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Sample records for phospholipase-c pi-plc family

  1. Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance.

    PubMed

    Vossen, Jack H; Abd-El-Haliem, Ahmed; Fradin, Emilie F; van den Berg, Grardy C M; Ekengren, Sophia K; Meijer, Harold J G; Seifi, Alireza; Bai, Yuling; ten Have, Arjen; Munnik, Teun; Thomma, Bart P H J; Joosten, Matthieu H A J

    2010-04-01

    The perception of pathogen-derived elicitors by plants has been suggested to involve phosphatidylinositol-specific phospholipase-C (PI-PLC) signalling. Here we show that PLC isoforms are required for the hypersensitive response (HR) and disease resistance. We characterised the tomato [Solanum lycopersicum (Sl)] PLC gene family. Six Sl PLC-encoding cDNAs were isolated and their expression in response to infection with the pathogenic fungus Cladosporium fulvum was studied. We found significant regulation at the transcriptional level of the various SlPLCs, and SlPLC4 and SlPLC6 showed distinct expression patterns in C. fulvum-resistant Cf-4 tomato. We produced the encoded proteins in Escherichia coli and found that both genes encode catalytically active PI-PLCs. To test the requirement of these Sl PLCs for full Cf-4-mediated recognition of the effector Avr4, we knocked down the expression of the encoding genes by virus-induced gene silencing. Silencing of SlPLC4 impaired the Avr4/Cf-4-induced HR and resulted in increased colonisation of Cf-4 plants by C. fulvum expressing Avr4. Furthermore, expression of the gene in Nicotiana benthamiana enhanced the Avr4/Cf-4-induced HR. Silencing of SlPLC6 did not affect HR, whereas it caused increased colonisation of Cf-4 plants by the fungus. Interestingly, Sl PLC6, but not Sl PLC4, was also required for resistance to Verticillium dahliae, mediated by the transmembrane Ve1 resistance protein, and to Pseudomonas syringae, mediated by the intracellular Pto/Prf resistance protein couple. We conclude that there is a differential requirement of PLC isoforms for the plant immune response and that Sl PLC4 is specifically required for Cf-4 function, while Sl PLC6 may be a more general component of resistance protein signalling.

  2. Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity.

    PubMed

    Abd-El-Haliem, Ahmed M; Vossen, Jack H; van Zeijl, Arjan; Dezhsetan, Sara; Testerink, Christa; Seidl, Michael F; Beck, Martina; Strutt, James; Robatzek, Silke; Joosten, Matthieu H A J

    2016-09-01

    Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

  3. Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity.

    PubMed

    Abd-El-Haliem, Ahmed M; Vossen, Jack H; van Zeijl, Arjan; Dezhsetan, Sara; Testerink, Christa; Seidl, Michael F; Beck, Martina; Strutt, James; Robatzek, Silke; Joosten, Matthieu H A J

    2016-09-01

    Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:26825689

  4. Leishmania amazonensis: heme stimulates (Na(+)+K(+))ATPase activity via phosphatidylinositol-specific phospholipase C/protein kinase C-like (PI-PLC/PKC) signaling pathways.

    PubMed

    Almeida-Amaral, Elmo Eduardo; Cardoso, Viviane Carrozino; Francioli, Fernanda Gomes; Meyer-Fernandes, José Roberto

    2010-04-01

    In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na(+)+K(+))ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH(3) and U73122, specific inhibitors of PI-PLC. (Na(+)+K(+))ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50nM. This effect was completely reversed by 10nM calphostin C, an inhibitor of PKC. Thus, the effect of 50nM heme on (Na(+)+K(+))ATPase activity is completely abolished by ET-18-OCH(3) and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na(+)+K(+))ATPase activity through a PI-PLC/PKC signaling pathway. PMID:20045694

  5. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    PubMed

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  6. Genome-Wide Analysis and Expression Profiling of the Phospholipase C Gene Family in Soybean (Glycine max)

    PubMed Central

    Zhou, Yonggang; Dong, Jinye; Chen, Huan; Dong, Yuanyuan; Wang, Nan; Li, Xiaowei; Li, Haiyan

    2015-01-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate to produce diacylglycerol and inositol 1,4,5-trisphosphate. It plays an important role in plant development and abiotic stress responses. However, systematic analysis and expression profiling of the phospholipase C (PLC) gene family in soybean have not been reported. In this study, 12 putative PLC genes were identified in the soybean genome. Soybean PLCs were found on chromosomes 2, 11, 14 and 18 and encoded 58.8–70.06 kD proteins. Expression pattern analysis by RT-PCR demonstrated that expression of the GmPLCs was induced by PEG, NaCl and saline-alkali treatments in roots and leaves. GmPLC transcripts accumulated specifically in roots after ABA treatment. Furthermore, GmPLC transcripts were analyzed in various tissues. The results showed that GmPLC7 was highly expressed in most tissues, whereas GmPLC12 was expressed in early pods specifically. In addition, subcellular localization analysis was carried out and confirmed that GmPLC10 was localized in the plasma membrane in Nicotiana benthamiana. Our genomic analysis of the soybean PLC family provides an insight into the regulation of abiotic stress responses and development. It also provides a solid foundation for the functional characterization of the soybean PLC gene family. PMID:26421918

  7. Genome-Wide Analysis and Expression Profiling of the Phospholipase C Gene Family in Soybean (Glycine max).

    PubMed

    Wang, Fawei; Deng, Yu; Zhou, Yonggang; Dong, Jinye; Chen, Huan; Dong, Yuanyuan; Wang, Nan; Li, Xiaowei; Li, Haiyan

    2015-01-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate to produce diacylglycerol and inositol 1,4,5-trisphosphate. It plays an important role in plant development and abiotic stress responses. However, systematic analysis and expression profiling of the phospholipase C (PLC) gene family in soybean have not been reported. In this study, 12 putative PLC genes were identified in the soybean genome. Soybean PLCs were found on chromosomes 2, 11, 14 and 18 and encoded 58.8-70.06 kD proteins. Expression pattern analysis by RT-PCR demonstrated that expression of the GmPLCs was induced by PEG, NaCl and saline-alkali treatments in roots and leaves. GmPLC transcripts accumulated specifically in roots after ABA treatment. Furthermore, GmPLC transcripts were analyzed in various tissues. The results showed that GmPLC7 was highly expressed in most tissues, whereas GmPLC12 was expressed in early pods specifically. In addition, subcellular localization analysis was carried out and confirmed that GmPLC10 was localized in the plasma membrane in Nicotiana benthamiana. Our genomic analysis of the soybean PLC family provides an insight into the regulation of abiotic stress responses and development. It also provides a solid foundation for the functional characterization of the soybean PLC gene family. PMID:26421918

  8. Plant phosphoinositide-specific phospholipase C

    PubMed Central

    Rupwate, Sunny D.; Rajasekharan, Ram

    2012-01-01

    Phosphoinositide-specific phospholipase C (PI-PLC) belongs to an important class of enzymes involved in signaling related to lipids. They hydrolyze a membrane-associated phospholipid, phosphatidylinositol-4,5-bisphosphate, to produce inositol-1,4,5-trisphosphate and diacylglycerol. The role of PI-PLC and the mechanism behind its functioning is well studied in animal system; however, mechanism of plant PI-PLC functioning remains largely obscure. Here, we attempted to summarize the understanding regarding plant PI-PLC mechanism of regulation, localization, and domain association. Using sedimentation based phospholipid binding assay and surface plasmon resonance spectroscopy, it was demonstrated that C2 domain of plant PI-PLC alone is capable of targeting membranes. Moreover, change in surface hydrophobicity upon calcium stimulus is the key element in targeting plant PI-PLC from soluble fractions to membranes. This property of altering surface hydrophobicity plays a pivot role in regulation of PI-PLC activity. PMID:22902702

  9. 1p36.32 rearrangements and the role of PI-PLC η2 in nervous tumours.

    PubMed

    Lo Vasco, Vincenza Rita

    2011-07-01

    Deletions in the distal region of the short arm of chromosome 1 (1p36) are widely diffuse, both in congenital 1p36 Deletion Syndrome and as somatic abnormalities in tumours. Rearrangements in 1p36 have been described in a broad spectrum of human neoplasias in addition to other chromosomal abnormalities. In neuroblastomas, wide hemizygous deletions in 1p36.23-1p36.32 have been described suggesting that the 1p36 region contains a tumour-suppressor gene involved in malignancy. A role for phosphoinositide (PI)-specific phospholipase C (PLC) η2, whose gene maps on 1p36.32, was suggested. PI-PLC η2 belongs to a family of enzymes related to the phosphoinositide signalling pathway, which provide an important intracellular signalling system involved in a variety of cell functions such as hormone secretion, neurotransmitter signal transduction, cell growth, membrane trafficking, ion channel activity, regulation of the cytoskeleton, cell cycle control and apoptosis. Expression of PI-PLC η2 occurs after birth and continues throughout the life. Synapse formation occurs during a short period of postnatal development. Thus, it is likely that PI-PLC η2 acts in formation and maintenance of the neuronal network in the brain. The fact that PI-PLC η2, a highly neuron-specific isozyme, is abundantly expressed in the postnatal brain suggests the importance of PI-PLC η2 in formation and maintenance of the neuronal network in the postnatal brain. Further studies are required to verify the possible involvement of PI-PLC η2 mutation/deletion in central nervous tumour tissues presenting abnormalities of the 1p36 chromosomal band.

