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)

Fertilization is accompanied by a rapid and transient calcium release in eggs, which is required for the onset of zygotic developmental program or 'egg activation'. Recently, it was found that Src family tyrosine kinase (SFK)-dependent phospholipase C (PLC) activity is necessary for the calcium transience in fertilized Xenopus eggs. The present study demonstrates that hydrogen peroxide (H2O2) stimulates protein-tyrosine phosphorylation in Xenopus eggs, which occurs primarily in the egg cortex of the animal hemisphere as revealed by indirect immunofluorescence study. Egg SFK was found to be upregulated by H2O2 while the SFK-specific inhibitor PP1 effectively blocked H2O2-induced tyrosine phosphorylation. As in fertilized eggs, PLCgamma, but not Shc, was tyrosine-phosphorylated in H2O2-treated eggs. H2O2 also caused inositol 1,4,5-trisphosphate (IP3) production and sustained calcium release. After limited application of H2O2, elevated SFK activity and tyrosine phosphorylation were quickly reversed. Under such conditions, eggs showed cortical contraction and dephosphorylation of p42 MAP kinase, both of which are indicative of egg activation. These egg activation events, as well as H2O2-induced IP3 production and calcium release, were sensitive to PP1 and PLC inhibitor U-73122. Together, the present study demonstrated that H2O2 can mimic, at least in part, early events of Xenopus egg activation that require an SFK-dependent PLC pathway.
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PMID:Hydrogen peroxide induces Src family tyrosine kinase-dependent activation of Xenopus eggs. 1114 52

The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.
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PMID:Differential roles of the NPXXY motif in formyl peptide receptor signaling. 1123 59

In the present study, the release of secreted beta-amyloid precursor protein (AbetaPPs) in response to thrombin stimulation in platelets has been investigated. Incubation of platelets with thrombin produced a concentration-dependent release of AbetaPPs with a concomitant reduction in the AbetaPP remaining in the lysates. The response to thrombin was not affected by pretreatment for 15 min with the phospholipase C inhibitor U-73122, with the protein kinase C inhibitor staurosporine, or with hydrogen peroxide (which at the concentrations used affects the phosphoinositide signalling system in human platelets). In contrast, pretreatment with wortmannin and sodium azide reduced the responses to thrombin. These data would suggest that thrombin may cause the release of AbetaPPs from human platelets via an activation of a phospholipase C-independent pathway. Thrombin-stimulated AbetaPPs release was also reduced by 4-hydroxynonenal. This finding, if it is a phenomenon also found for CNS cells, could be of relevance to the pathogenesis of Alzheimer's disease, given that an accumulation of 4-hydroxynonenal is found in this disease.
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PMID:Effects of staurosporine, U-73122, wortmannin, 4-hydroxynonenal and sodium azide upon the release of secreted beta-amyloid precursor protein from human platelets in response to thrombin stimulation. 1135 46

In Jurkat T lymphocytes, hydrogen peroxide (H(2)O(2)) potentiates the phosphorylation level of extracellular signal-regulated kinase 1 and 2 (ERK1/2) caused by T cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 or anti-CD3 alone. Submillimolar concentrations of H(2)O(2)-induced phosphorylation of ERK1/2 and MAP/ERK kinase 1 and 2 (MEK1/2) without antigenic stimulation. H(2)O(2) also induced the electrophoretic mobility shift of Lck from 56 to 60 kDa. The MEK inhibitor, PD98059 attenuated ERK1/2 and MEK1/2 phosphorylation, as well as the migration shift of Lck induced by H(2)O(2). The phospholipase C (PLC) inhibitor, U73122, and EGTA reduced the phosphorylation of both ERK1/2 and MEK1/2 induced by H(2)O(2). Interestingly, an increase of intracellular cAMP level with forskolin or 8-(4-chlorophenylthio)-cAMP augmented ERK1/2 phosphorylation by H(2)O(2), while inhibiting MEK1/2 phosphorylation by H(2)O(2). These results demonstrate an alternative pathway that results in augmentation of ERK1/2 phosphorylation without concomitant MEK1/2 phosphorylation in T cells.
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PMID:cAMP potentiates H(2)O(2)-induced ERK1/2 phosphorylation without the requirement for MEK1/2 phosphorylation. 1149 22

