic healthy donors and from patients with hypereosinophilia. Flow cytometric analysis using different anti-CD52 monoclonal antibodies (MoAbs) (mouse IgG3, humanized IgG1, and rat IgM) showed significant and homogeneous expression of CD52 on human eosinophils, but not on neutrophils. In addition, reverse transcription-polymerase chain reaction and Northern blot analysis showed that CD52 mRNA was constitutively expressed in eosinophils but not in neutrophils. Furthermore, expression of CD52 could be diminished in a dose-dependent manner by preincubation of eosinophils with phosphatidylinositol-specific phospholipase C, suggesting that CD52 on eosinophils is anchored to the membrane through a glycosylphosphatidylinositol (GPI) molecule. Whereas the phorbolester phorbol myristate acetate was able to downregulate the expression of CD52 on eosinophils in a dose-dependent manner, different eosinophil activating cytokines and chemotaxins had no effect. Cross-linking of CD52 by mouse anti-CD52 MoAb (IgG3) and humanized anti-CD52 MoAb (IgG1) with goat antimouse antibody and mouse antihuman antibody, respectively, dose-dependently resulted in an inhibition of reactive oxygen species production of eosinophils after stimulation with C5a, platelet-activating factor, and granulocyte-macrophage colony-stimulating factor. In summary, this study shows that the GPI-anchored antigen CD52 is not only a useful marker to distinguish eosinophils from neutrophils. The data point out a novel role of the CD52 antigen on human eosinophils that might be of clinical relevance, because cross-linking of this molecule will stop the destructive power of human eosinophils in the inflammatory tissue.
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PMID:Surface and mRNA expression of the CD52 antigen by human eosinophils but not by neutrophils. 897 62

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.
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PMID:Stimulation of intracellular Ca2+ levels in human neutrophils by soluble immune complexes. Functional activation of FcgammaRIIIb during priming. 921 19

The small GTPase Rap1 is highly expressed in human neutrophils, but its function is largely unknown. Using the Rap1-binding domain of RalGDS (RalGDS-RBD) as an activation-specific probe for Rap1, we have investigated the regulation of Rap1 activity in primary human neutrophils. We found that a variety of stimuli involved in neutrophil activation, including fMet-Leu-Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgG-coated particles, induce a rapid and transient Rap1 activation. In addition, we found that Rap1 is normally activated in neutrophils from chronic granulomatous disease patients that lack cytochrome b558 or p47phox and have a defective NADPH oxidase system. From these results we conclude that in neutrophils Rap1 is activated independently of respiratory burst induction. Finally, we found that Rap1 is activated by both the Ca2+ ionophore ionomycin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating that phospholipase C (PLC) activation leading to elevated levels of intracellular free Ca2+ and diacylglycerol (DAG) can mediate Rap1 activation. However, inhibition of PLC and Ca2+ depletion only marginally affected fMLP-induced Rap1 activation, suggesting that additional pathways may control Rap1 activation.
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PMID:Activation of the small GTPase rap1 in human neutrophils. 973 Oct 72


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