  10. Revealing Transient Interactions between Phosphatidylinositol-specific Phospholipase C and Phosphatidylcholine--Rich Lipid Vesicles

    NASA Astrophysics Data System (ADS)

    Yang, Boqian; He, Tao; Grauffel, Cédric; Reuter, Nathalie; Roberts, Mary; Gershenson, Anne

    2013-03-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes transiently interact with target membranes. Previous fluorescence correlation spectroscopy (FCS) experiments showed that Bacillus thuringiensis PI-PLC specifically binds to phosphatidylcholine (PC)-rich membranes and preferentially interacts with unilamellar vesicles that show larger curvature. Mutagenesis studies combined with FCS measurements of binding affinity highlighted the importance of interfacial PI-PLC tyrosines in the PC specificity. All-atom molecular dynamics simulations of PI-PLC performed in the presence of a PC membrane indicate these tyrosines are involved in specific cation-pi interactions with choline headgroups. To further understand those transient interactions between PI-PLC and PC-rich vesicles, we monitor single fluorescently labeled PI-PLC proteins as they cycle on and off surface-tethered small unilamellar vesicles using total internal reflection fluorescent microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of PI-PLC membrane interactions as well as the curvature dependence. The PC specificity and the vesicle curvature dependence of this PI-PLC/membrane interaction provide insight into how the interface modulates protein-membrane interactions. This work was supported by the National Institute of General Medical Science of the National Institutes of Health (R01GM060418).

  11. The ability of Listeria monocytogenes PI-PLC to facilitate escape from the macrophage phagosome is dependent on host PKCbeta.

    PubMed

    Poussin, Mathilde A; Leitges, Michael; Goldfine, Howard

    2009-01-01

    Listeria monocytogenes are facultative intracellular pathogenic bacteria that can infect macrophages as well as non-professional phagocytes. After entry in the host cell, the bacteria escape from the phagosome into the cytoplasm. In murine macrophages and in cell lines derived from these cells, escape of L. monocytogenes from the phagosome is absolutely dependent on listeriolysin O (LLO) and facilitated by a secreted phosphatidylinositol-specific phospholipase C (PI-PLC). Work in this laboratory has previously demonstrated a LLO and PI-PLC-dependent translocation of host PKCbeta isoforms. Pharmacological inhibition of PKCbeta resulted in a significant reduction in permeabilization of the phagosome, and in the number of bacteria reaching the cytosol. These findings led to the prediction that the bacterial PI-PLC promotes escape through the production of diacylglycerol leading to the activation of host PKCbeta. To test this hypothesis, bone marrow-derived macrophages (BMMf) obtained from PKCbeta knockout (PKCbetaKO) or C57Bl/6 mice were infected with L. monocytogenes. We observed that wild-type L. monocytogenes escapes from the phagosome of PKCbetaKO BMMf as well as from C57Bl/6 BMMf. However, in PKCbetaKO BMMf, L. monocytogenes uses a PI-PLC-independent, but phosphatidylcholine-preferring PLC (PC-PLC)-dependent pathway to facilitate escape. These findings strongly support the hypothesis that PI-PLC promotes escape through mobilization of host PKCbeta.

  12. Cloning, expression, and mutagenesis of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: a potential staphylococcal virulence factor.

    PubMed Central

    Daugherty, S; Low, M G

    1993-01-01

    Staphylococcus aureus secretes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) which is able to hydrolyze the membrane lipid PI and membrane protein anchors containing glycosyl-PI. The gene for PI-PLC (plc) was cloned from S. aureus into Escherichia coli. Oligonucleotide probes based on partial protein sequence and polyclonal antibodies raised against the purified protein were used to identify positive clones. E. coli transformed with a plasmid containing the plc gene expressed PI-PLC enzyme activity which was abolished by mutagenesis with a tetracycline resistance gene. The plc gene was present in all 15 S. aureus strains examined but not in any of 6 coagulase-negative staphylococcal species. The plc gene contained 984 bp and coded for a mature protein with a calculated molecular mass of 34,107 Da. Amino acid sequence comparisons indicated that the staphylococcal plc gene was similar (51 to 56%) to the PI-PLCs from Bacillus cereus, Bacillus thuringiensis, and Listeria monocytogenes. The recombinant PI-PLC expressed in E. coli was purified and exhibited biochemical properties identical to those of the native PI-PLC from S. aureus. PI-PLC production was decreased in agr mutant strains of S. aureus. However, PI-PLC production by both agr+ and agr mutant strains exhibited a similar dependence on the type of medium used. These data suggested that PI-PLC production was regulated by both agr-dependent and agr-independent mechanisms. Images PMID:8225585

  13. Does Changing the Predicted Dynamics of a Phospholipase C Alter Activity and Membrane Binding?

    PubMed Central

    Cheng, Jiongjia; Karri, Sashank; Grauffel, Cédric; Wang, Fang; Reuter, Nathalie; Roberts, Mary F.; Wintrode, Patrick L.; Gershenson, Anne

    2013-01-01

    The enzymatic activity of secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes is associated with bacterial virulence. Although the PI-PLC active site has no obvious lid, molecular-dynamics simulations suggest that correlated loop motions may limit access to the active site, and two Pro residues, Pro245 and Pro254, are associated with these correlated motions. Whereas the region containing both Pro residues is quite variable among PI-PLCs, it shows high conservation in virulence-associated, secreted PI-PLCs that bind to the surface of cells. These regions of the protein are also associated with phosphatidylcholine binding, which enhances PI-PLC activity. In silico mutagenesis of Pro245 disrupts correlated motions between the two halves of Bacillus thuringiensis PI-PLC, and Pro245 variants show significantly reduced enzymatic activity in all assay systems. PC still enhanced activity, but not to the level of wild-type enzyme. Mutagenesis of Pro254 appears to stiffen the PI-PLC structure, but experimental mutations had minor effects on activity and membrane binding. With the exception of P245Y, reduced activity was not associated with reduced membrane affinity. This combination of simulations and experiments suggests that correlated motions between the two halves of PI-PLC may be more important for enzymatic activity than for vesicle binding. PMID:23332071

  14. Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins.

    PubMed Central

    Gandhi, A J; Perussia, B; Goldfine, H

    1993-01-01

    The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins. The L. monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes. The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B. thuringiensis. Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low. Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins. Our data show that L. monocytogenes PI-PLC has low activity on GPI-anchored proteins. PMID:8253689

  15. Bacterial phospholipases C.

    PubMed Central

    Titball, R W

    1993-01-01

    A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C. PMID:8336671

  16. End-product diacylglycerol enhances the activity of PI-PLC through changes in membrane domain structure.

    PubMed

    Ahyayauch, Hasna; Sot, Jesús; Collado, M Isabel; Huarte, Nerea; Requejo-Isidro, José; Alonso, Alicia; Goñi, Félix M

    2015-04-01

    Diacylglycerol (DAG)-induced activation of phosphatidylinositol-phospholipase C (PI-PLC) was studied with vesicles containing PI, either pure or in mixtures with dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, sphingomyelin, or galactosylceramide, used as substrates. At 22°C, DAG at 33 mol % increased PI-PLC activity in all of the mixtures, but not in pure PI bilayers. DAG also caused an overall decrease in diphenylhexatriene fluorescence polarization (decreased molecular order) in all samples, and increased overall enzyme binding. Confocal fluorescence microscopy of giant unilamellar vesicles of all of the compositions under study, with or without DAG, and quantitative evaluation of the phase behavior using Laurdan generalized polarization, and of enzyme binding to the various domains, indicated that DAG activates PI-PLC whenever it can generate fluid domains to which the enzyme can bind with high affinity. In the specific case of PI/dimyristoyl phosphatidylcholine bilayers at 22°C, DAG induced/increased enzyme binding and activation, but no microscopic domain separation was observed. The presence of DAG-generated nanodomains, or of DAG-induced lipid packing defects, is proposed instead for this system. In PI/galactosylceramide mixtures, DAG may exert its activation role through the generation of small vesicles, which PI-PLC is known to degrade at higher rates. In general, our results indicate that global measurements obtained using fluorescent probes in vesicle suspensions in a cuvette are not sufficient to elucidate DAG effects that take place at the domain level. The above data reinforce the idea that DAG functions as an important physical agent in regulating membrane and cell properties.

  17. End-Product Diacylglycerol Enhances the Activity of PI-PLC through Changes in Membrane Domain Structure

    PubMed Central

    Ahyayauch, Hasna; Sot, Jesús; Collado, M. Isabel; Huarte, Nerea; Requejo-Isidro, José; Alonso, Alicia; Goñi, Félix M.