Substrate analogues of phosphatidylinositol (1) were synthesized and evaluated as potential inhibitors of the bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. The chiral analogues of the water-soluble phospholipid substrate 5 were designed to probe the effects of varying the inositol C-2 hydroxyl group, which is generally believed to serve as the nucleophile in the first step of the hydrolysis of phosphatidylinositols by PI-PLC. In the analogues 6-9, the C-2 hydroxyl group on the inositol ring of the phosphatidylinositol derivatives was rationally altered in several ways. Inversion of the stereochemistry at C-2 of the inositol ring led to the scyllo derivative 6. The inositol C-2 hydroxy group was replaced with inversion by a fluorine to produce the scyllo-fluoro inositol 7 and with a hydrogen atom to furnish the 2-deoxy compound 8. The C-2 hydroxyl group was O-methylated to prepare the methoxy derivative 9. The natural inositol configuration at C-2 was retained in the nonhydrolyzable phosphorodithioate analogue 10. The inhibition of PI-PLC by each of these analogues was then analyzed in a continuous assay using D-myo-inositol 1-(4-nitrophenyl phosphate) (25) as a chromogenic substrate. The kinetic parameters for each of these phosphatidylinositol derivatives were determined, and each was found to be a competitive inhibitor with K(i)'s as follows: 6, 0.2 mM; 10, 0.6 mM; 8, 2.6 mM; 9, 6.6 mM; and 7, 8.8 mM. This study further establishes that the hydrolysis of phosphatidylinositol analogues by bacterial PI-PLC requires not only the presence of a C-2 hydroxyl group on the inositol ring, but the stereochemistry at this position must also correspond to the natural myo-configuration. For future inhibitor design, it is perhaps noteworthy that the best inhibitors 6 and 10 each possess a hydroxyl group at the C-2 position. Several of the inhibitors identified in this study are now being used to obtain crystallographic information for an enzyme-inhibitor complex to gain further insights regarding the mechanism of hydrolysis of phosphatidylinositides by this PI-PLC.
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PMID:Synthesis and Kinetic Evaluation of Inhibitors of the Phosphatidylinositol-Specific Phospholipase C from Bacillus cereus. 1166 84

Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitric-oxide synthase inhibitors diphenyleneiodonium and N(G)methyl-l-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-gamma-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.
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PMID:Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR. 1171 8

We have studied the effect of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)), two reactive oxygen species (ROS) on histamine release (HR) from RBL-2H3 cells, a rat mucosal-type mast cell line. Marked HR was elicited by antigen (DNP-HSA), calcium ionophore A23187, sodium fluoride or phospholipase C, but not with compound 48/80 or 1,2-dioctanoyl-sn-glycerol. The NO-synthase substrate L-arginine and its inactive enantiomer (D-arginine), each on its own, induced a small but significant increase in HR above the basal level. However, the NO-donors (sodium nitroprusside or NaNO(3)) or the NO-synthase inducer lipopolysaccharide did not induce HR. Moreover, methylene blue (MB), which inhibits guanylate cyclase and N(omega)-nitro-L-arginine (L-NA), an inhibitor of NO synthase, were also without effect on either the basal HR or the L-arginine-induced HR. HR induced by A23187, DNP-HSA, sodium fluoride or phospholipase C was markedly reduced by MB, but mildly by L-NA (both at 1-100 microM). H(2)O(2) (0.01-1.0 mM) on its own did not induce HR, but it had a potent inhibitory effect on DNP-HSA- or A23187-induced HR, which was not reversed by L-NA (1-100 microM). Taken together, it seems that neither the stimulatory nor the inhibitory effects of the NO-related compounds on HR can be attributed to NO, but rather to other mechanisms. The inhibition of HR by H(2)O(2) also does not involve NO and suggests a negative feedback regulatory role for the peroxide in the allergic inflammation.
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PMID:Effects of nitric oxide and hydrogen peroxide on histamine release from RBL-2H3 cells. 1172 90