    2015-01-01

    Diacylglycerol (DAG)-induced activation of phosphatidylinositol-phospholipase C (PI-PLC) was studied with vesicles containing PI, either pure or in mixtures with dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, sphingomyelin, or galactosylceramide, used as substrates. At 22°C, DAG at 33 mol % increased PI-PLC activity in all of the mixtures, but not in pure PI bilayers. DAG also caused an overall decrease in diphenylhexatriene fluorescence polarization (decreased molecular order) in all samples, and increased overall enzyme binding. Confocal fluorescence microscopy of giant unilamellar vesicles of all of the compositions under study, with or without DAG, and quantitative evaluation of the phase behavior using Laurdan generalized polarization, and of enzyme binding to the various domains, indicated that DAG activates PI-PLC whenever it can generate fluid domains to which the enzyme can bind with high affinity. In the specific case of PI/dimyristoyl phosphatidylcholine bilayers at 22°C, DAG induced/increased enzyme binding and activation, but no microscopic domain separation was observed. The presence of DAG-generated nanodomains, or of DAG-induced lipid packing defects, is proposed instead for this system. In PI/galactosylceramide mixtures, DAG may exert its activation role through the generation of small vesicles, which PI-PLC is known to degrade at higher rates. In general, our results indicate that global measurements obtained using fluorescent probes in vesicle suspensions in a cuvette are not sufficient to elucidate DAG effects that take place at the domain level. The above data reinforce the idea that DAG functions as an important physical agent in regulating membrane and cell properties. PMID:25863059

  18. Biosynthesis of glycosylphosphatidylinositol-anchored human placental alkaline phosphatase: evidence for a phospholipase C-sensitive precursor and its post-attachment conversion into a phospholipase C-resistant form.

    PubMed Central

    Wong, Y W; Low, M G

    1994-01-01

    Previous studies have shown that some cells (e.g. SKG3a) express human placental alkaline phosphatase (AP) in a form which can be released from the membrane by bacterial PtdIns-specific phospholipase C (PI-PLC) while others (e.g. HeLa) are relatively resistant to this enzyme. Chemical and enzymic degradation studies have suggested that the PI-PLC resistance of AP is due to inositol acylation of its glycosylphosphatidylinositol (GPI) anchor. In order to identify the biosynthetic origin of PI-PLC resistance we determined the PI-PLC sensitivity of AP in 35S-labelled cells (10 min pulse; 0-60 min chase) by Triton X-114 phase separation. At the beginning of the chase period, the majority of the AP synthesized was hydrophilic, indicating that it had not acquired a GPI anchor. The concentration of hydrophilic AP species decreased with a t1/2 of 30-60 min but was not processed to an endoglycosidase H-resistant species or secreted into the medium. In both SKG3a and HeLa cells all of the hydrophobic, GPI-anchored AP detectable at the beginning of the chase was PI-PLC sensitive. PI-PLC-resistant species of AP were only observed in HeLa cells and these only appeared after about 30 min. The delayed appearance of PI-PLC resistance was unexpected as previous studies have suggested that candidate GPI-anchor precursors are PI-PLC-resistant as a result of inositol acylation. This work reveals unanticipated complexities in the biosynthesis of AP and its GPI anchor. Images Figure 1 Figure 2 Figure 3 PMID:8037672

  19. Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils

    PubMed Central

    White, Mark J.; Boyd, Jeffrey M.

    2014-01-01

    Staphylococcus aureus is an important human pathogen that employs a large repertoire of secreted virulence factors to promote disease pathogenesis. Many strains of S. aureus possess a plc gene that encodes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) capable of hydrolyzing PI and cleaving glycosyl-PI (GPI)-linked proteins from cell surfaces. Despite being secreted by virulent staphylococci, the contribution of PI-PLC to the capacity of S. aureus to cause disease remains undefined. Our goal in these studies was to understand PI-PLC in the context of S. aureus biology. Among a collection of genetically diverse clinical isolates of S. aureus, community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 secreted the most PI-PLC. Screening a collection of two-component system (TCS) mutants of S. aureus, we identified both the agr quorum-sensing system and the SrrAB TCS to be positive regulators of plc gene expression. Real-time PCR and PI-PLC enzyme assays of the TCS mutants, coupled with SrrA promoter binding studies, demonstrated that SrrAB was the predominant transcriptional activator of plc. Furthermore, plc regulation was linked to oxidative stress both in vitro and in vivo in a SrrAB-dependent manner. A Δplc mutant in a CA-MRSA USA300 background exhibited a survival defect in human whole blood and in isolated neutrophils. However, the same mutant strain displayed no survival defect in murine models of infection or murine whole blood. Overall, these data identify potential links between bacterial responses to the host innate immune system and to oxidative stress and suggest how PI-PLC could contribute to the pathogenesis of S. aureus infections. PMID:24452683

  20. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids

    SciTech Connect

    Mayor, S.; Menon, A.K.; Cross, G.A. )

    1990-04-15

    A common diagnostic feature of glycosylinositol phospholipid (GPI)-anchored proteins is their release from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). However, some GPI-anchored proteins are resistant to this enzyme. The best characterized example of this subclass is the human erythrocyte acetylcholinesterase, where the structural basis of PI-PLC resistance has been shown to be the acylation of an inositol hydroxyl group(s). Both PI-PLC-sensitive and resistant GPI-anchor precursors (P2 and P3, respectively) have been found in Trypanosoma brucei, where the major surface glycoprotein is anchored by a PI-PLC-sensitive glycolipid anchor. The accompanying paper shows that P2 and P3 have identical glycans, indistinguishable from the common core glycan found on all the characterized GPI protein anchors. This paper shows that the single difference between P2 and P3, and the basis for the PI-PLC insusceptibility of P3, is a fatty acid, ester-linked to the inositol residue in P3. The inositol-linked fatty acid can be removed by treatment with mild base to restore PI-PLC sensitivity. Biosynthetic labeling experiments with (3H)palmitic acid and (3H)myristic acid show that (3H)palmitic acid specifically labels the inositol residue in P3 while (3H)myristic acid labels the diacylglycerol portion. Possible models to account for the simultaneous presence of PI-PLC-resistant and sensitive glycolipids are discussed in the context of available information on the biosynthesis of GPI-anchors.

  1. Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity.

    PubMed

    Wehbi, Hania; Feng, Jianwen; Roberts, Mary F

    2003-06-27

    Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca(2+)-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T(m) for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 degrees C (well below the T(m) for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little

  2. Crystallization, optimization and preliminary X-ray characterization of a metal-dependent PI-PLC from Streptomyces antibioticus

    SciTech Connect

    Jackson, Michael R.; Selby, Thomas L.

    2012-10-30

    A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) fromStreptomyces antibioticushas been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space groupP222, with unit-cell parametersa= 41.26,b= 51.86,c = 154.78 Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23 Å resolution.

  3. Release of renal dipeptidase from glycosylphosphatidylinositol anchor by insulin-triggered phospholipase C/intracellular Ca2+.

    PubMed

    Yoon, Hyun Joong; Park, Sung Wook; Lee, Hwanghee Blaise; Im, Shun Young; Hooper, Nigel M; Park, Haeng Soon

    2007-05-01

    Glycosylphosphatidylinositol (GPI) anchored proteins appear to be released from the plasma membrane due to various extracellular stimuli. To determine the signaling pathway from insulin to GPI-protein, the release of GPI-renal dipeptidase (RDPase, EC 3.4.13.19) from porcine proximal tubules, stimulated by insulin, was explored. Insulin stimulated the release of RDPase in a concentration-dependent manner (half maximal release at 0.58 nM), which peaked at 10-20 min. Western blot analysis, with antibody against the cross-reacting determinant (CRD), revealed that RDPase was released by a GPI-specific phospholipase C (GPI-PLC), and was shown to be Ca2+-dependent. A PI-PLC inhibitor, U73122, effectively blocked the effect of insulin on the release of RDPase, suggesting insulin is associated with an intracellular PI-PLC. Insulin treatment increased the production of intracellular Ca2+ from porcine proximal tubules. Intracellular Ca2+, coupled with insulin, facilitated the releases of RDPase, an inhibitor of inositol trisphosphate-dependent Ca2+ from the endoplasmic reticulum, and a Ca2+ channel blocker that blocked the effect of insulin. Taken together, these results suggest that insulin, in part, may activate a GPI-PLC, via PI-PLC/intracellular Ca2+, which may consequently stimulate the release of RDPase.

  4. Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes.

    PubMed Central

    Higgins, J A; Hitchin, B W; Low, M G

    1989-01-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles. PMID:2543374

  5. Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C.

    PubMed

    Brewis, I A; Turner, A J; Hooper, N M

    1994-10-15

    Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.

  6. Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.

    PubMed Central

    Flick, J S; Thorner, J

    1993-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses. Images PMID:8395015

  7. The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

    PubMed

    Chakraborty, Sandeep; Rendón-Ramírez, Adela; Ásgeirsson, Bjarni; Dutta, Mouparna; Ghosh, Anindya S; Oda, Masataka; Venkatramani, Ravindra; Rao, Basuthkar J; Dandekar, Abhaya M; Goñi, Félix M

    2013-01-01

    The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that

  8. Phospholipase C β3 is a key component in the Gβγ/PKCη/PKD-mediated regulation of trans-Golgi network to plasma membrane transport

    PubMed Central

    Díaz Añel, Alberto M.

    2007-01-01

    The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gβγ (GTP-binding protein βγ subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCη (protein kinase Cη) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCη, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gβγ, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that βγ-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCβ3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCβ3, which is necessary to activate PKCη and PKD in that Golgi compartment, via DAG production. PMID:17492941

  9. Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

    PubMed Central

    Lehto, M T; Sharom, F J

    1998-01-01

    Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion

  10. Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant

    PubMed Central

    Runkel, Fabian; Hintze, Maik; Griesing, Sebastian; Michels, Marion; Blanck, Birgit; Fukami, Kiyoko; Guénet, Jean-Louis; Franz, Thomas

    2012-01-01

    Background Inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG) are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia), whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. Methodology/Principal Findings We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3mNab) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3mNab alleles are phenotypically normal. However, the presence of one Plcd3mNab allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the Plcd3mNab mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. Conclusions/Significance The Plcd3mNab mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface. PMID:22723964

  11. Nuclear phospholipase C in biological control and cancer.

    PubMed

    Ramazzotti, Giulia; Faenza, Irene; Follo, Matilde Y; Fiume, Roberta; Piazzi, Manuela; Giardino, Roberto; Fini, Milena; Cocco, Lucio

    2011-01-01

    Inositol lipids are key regulators of several cellular functions. The identification of an independent nuclear polyphosphoinositides signaling machinery has led the way to find new roles for these molecules. PI-PLC-β1 is the most extensively studied PLC isoform in the nuclear compartment and a key player in the regulation of nuclear lipid signaling. Nuclear PI-PLC-β1 is involved in cell cycle progression and differentiation in response to growth factor stimulation. A growing body of evidence has demonstrated that nuclear phosphoinositides are also involved in cancer cell generation, proliferation, and resistance to apoptosis. Evidence on ex vivo human cancer cells from patients with myelodysplastic syndromes (MDS) confirmed these observations, suggesting the involvement of PI-PLC-β1 both in the pathogenesis of the disease and in the progression of MDS to acute myeloid leukemia. These studies have offered new targets for the development of novel therapeutic strategies as well as new prognostic tools.

  12. Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

    PubMed Central

    Liu, T.; Ryan, M.; Dahlquist, F. W.; Griffith, O. H.

    1997-01-01

    Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa. PMID:9300493

  13. Primary phospholipase C and brain disorders.

    PubMed

    Yang, Yong Ryoul; Kang, Du-Seock; Lee, Cheol; Seok, Heon; Follo, Matilde Y; Cocco, Lucio; Suh, Pann-Ghill

    2016-05-01

    In the brain, the primary phospholipase C (PLC) proteins, PLCβ, and PLCγ, are activated primarily by neurotransmitters, neurotrophic factors, and hormones through G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). Among the primary PLC isozymes, PLCβ1, PLCβ4, and PLCγ1 are highly expressed and differentially distributed, suggesting a specific role for each PLC subtype in different regions of the brain. Primary PLCs control neuronal activity, which is important for synapse function and development. In addition, dysregulation of primary PLC signaling is linked to several brain disorders including epilepsy, schizophrenia, bipolar disorder, Huntington's disease, depression and Alzheimer's disease. In this review, we included current knowledge regarding the roles of primary PLC isozymes in brain disorders. PMID:26639088

  14. Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C.

    PubMed

    Müller, Alexandra; Klöppel, Christine; Smith-Valentine, Megan; Van Houten, Judith; Simon, Martin

    2012-01-01

    Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content.

  15. Evaluation and interlaboratory validation of a selective agar for phosphatidylinositol-specific phospholipase C activity using a chromogenic substrate to detect Listeria monocytogenes from foods.

    PubMed

    Jinneman, Karen C; Hunt, Jan M; Eklund, Cheryl A; Wernberg, Jane S; Sado, Patricia N; Johnson, Janelle M; Richter, Richelle S; Torres, Selene T; Ayotte, Eugene; Eliasberg, Stacey J; Istafanos, Phillip; Bass, Deborah; Kexel-Calabresa, Nancy; Lin, Wen; Barton, Curtis N

    2003-03-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) activity is a potential virulence factor and is exhibited only by the Listeria species Listeria monocytogenes and Listeria ivanovii. A chromogenic substrate for the direct detection of PI-PLC activity is available in a new medium (BCM L. monocytogenes plating agar). The use of a chromogenic substrate offers a mechanism with which to directly screen for L. monocytogenes and L. ivanovii other than the esculin used in Oxford (OXF) and Palcam (PAL) agars, which screen for all Listeria species. The specificity levels of BCM plating agar and of BCM confirmation and rhamnose agars were evaluated with 107 Listeria and 10 Bacillus species isolates. In addition, BCM L. monocytogenes plating agar was compared with standard Listeria selective agars (OXF and PAL agars) with regard to the recovery of L. monocytogenes from 2,000 food and environmental samples obtained from eight participating laboratories. A Listeria species was isolated from at least one of the agars in 209 analyses, and L. monocytogenes was isolated in 135 of these analyses. In 27 of the analyses in which L. monocytogenes was isolated, one or more of the selective differential agars used failed to isolate L. monocytogenes, and therefore the results of these analyses were discrepant. Relative to a reference method involving the use of all three agars (OXF, PAL, and BCM agars), the OXF-BCM, PAL-BCM, and OXF-PAL combinations had sensitivities of 99.3, 99.2, and 90.2%, respectively. In statistical analyses of the different combinations of agars, the OXF-BCM and BCM-PAL combinations were found to be superior to the OXF-PAL combination for the detection of L. monocytogenes.

  16. Isolation and detection of Listeria monocytogenes using fluorogenic and chromogenic substrates for phosphatidylinositol-specific phospholipase C.

    PubMed

    Restaino, L; Frampton, E W; Irbe, R M; Schabert, G; Spitz, H

    1999-03-01

    The BCM Listeria monocytogenes detection system (LMDS) consists of a selective preenrichment broth (LMPEB), selective enrichment broth (LMSEB), selective/differential plating medium (LMPM), and identification on a confirmatory plating medium (LMCM). The efficacy of the BCM LMDS was determined using pure cultures and naturally and artificially contaminated environmental sponges. The BCM LMPEB allowed the growth of Listeria and resuscitation of heat-injured L. monocytogenes. The BCM LMSEB, which contains the fluorogenic substrate 4-methylumbelliferyl-myo-inositol-1-phosphate and detects phosphatidylinositol phospholipase C (PI-PLC) activity, provided a presumptive positive test for the presence of pathogenic Listeria (L. monocytogenes and L. ivanovii) after 24 h at 35 degrees C. An initial inoculum of 10 to 100 CFU/ml of L. monocytogenes in BCM LMSEB yielded a fluorogenic response after 24 h. On BCM LMPM, L. monocytogenes and L. ivanovii were the two Listeria species forming turquoise convex colonies (1.0 to 2.5 mm in diameter) from PI-PLC activity on the chromogenic substrate, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate. L. monocytogenes was distinguished from L. ivanovii by either its fluorescence on BCM LMCM or acid production from rhamnose. False-positive organisms (Bacillus cereus, Staphylococcus aureus, Bacillus thuringiensis, and yeasts) were eliminated by at least one of the media in the BCM LMDS. Using a pure culture system, the BCM LMDS detected one to two L. monocytogenes cells from a sponge rehydrated in 10 ml of DE neutralizing broth. In an analysis of 162 environmental sponges from facilities inspected by the U.S. Department of Agriculture (USDA), the values for identification of L. monocytogenes by BCM LMDS and the USDA method were 30 and 14 sites, respectively, with sensitivity and specificity values of 85.7 and 100.0% versus 40.0 and 66.1%, respectively. No false-positive organisms were isolated by BCM LMDS, whereas 26.5% of the sponges

  17. Differential inhibitory effects of 2-azafluorenones on PI-PLC activation but not on PC-PLC- or PC-PLD-activation induced by histamine, PAF, PMA or A23187 in C6 glioma cells.

    PubMed

    Wang, Hai-Long; Wang, Li-Chuan; Wei, Jiann-Wu

    2013-02-28

    In this study, C6 glioma cells were used to test the effects of 2-azafluorenone and its related compounds on membrane phosphatidylinositol (PI) and phosphatidylcholine (PC) turnover. An increase of [³H]-labeled inositol phosphate (IP1) formation by histamine (100 μM) or A23187 (100 nM) via the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) to breakdown labeled substrate was observed, and this effect could be partially blocked by about half at 100 μM of 2-azafluorenones. Histamine induced the increase of IP1 formation, but failed to cause an increase in extracellularly releasing of [3H]choline metabolites, or intracellular accumulation of [³H]phosphscholine. However, platelet activation factor (PAF) from 0.2 to 1 μM, and phorbol 12-myristate-13-acetate (PMA) at 1 μM caused an increase in extracellularly releasing of [³H]choline metabolites, and intracellular accumulation of [³H]phosphocholine via the activation on phosphatidylcholine (PC)-PLC. These responses of PAF and PMA were not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at high concentration (10⁻⁴ M). A23187 induced an increase of intracellular [³H]choline release via the activation of PCphospholipase D (PLD). This increasing effect of 100 nM A23187 was not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at a high concentration of 10⁻⁴ M. In summary, the inhibitory effect of 2-azafluorenone and its related compound 4-methyl-2-azafluorenone was observed selectively on PIPLC, but not on PC-PLC or PC-PLD based on changes of products after the activation of these enzymes.