Calcium influx is required for the mammalian sperm acrosome reaction, an exocytotic event occurring in the sperm head after binding to the egg. Prior to this binding, the spermatozoon undergo, in the female reproductive tract, a series of biochemical transformations, collectively called capacitation. The first event in capacitation is the elevation of intracellular calcium, bicarbonate and hydrogen peroxide, which collectively activate adenylyl cyclase to produce cyclic-AMP, which activates protein kinase A. During capacitation, there is an increase in the membrane-bound phospholipase C, and this binding is highly stimulated by adding epidermal growth factor to the cells. We suggest that zona-pellucida binds to at least two different receptors in the sperm head plasma membrane. One is a G(i)-coupled receptor that can activate phospholipase Cbeta(1) and might regulate adenylyl cyclase to further enhance cyclic-AMP levels. The cyclic AMP activates protein kinase A to open a calcium channel in the outer acrosomal membrane, resulting in a relatively small rise in cytosolic calcium. This rise in Ca(2+) leads to activation of phospholipase Cgamma, which is coupled to the second tyrosine kinase receptor. The products of phospholipase C activity, diacylglycerol and inositol-trisphosphate (IP(3)), will lead to activation of protein kinase C (PKC) and IP(3)-receptor. PKC will open a calcium channel in the plasma membrane and IP(3) will activate the calcium channel in the outer acrosomal membrane, leading to a higher increase in cytosolic calcium. In addition, the depletion of calcium in the acrosome will activate a store-operated Ca(2+) channel, resulting in a very fast increase in cytosolic calcium (300-500 nM), leading to membrane fusion and completing the acrosome reaction.
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PMID:Role and regulation of intracellular calcium in acrosomal exocytosis. 1173 Sep 12

Clostridium perfringens biotype A strains are the causative agents of gas-gangrene in man and are also implicated as etiological agents in sudden death syndrome in young domestic livestock. The main virulence factor produced by these strains is a zinc-dependent, phosphatidylcholine-preferring phospholipase C (alpha-toxin). The crystal structure of alpha-toxin, at pH 7.5, with the active site open and therefore accessible to substrate has previously been reported, as has calcium-binding to the C-terminal domain of the enzyme at pH 4.7. Here we focus on conformation changes in the N-terminal domain of alpha-toxin in crystals grown at acidic pH. These changes result in both the obscuring of the toxin active site and the loss of one of three zinc ions from it. Additionally, this "closed" form contains a small alpha helix, not present in the open structure, which hydrogen bonds to both the N and C-terminal domains. In conjunction with the previously reported findings that alpha-toxin can exist in active and inactive forms and that Thr74Ile and Phe69Cys substitutions markedly reduced the haemolytic activity of the enzyme, our work suggests that these loop conformations play a critical role in the activity of the toxin.
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PMID:Crystal structure of the C. perfringens alpha-toxin with the active site closed by a flexible loop region. 1205 5

Proton efflux from chondrocytes alters the extracellular pH and ionic composition of cartilage, and influences the synthesis and degradation of extracellular matrix. Epidermal growth factor (EGF) promotes chondrocyte proliferation during skeletal development and accumulates in the synovial fluid in rheumatoid arthritis. The purpose of this study was to investigate the effect of EGF on proton efflux from chondrocytes. When monitored using a Cytosensor microphysiometer, EGF was found to rapidly activate proton efflux from CFK2 chondrocytic cells and rat articular chondrocytes. The actions of EGF were concentration-dependent with half-maximal effects at 0.3-0.7 ng/ml. Partial desensitization and time-dependent recovery of the response were observed following repeated exposures to EGF. EGF-induced proton efflux was dependent on extracellular glucose, and inhibitors of Na(+)/H(+) exchange (NHE) markedly attenuated the initial increase in proton efflux. The response was diminished by inhibitors of phosphatidylinositol 3-kinase and phospholipase C, but not by inhibitors of MEK (MAPK/ERK kinase) or protein kinase A or C. Thus, EGF-induced proton efflux involves glucose metabolism and NHE, and is regulated by a discrete subset of EGF-activated signaling pathways. In vivo, proton efflux induced by EGF may lead to an acidic environment, enhancing turnover of cartilage matrix during development and in rheumatoid arthritis.
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PMID:Epidermal growth factor stimulates proton efflux from chondrocytic cells. 1211 41


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