  18. Analysis and pharmacological targeting of phospholipase C beta interactions with G proteins.

    PubMed

    Lehmann, David M; Yuan, Chujun; Smrcka, Alan V

    2007-01-01

    Phosphatidylinositol-specific phospholipase C enzymes (PLC) catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate generating the second messengers diacylglycerol and inositol 1,4,5-triphosphate. Mammalian phosphoinositide-specific phospholipase C beta (PLCbeta) activity is regulated by the alpha(q) family of G-protein alpha subunits and by Gbetagamma subunits. Regulation of PLCbeta enzymatic activity can be assayed by reconstituting purified G-protein subunits with purified PLCbeta in the presence of phospholipid vesicles containing the substrate phosphatidylinositol 4,5-bisphosphate. This chapter describes methods for expression and purification of PLCbeta and Gbetagamma from insect cells, assay of G-protein-dependent regulation of PLC activity, and assessment of G-protein-PLC direct binding interactions. This combination of functional and direct binding analysis provides a powerful approach to characterizing PLC and G-protein interfaces, identifying inhibitors of this interaction, and potentially uncovering new modes of PLC regulation.

  19. Arabidopsis non-specific phospholipase C1: characterization and its involvement in response to heat stress

    PubMed Central

    Krčková, Zuzana; Brouzdová, Jitka; Daněk, Michal; Kocourková, Daniela; Rainteau, Dominique; Ruelland, Eric; Valentová, Olga; Pejchar, Přemysl; Martinec, Jan

    2015-01-01

    The Arabidopsis non-specific phospholipase C1 (NPC) protein family is encoded by the genes NPC1 – NPC6. It has been shown that NPC4 and NPC5 possess phospholipase C activity; NPC3 has lysophosphatidic acid phosphatase activity. NPC3, 4 and 5 play roles in the responses to hormones and abiotic stresses. NPC1, 2 and 6 has not been studied functionally yet. We found that Arabidopsis NPC1 expressed in Escherichia coli possesses phospholipase C activity in vitro. This protein was able to hydrolyse phosphatidylcholine to diacylglycerol. NPC1-green fluorescent protein was localized to secretory pathway compartments in Arabidopsis roots. In the knock out T-DNA insertion line NPC1 (npc1) basal thermotolerance was impaired compared with wild-type (WT); npc1 exhibited significant decreases in survival rate and chlorophyll content at the seventh day after heat stress (HS). Conversely, plants overexpressing NPC1 (NPC1-OE) were more resistant to HS compared with WT. These findings suggest that NPC1 is involved in the plant response to heat. PMID:26581502

  20. Arabidopsis non-specific phospholipase C1: characterization and its involvement in response to heat stress.

    PubMed

    Krčková, Zuzana; Brouzdová, Jitka; Daněk, Michal; Kocourková, Daniela; Rainteau, Dominique; Ruelland, Eric; Valentová, Olga; Pejchar, Přemysl; Martinec, Jan

    2015-01-01

    The Arabidopsis non-specific phospholipase C (NPC) protein family is encoded by the genes NPC1 - NPC6. It has been shown that NPC4 and NPC5 possess phospholipase C activity; NPC3 has lysophosphatidic acid phosphatase activity. NPC3, 4 and 5 play roles in the responses to hormones and abiotic stresses. NPC1, 2 and 6 has not been studied functionally yet. We found that Arabidopsis NPC1 expressed in Escherichia coli possesses phospholipase C activity in vitro. This protein was able to hydrolyse phosphatidylcholine to diacylglycerol. NPC1-green fluorescent protein was localized to secretory pathway compartments in Arabidopsis roots. In the knock out T-DNA insertion line NPC1 (npc1) basal thermotolerance was impaired compared with wild-type (WT); npc1 exhibited significant decreases in survival rate and chlorophyll content at the seventh day after heat stress (HS). Conversely, plants overexpressing NPC1 (NPC1-OE) were more resistant to HS compared with WT. These findings suggest that NPC1 is involved in the plant response to heat. PMID:26581502

  1. The action of phosphatidylinositol-specific phospholipases C on membranes.

    PubMed

    Low, M G; Finean, J B

    1976-01-15

    A phospholipase C prepared from lymphocytes readily hydrolysed pure phosphatidyl-inositol but was relatively ineffective against phosphatidylinositol in erythrocyte "ghosts" and rat liver microsomal fraction and also against sonicated lipid extracts from these membranes. In contrast, a phospholipase C prepared from Staphylcoccus aureus readily hydrolysed phosphatidylinositol in sonicated lipid extracts but had only low activity against purified phosphatidylinositol. Unlike the enzyme from lymphocytes, the S. aureus phospholipase C did not require Ca2+ for its activity and was inhibited by cations. The previously reported specificity of this enzyme was confirmed by our observation of hydrolysis of approx. 75% of the phosphatidylinositol in ox, sheep and cat erythrocyte "ghosts" together with no detectable effect on the major erythrocyte membrane phospholipids. The phosphatidylinositol of rat liver microsomal fraction was hydrolysed only to a maximum of 15%. Some preliminary experiments showed that approx. 60% of the phosphatidylinositol of ox or sheep erythrocytes could be hydrolysed without causing substantial haemolysis.

  2. Phospholipase C/diacylglycerol kinase-mediated signalling is required for benzothiadiazole-induced oxidative burst and hypersensitive cell death in rice suspension-cultured cells.

    PubMed

    Chen, Jie; Zhang, Weidong; Song, Fengming; Zheng, Zhong

    2007-01-01

    The involvement of phospholipase C/diacylglycerol kinase (PLC/DGK)-mediated signalling in oxidative burst and hypersensitive cell death was studied in rice suspension-cultured cells treated with benzothiadiazole (BTH) and infected by Xanthomonas oryza pv. oryza (Xoo), the causal agent of rice leaf blight disease. Treatment of rice suspension cells with BTH resulted in a significant oxidative burst, as indicated by accumulation of superoxide anion and H(2)O(2), and hypersensitive cell death, as determined by Evans blue staining. A peak in oxidative burst was detected 3-4 h after BTH treatment and hypersensitive cell death was observed 8 h after treatment. In addition, significant oxidative burst and hypersensitive cell death were detected in BTH-treated suspension cells, but not in untreated control cells, after Xoo infection. Scavengers and antioxidants of active oxygen species, e.g., superoxide dismutase, catalase, N-acetylcysteine, and flavone, reduced significantly the BTH-induced oxidative burst and hypersensitive cell death, indicating that oxidative burst is required for BTH-induced hypersensitive cell death. Expression of the PLC/DGK pathway genes, a diacylglycerol kinase gene, OsDAGK1, and a phosphoinositide-specific phospholipase C gene, OsPI-PLC1, and a defence-related EREBP transcriptional factor gene, OsBIERF3, was activated in rice cells after BTH treatment and in the BTH-treated cells after Xoo infection. Treatment of rice cells with phosphatidic acid, a phospholipid signalling molecule, resulted in the production of oxidative burst and hypersensitive cell death. However, neomycin, a PLC inhibitor, inhibited partially but not completely the production of oxidative burst, hypersensitive cell death, and expression of OsBIERF3 and OsDAGK1 induced by BTH in rice cells. These results suggest that PLC/DGK-mediated signalling plays an important role in BTH-induced oxidative burst, hypersensitive response, and activation of defence response in rice.

  3. Phosphatidylinositol Specific Phospholipase C of Plant Stems 1

    PubMed Central

    Pfaffmann, Helmut; Hartmann, Elmar; Brightman, Andrew O.; Morré, D. James

    1987-01-01

    A phosphatidylinositol-specific phospholipase C of plant stems (EC 3.1.4.10) assayed at pH 6.6 and at 30°C cleaved phosphatidylinositol such that more than 85% of the product was inositol-1-phosphate. Other phospholipids were cleaved 5 to 10% or less under these conditions. The phospholipase had both a soluble and a membrane-associated form. The soluble activity accounted for approximately 85 to 90% of the activity and 15% was associated with membranes. The membrane-associated activity was most concentrated in the plasma membranes of hypocotyl segments of both soybean (Glycine max) and bushbean (Phaseolus vulgaris). The plasma membrane location was verified by analysis of highly purified plasma membranes prepared both by aqueous two-phase partitioning and by preparative free-flow electrophoresis and from the quantitation of the activity in all major cell fractions. Internal membranes also contained phospholipase C activity but at specific activity levels of about 0.1 those present in plasma membranes. Golgi apparatus-enriched fractions from which plasma membrane contaminants were removed by two-phase partition contained the activity at specific activity levels 0.2 those of plasma membrane. Both the soluble and the membrane-associated activity was stimulated by calcium but not by calmodulin, either alone or in the presence of calcium. PMID:16665820

  4. Defective phosphatidic acid-phospholipase C signaling in diabetic cardiomyopathy.

    PubMed

    Tappia, Paramjit S; Maddaford, Thane G; Hurtado, Cecilia; Dibrov, Elena; Austria, J Alejandro; Sahi, Nidhi; Panagia, Vincenzo; Pierce, Grant N

    2004-03-26

    The effects of exogenous phosphatidic acid (PA) on Ca2+ transients and contractile activity were studied in cardiomyocytes isolated from chronic streptozotocin-induced diabetic rats. In control cells, 25 microM PA induced a significant increase in active cell shortening and Ca2+ transients. PA increased IP3 generation in the control cardiomyocytes and its inotropic effects were blocked by a phospholipase C inhibitor. In cardiomyocytes from diabetic rats, PA induced a 25% decrease in active cell shortening and no significant effect on Ca2+ transients. Basal and PA-induced IP3 generation in diabetic rat cardiomyocytes was 3-fold lower as compared to control cells. Sarcolemmal membrane PLC activity was impaired. Insulin treatment of the diabetic animals resulted in a partial recovery of PA responses. Our results, therefore, identify an important defect in the PA-PLC signaling pathway in diabetic rat cardiomyocytes, which may have significant implications for heart dysfunction during diabetes. PMID:15003542

  5. Aberrant accumulation of phospholipase C-delta in Alzheimer brains.

    PubMed Central

    Shimohama, S.; Homma, Y.; Suenaga, T.; Fujimoto, S.; Taniguchi, T.; Araki, W.; Yamaoka, Y.; Takenawa, T.; Kimura, J.

    1991-01-01

    Since phosphoinositide-specific phospholipase C (PLC) is one of the key molecules in signal transduction, the authors assessed its involvement in Alzheimer's disease (AD). Immunostaining of a specific antibody against the PLC isozyme, PLC-delta, demonstrated that this enzyme was abnormally accumulated in neurofibrillary tangles (NFT), the neurites surrounding senile plaque (SP) cores, and neuropil threads in AD brains. Western blot analysis confirmed that PLC-delta was concentrated in the paired helical filament (PHF)-rich fraction of AD brains. Antibodies to other PLC isozymes did not produce positive immunostaining of these pathologic structures. Moreover, diffuse and amorphous deposits of PLC-delta were found to precede the accumulation of fibrillary deposits. These results suggest that PLC-delta accumulation is a crucial event that ultimately may contribute to the formation of PHF. Images Figure 1 Figure 2 Figure 3 PMID:1928298

  6. Defective phosphatidic acid-phospholipase C signaling in diabetic cardiomyopathy.

    PubMed

    Tappia, Paramjit S; Maddaford, Thane G; Hurtado, Cecilia; Dibrov, Elena; Austria, J Alejandro; Sahi, Nidhi; Panagia, Vincenzo; Pierce, Grant N

    2004-03-26

    The effects of exogenous phosphatidic acid (PA) on Ca2+ transients and contractile activity were studied in cardiomyocytes isolated from chronic streptozotocin-induced diabetic rats. In control cells, 25 microM PA induced a significant increase in active cell shortening and Ca2+ transients. PA increased IP3 generation in the control cardiomyocytes and its inotropic effects were blocked by a phospholipase C inhibitor. In cardiomyocytes from diabetic rats, PA induced a 25% decrease in active cell shortening and no significant effect on Ca2+ transients. Basal and PA-induced IP3 generation in diabetic rat cardiomyocytes was 3-fold lower as compared to control cells. Sarcolemmal membrane PLC activity was impaired. Insulin treatment of the diabetic animals resulted in a partial recovery of PA responses. Our results, therefore, identify an important defect in the PA-PLC signaling pathway in diabetic rat cardiomyocytes, which may have significant implications for heart dysfunction during diabetes.

  7. Recent research progress with phospholipase C from Bacillus cereus.

    PubMed

    Lyu, Yan; Ye, Lidan; Xu, Jun; Yang, Xiaohong; Chen, Weiwei; Yu, Hongwei

    2016-01-01

    Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLC Bc , a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLC Bc remain challenging. Herein, we summarize the research progress regarding PLC Bc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLC Bc . This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLC Bc and its application. PMID:26437973

  8. Down-regulation of phospholipase C-beta1 following chronic muscarinic receptor activation.

    PubMed

    Sorensen, S D; Linseman, D A; Fisher, S K

    1998-04-01

    To determine whether prolonged activation of a phospholipase C-coupled receptor can lead to a down-regulation of its effector enzyme, SH-SY5Y neuroblastoma cells were incubated for 24 h with the muscarinic receptor agonist, oxotremorine-M. Under these conditions, significant reductions (46-53%) in muscarinic cholinergic receptor density, G(alphaq/11) and phospholipase C-beta1 (but not the beta3-or gamma1 isoforms) were observed. These results suggest that a selective down-regulation of phospholipase C-beta1 may play a role in adaptation to chronic muscarinic receptor activation. PMID:9617763

  9. Stimulation and binding of myocardial phospholipase C by phosphatidic acid.

    PubMed

    Henry, R A; Boyce, S Y; Kurz, T; Wolf, R A

    1995-08-01

    Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.

  10. Structure, function, and control of phosphoinositide-specific phospholipase C.

    PubMed

    Rebecchi, M J; Pentyala, S N

    2000-10-01

    Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol. The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs. New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies. Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response. Clues to their true biological roles were also obtained. Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development. In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function. PMID:11015615

  11. Effects of Phospholipase C on Fusarium graminearum Growth and Development.

    PubMed

    Zhu, Qili; Zhou, Benguo; Gao, Zhengliang; Liang, Yuancun

    2015-12-01

    Phospholipase C (PLC) plays important roles in regulating various biological processes in eukaryotes. Currently, little is known about the function of PLC in filamentous fungi, especially the plant pathogenic fungi. Fusarium graminearum is the causal agent of Fusarium head blight in many cereal crops. BLAST search revealed that Fusarium genome contains six FgPLC genes. Using quantitative RT-PCR, different FgPLC gene expressions in mycelia were analyzed. To investigate the role of FgPLC in F. graminearum biology, a pharmacological study using a known inhibitor of PLC (U73122) was conducted. Results showed that inhibition of FgPLC resulted in significant alterations of mycelial growth, conidiation, conidial germination, perithecium formation, and expressions of Tri5 and Tri6 genes. As expected, the treatment of F. graminearum with U73343, an inactive analog of U73122, showed no effect on F. graminearum biology. Our results suggested strongly that FgPLC plays important roles in F. graminearum growth and development. PMID:26316232

  12. Effects of Phospholipase C on Fusarium graminearum Growth and Development.

    PubMed

    Zhu, Qili; Zhou, Benguo; Gao, Zhengliang; Liang, Yuancun

    2015-12-01

    Phospholipase C (PLC) plays important roles in regulating various biological processes in eukaryotes. Currently, little is known about the function of PLC in filamentous fungi, especially the plant pathogenic fungi. Fusarium graminearum is the causal agent of Fusarium head blight in many cereal crops. BLAST search revealed that Fusarium genome contains six FgPLC genes. Using quantitative RT-PCR, different FgPLC gene expressions in mycelia were analyzed. To investigate the role of FgPLC in F. graminearum biology, a pharmacological study using a known inhibitor of PLC (U73122) was conducted. Results showed that inhibition of FgPLC resulted in significant alterations of mycelial growth, conidiation, conidial germination, perithecium formation, and expressions of Tri5 and Tri6 genes. As expected, the treatment of F. graminearum with U73343, an inactive analog of U73122, showed no effect on F. graminearum biology. Our results suggested strongly that FgPLC plays important roles in F. graminearum growth and development.

  13. Incidence and identification of phospholipase C-producing bacteria in fresh and spoiled homogenized milk.

    PubMed

    Fox, C W; Chrisope, G L; Marshall, R T

    1976-11-01

    Bacteria which produced phospholipase C were isolated from 13 of 34 fresh and 15 of 35 spoiled samples of homogenized milk. No single off flavor was assigned consistently to samples with phospholipase producers, but 75% of them were bitter. Pseudomonads constituted 62% of the isolates. Other phospholipase C-producing genera and their numbers were Acinetobacter, two; Alcaligenes, three; Bacillus, two; Citrobacter, one; Enterobacter, three; and Flavobacterium, two. Two unidentified yeasts also were isolated.

  14. Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C.

    PubMed

    Low, M G; Finean, J B

    1977-10-01

    Purified phosphatidylinositol-specific phospholipase C from Staphylococcus aureus released a substantial proportion of the total alkaline phosphatase activity from a wide range of tissues from several mammalian species. Co-purification of the phospholipase C and alkaline phosphatase-releasing activities and the inhibition of both these activities by iso-osmotic salt solutions suggested that the releasing effect was unlikely to be due to a contaminant.

  15. Effects of intra- and extracellularly applied phospholipases C on excitability of squid giant axons.

    PubMed

    Nakajima, M; Ikezawa, H; Yamagishi, S

    1986-01-01

    The effects of phospholipases C on the membrane excitability of the squid giant axon were investigated using phosphatidylcholine-hydrolyzing phospholipase C and sphingomyelinase C of Bacillus cereus, and phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis. When the squid axon was perfused internally with phosphatidylcholine-hydrolyzing phospholipase C in KF or K-glutamate solution, the action potential was blocked in 4-7 min and membrane resistance decreased with time to a level less than one-tenth that of control. These effects were irreversible. When the axon was perfused internally with sphingomyelinase C in KF solution, the action potential was decreased to 30% in 3 min. Perfusion with enzyme-free KF solution fully restored the action potential. When the axon was perfused internally with phosphatidylinositol-specific phospholipase C in K - glutamate solution, the action potential was gradually decreased and blocked after 10 min. Perfusion with enzyme-free KF solution restored the action potential by 70%. When phosphatidylcholine-hydrolyzing phospholipase C was applied externally to the squid axon, the action potential and the membrane resistance were slowly but irreversibly decreased. These results suggest that membrane phospholipids, such as phosphatidylcholine and phosphatidylinositol, may be associated with the excitability of the membrane of the squid axon.

  16. Phosphatidylinositol 4,5-bisphosphate phospholipase C and phosphomonoesterase in Dunaliella salina membranes

    SciTech Connect

    Einspahr, K.J.; Peeler, T.C.; Thompson, G.A. Jr. )

    1989-07-01

    In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous ({sup 3}H)phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}). Based on release of ({sup 3}H)inositol phosphates, the plasma membrane exhibited a PIP{sub 2}-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast bottom phase (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of ({sup 3}H)inositol phosphates released were recovered as ({sup 3}H)inositol trisphosphate (IP{sub 3}). These results suggest a plausible mechanism for the rapid breakdown of PIP{sub 2} and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. The authors have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive, reaching maximal activity at 10 micromolar free (Ca{sup 2+}). They also report here that 100 micromolar GTP{gamma}S stimulates phospholipase C activity over a range of free (Ca{sup 2+}). Together, these results provide evidence that the plasma membrane PIP{sub 2}-phospholipase C of D. salina may be subject to Ca{sup 2+} and G-protein regulation.

  17. PLC-δ1-Lf, a novel N-terminal extended phospholipase C-δ1.

    PubMed

    Kim, Na Young; Ahn, Sang Jung; Kim, Moo-Sang; Seo, Jung Soo; Kim, Bo Seong; Bak, Hye Jin; Lee, Jin Young; Park, Myoung-Ae; Park, Ju Hyeon; Lee, Hyung Ho; Chung, Joon Ki

    2013-10-10

    Phospholipase C-δ (PLC-δ), a key enzyme in phosphoinositide turnover, is involved in a variety of physiological functions. The widely expressed PLC-δ1 isoform is the best characterized and the most well understood phospholipase family member. However, the functional and molecular mechanisms of PLC-δ1 remain obscure. Here, we identified that the N-terminal region of mouse PLC-δ1 gene has two variants, a novel alternative splicing form, named as long form (mPLC-δ1-Lf) and the previously reported short form (mPLC-δ1-Sf), having exon 2 and exon 1, respectively, while both the gene variants share exons 3-16 for RNA transcription. Furthermore, the expression, identification and enzymatic characterization of the two types of PLC-δ1 genes were compared. Expression of mPLC-δ1-Lf was found to be tissue specific, whereas mPLC-δ1-Sf was widely distributed. The recombinant mPLC-δ1-Sf protein exhibited higher activity than recombinant mPLC-δ1-Lf protein. Although, the general catalytic and regulatory properties of mPLC-δ1-Lf are similar to those of PLC-δ1-Sf isozyme, the mPLC-δ1-Lf showed some distinct regulatory properties, such as tissue-specific expression and lipid binding specificity, particularly for phosphatidylserine.

  18. Vascular smooth muscle cell spreading onto fibrinogen is regulated by calpains and phospholipase C.

    PubMed

    Paulhe, F; Bogyo, A; Chap, H; Perret, B; Racaud-Sultan, C

    2001-11-01

    Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of (32)P-labeled phosphatidic acid and phosphatidylinositol-3,4-bisphosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4-bisphosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains.

  19. Regulation of phosphatidylcholine biosynthesis in cultured chick embryonic muscle treated with phospholipase C.

    PubMed

    Sleight, R; Kent, C

    1980-11-25

    Cultures of embryonic chick muscle cells grown in medium containing phospholipase C from Clostridium perfringens incorporated [3H]choline into lipid at a rate 3- to 5-fold higher than control cultures. To determine the mechanism by which stimulation of phosphatidylcholine synthesis occurred in phospholipase C-treated cells, activities of enzymes and levels of intermediates in the biosynthetic pathway for phosphatidylcholine were examined. Activities of choline kinase, choline phosphotransferase, glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, acylglycerol-3-phosphate acyltransferase, and phosphatidic acid phosphatase in phospholipase C-treated cells were the same or only slightly higher than in control cells. CTP:phosphocholine cytidylyltransferase, on the other hand, was 3 times as active in homogenates from phospholipase C-treated cells. Levels of phosphocholine decreased and levels of CDP-choline increased in phospholipase C-treated cells, and a calculation of the disequilibrium ratio indicated that the cytidylyltransferase reaction was not at equilibrium. The cytidylyltransferase was, thus, identified as the regulatory enzyme for choline flux in these cells. The cytidylyltransferase was located in both the cytosolic and particulate fractions from cultured muscle cells and a much larger portion of enzyme activity was associated with the particulate fraction in cells treated with phospholipase C. Sonicated preparations of total chick lipids, phosphatidylethanolamine, and phosphatidylserine greatly stimulated the cytosolic cytidylyltransferase activity but had no effect on the particulate enzyme. Neither stimulation of incorporation of [3H]choline into lipid nor activation of the cytidylyltransferase was dependent on protein synthesis. A model for the mechanism of regulation of phosphatidylcholine synthesis in embryonic chick muscle is presented.

  20. Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1.

    PubMed

    Taguchi, R; Ikezawa, H

    1987-10-01

    The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Lipid vesicle fusion induced by phospholipase C activity in model bile.

    PubMed

    Little, T E; Madani, H; Lee, S P; Kaler, E W

    1993-02-01

    Using a system of phosphatidylcholine-cholesterol vesicles to model the vesicle phase of mammalian bile (1:1 molar ratio) we evaluated whether very small amounts of C. perfringens phospholipase C activity (0.5-6.5 nmol/min per ml) could lead to vesicle fusion, a precursor step for cholesterol precipitation in gallbladder bile. Quasielastic light scattering spectroscopy (QLS) was used to monitor vesicle growth and aggregation in model bile (0.89 mM total lipid) in the presence of phospholipase C. Vesicle growth over 2 h could be detected with phospholipase activity as little as 0.5 nmol/min per ml. Vesicle growth was sustainable over days in the absence of Ca2+ once as little as 3-7 mol% diacylglycerol had been generated as a result of the initial phospholipase C treatment. The presence of fusion intermediates was confirmed using transmission electron microscopy. In addition, kinetically slow vesicle fusion with intravesicle content mixing and minimal leakage was also confirmed by fluorescence spectroscopy using two populations of vesicles containing 5 mM TbCl3 or 50 mM dipicolinic acid. Efficient fusion (40% maximum fluorescence) was obtained at 30 min at 25 degrees C with phospholipase C activity. This level of enzyme activity approximates that found in human gallbladder bile (1.2 nmol/min per ml). We conclude that the hydrolysis products of phospholipase C activity can, in very small amounts (3-7 mol% diacylglycerol), lead to destabilization and fusion of cholesterol-saturated biliary vesicles. A reappraisal of the importance of phospholipase C hydrolysis products in the pathogenesis of cholesterol gallstones is warranted based on these observations.

  2. A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility

    PubMed Central

    Kashir, Junaid; Konstantinidis, Michalis; Jones, Celine; Lemmon, Bernadette; Chang Lee, Hoi; Hamer, Rebecca; Heindryckx, Bjorn; Deane, Charlotte M.; De Sutter, Petra; Fissore, Rafael A.; Parrington, John; Wells, Dagan; Coward, Kevin

    2012-01-01

    BACKGROUND Male factor and idiopathic infertility contribute significantly to global infertility, with abnormal testicular gene expression considered to be a major cause. Certain types of male infertility are caused by failure of the sperm to activate the oocyte, a process normally regulated by calcium oscillations, thought to be induced by a sperm-specific phospholipase C, PLCzeta (PLCζ). Previously, we identified a point mutation in an infertile male resulting in the substitution of histidine for proline at position 398 of the protein sequence (PLCζH398P), leading to abnormal PLCζ function and infertility. METHODS AND RESULTS Here, using a combination of direct-sequencing and mini-sequencing of the PLCζ gene from the patient and his family, we report the identification of a second PLCζ mutation in the same patient resulting in a histidine to leucine substitution at position 233 (PLCζH233L), which is predicted to disrupt local protein interactions in a manner similar to PLCζH398P and was shown to exhibit abnormal calcium oscillatory ability following predictive 3D modelling and cRNA injection in mouse oocytes respectively. We show that PLCζH233L and PLCζH398P exist on distinct parental chromosomes, the former inherited from the patient's mother and the latter from his father. Neither mutation was detected utilizing custom-made single-nucleotide polymorphism assays in 100 fertile males and females, or 8 infertile males with characterized oocyte activation deficiency. CONCLUSIONS Collectively, our findings provide further evidence regarding the importance of PLCζ at oocyte activation and forms of male infertility where this is deficient. Additionally, we show that the inheritance patterns underlying male infertility are more complex than previously thought and may involve maternal mechanisms. PMID:22095789

  3. Non-specific phospholipase C4 mediates response to aluminum toxicity in Arabidopsis thaliana.

    PubMed

    Pejchar, Přemysl; Potocký, Martin; Krčková, Zuzana; Brouzdová, Jitka; Daněk, Michal; Martinec, Jan

    2015-01-01

    Aluminum ions (Al) have been recognized as a major toxic factor for crop production in acidic soils. The first indication of the Al toxicity in plants is the cessation of root growth, but the mechanism of root growth inhibition is largely unknown. Here we examined the impact of Al on the expression, activity, and function of the non-specific phospholipase C4 (NPC4), a plasma membrane-bound isoform of NPC, a member of the plant phospholipase family, in Arabidopsis thaliana. We observed a lower expression of NPC4 using β-glucuronidase assay and a decreased formation of labeled diacylglycerol, product of NPC activity, using fluorescently labeled phosphatidylcholine as a phospholipase substrate in Arabidopsis WT seedlings treated with AlCl3 for 2 h. The effect on in situ NPC activity persisted for longer Al treatment periods (8, 14 h). Interestingly, in seedlings overexpressing NPC4, the Al-mediated NPC-inhibiting effect was alleviated at 14 h. However, in vitro activity and localization of NPC4 were not affected by Al, thus excluding direct inhibition by Al ions or possible translocation of NPC4 as the mechanisms involved in NPC-inhibiting effect. Furthermore, the growth of tobacco pollen tubes rapidly arrested by Al was partially rescued by the overexpression of AtNPC4 while Arabidopsis npc4 knockout lines were found to be more sensitive to Al stress during long-term exposure of Al at low phosphate conditions. Our observations suggest that NPC4 plays a role in both early and long-term responses to Al stress. PMID:25763003

  4. Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization.

    PubMed Central

    Berka, R M; Vasil, M L

    1982-01-01

    Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free. Images PMID:6811552

  5. Glycosyl-phosphatidylinositol (GPI)-anchored renal dipeptidase is released by a phospholipase C in vivo.

    PubMed

    Park, Sung Wook; Choi, Kyong; Lee, Hwanghee Blaise; Park, Sung Kwang; Turner, Anthony J; Hooper, Nigel M; Park, Haeng Soon

    2002-01-01

    The release mechanism of the glycosyl-phosphatidylinositol (GPI)-anchored renal dipeptidase (EC 3.4.13.19) in vivo has been investigated. Triton X-114 phase separation indicated that the dipeptidase is exclusively present as a hydrophilic form in urine from porcine, rat, rabbit and human. Western blot analysis of human and porcine purified dipeptidase and the urine concentrates with anti-(cross-reacting determinant) serum demonstrated the presence of inositol 1,2-cyclic monophosphate indicating that the renal dipeptidase had been released from the membrane by the action of a phospholipase C. This is the first direct evidence for cleavage of a human GPI-anchored protein by a responsible phospholipase C in vivo.

  6. Modification of erythrocyte membranes by a purified phosphatidylinositol-specific phospholipase C (Staphylococcus aureus).

    PubMed

    Low, M G; Finean, J B

    1977-02-15

    A phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was purified by a three-step procedure. The specific activity of the purified enzyme was approx. 6000 times that of the culture supernatant, with an overall recovery of approx. 10%. Estimation of the molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by gel filtration gave values of 33000 and 20000 respectively. A thiol group appears to be necessary for the activity of the enzyme. The purified enzyme had no detectable delta-haemolytic activity and was unable to hydrolyse S. aureus phospholipids. Phosphatidyl-inositol in erythrocyte 'ghosts' was readily hydrolysed by the purified phospholipase C. However, in contrast with our previous preliminary observations, phosphatidylinositol in intact erythrocytes was not significantly hydrolysed. These results suggest that at least 75-80% of the phosphatidylinositol is located at the inner leaflet of the membrane.

  7. Lysis of erythrocytes from stored human blood by phospholipase C (Bacillus cereus).

    PubMed Central

    Little, C; Rumsby, M G

    1980-01-01

    The ability of phospholipase C (Bacillus cereus) to lyse erythrocytes from human blood that had been stored under Transfusion Service conditions for up to 16 weeks has been examined. When incubated at 20 degrees C with enzyme (0.03 mg/ml, 55 units/ml) for up to 1 h fresh erythrocytes were not lysed. After about 4 weeks of storage a population of very readily lysed erythrocytes appeared. The morphological changes in erythrocytes from blood stored up to 16 weeks were examined by scanning electron microscopy. The proportion of very readily lysed erythrocytes correlated well with the proportion of spheroechinocytes I. This morphological form was shown to be preferentially removed by phospholipase C and before lysis a transient appearance of smooth spheres occurred. The decrease in blood ATP concentrations on storage was measured and found to correlate with the disappearance of discoid erythrocyte forms, but not directly with the increased susceptibility of the erythrocytes to lysis by the enzyme. However, erythrocytes of up to at least 15 weeks of age could be made less susceptible to lysis by pre-incubation in a medium designed to cause intracellular regeneration of ATP. During the lysis of spheroechinocytes I by electrophoretically pure recrystallized phospholipase C a rapid degradation of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphatidylinositol) occurred together with a slower degradation of sphingomyelin. Images PLATE 2 PLATE 1 PMID:6773524

  8. Enhanced bradykinin-stimulated phospholipase C activity in murine embryonic stem cells lacking the G-protein alphaq-subunit.

    PubMed Central

    Ricupero, D A; Polgar, P; Taylor, L; Sowell, M O; Gao, Y; Bradwin, G; Mortensen, R M

    1997-01-01

    The gene coding for the G-protein alphaq subunit was interrupted by homologous recombination in murine embryonic stem cells (alphaq-null ES cells) as detected by Southern analysis and reverse-transcriptase PCR. The bradykinin (BK) B2 receptor was stably transfected into wild-type (WT) alphai-2-null and alphaq-null ES cells. The B2 receptor bound BK with high affinity and mobilized Ca2+. BK also activated phospholipase C (PLC), as determined by total inositol phosphate (IP) accumulation in a Bordetella pertussis toxin- and genistein-insensitive manner. In WT and alphai-2-null ES cells, BK increased IP levels approx. 4-fold above baseline. Most interestingly, in alphaq-null ES cells, BK increased IP accumulation approx. 9-fold above baseline. Re-expression of alphaq in alphaq-null ES cells resulted in normalization of the BK-stimulated IP accumulation (4-fold above baseline). These results suggest that the B2 receptor activates PLC through more than one member of the Gq family. Additionally, the absence of alphaq alters the kinetics of IP generation, which may reflect intrinsic characteristics of individual members of the Gq family or a decreased susceptibility to heterologous regulation in the alphaq-null ES cells, thus allowing for a more sustained generation of IP. PMID:9581559

  9. The phenotype of a phospholipase C (plc-1) mutant in a filamentous fungus, Neurospora crassa.

    PubMed

    Lew, Roger R; Giblon, Rachel E; Lorenti, Miranda S H

    2015-09-01

    In the filamentous fungus Neurospora crassa, phospholipase C may play a role in hyphal extension at the growing tips as part of a growth-sensing mechanism that activates calcium release from internal stores to mediate continued expansion of the hyphal tip. One candidate for a tip-localized phospholipase C is PLC-1. We characterized morphology and growth characteristics of a knockout mutant (KO plc-1) and a RIP mutated strain (RIP plc-1) (missense mutations and a nonsense mutation render the gene product non-functional). Growth and hyphal cytology of wildtype and KO plc-1 were similar, but the RIP plc-1 mutant grew slower and exhibited abnormal membrane structures at the hyphal tip, imaged using the fluorescence dye FM4-64. To test for causes of the slower growth of the RIP plc-1 mutant, we examined its physiological poise compared to wildtype and the KO plc-1 mutant. The electrical properties of all three strains and the electrogenic contribution of the plasma membrane H(+)-ATPase (identified by cyanide inhibition) were the same. Responses to high osmolarity were also similar. However, the RIP plc-1 mutant had a significantly lower turgor, a possible cause of its slower growth. While growth of all three strains was inhibited by the phospholipase C inhibitor 3-nitrocoumarin, the RIP plc-1 mutant did not exhibit hyphal bursting after addition of the inhibitor, observed in both wildtype and the KO plc-1 mutant. Although the plc-1 gene is not obligatory for tip growth, the phenotype of the RIP plc-1 mutant - abnormal tip cytology, lower turgor and resistance to inhibitor-induced hyphal bursting - suggest it does play a role in tip growth. The expression of a dysfunctional plc-1 gene may cause a shift to alternative mechanism(s) of growth sensing in hyphal extension.

  10. Short-chain phosphatidylinositol conformation and its relevance to phosphatidylinositol-specific phospholipase C.

    PubMed

    Zhou, C; Garigapati, V; Roberts, M F

    1997-12-16

    The solution conformation of chiral diheptanoylphosphatidylinositol (D- and L-inositol isomers) has been characterized by NMR spectroscopy. A positive NOE between the inositol C2 proton and an sn-3 glycerol CH2 proton has been observed in the D- but not in the L-inositol isomer of diheptanoylphosphatidylinositol (PI). Computer modeling using QUANTA constrained by this NOE and ring coupling constants suggests that the inositol ring is nearly parallel to the chain packing direction, leaving the phosphate ester accessible to attack by phosphatidylinositol-specific phospholipase C enzymes. In this model, the hydroxyl groups in the 2- and 6-positions of inositol form hydrogen bonds with the pro-R and ester oxygens, respectively. Chemical shifts and 13C spin-lattice relaxation times were also used to assess conformation and lipid dynamics in monomer and micelle states. The 13C T1's of inositol C2 and C6 in monomeric phosphatidylinositol were markedly less than for other inositol ring carbons. These results are consistent with the hydrogen bonds to the phosphate constraining the motions of C2 and C6. Diheptanoylphosphatidyl-2-O-methylinositol is a good inhibitor of PI-specific phospholipase C because it blocks the initial phosphotransferase step in PI hydrolysis. Introduction of the methyl group on the C-2 hydroxyl group lowers the CMC of the derivative compared to diheptanoylphosphatidylinositol. However, an NOE between an sn-3 glycerol proton and the inositol C2 proton constrains the orientation of the inositol ring with respect to the glycerol backbone in a conformation similar to diheptanoylphosphatidylinositol. Modeling of the 2-O-methylinositol derivative suggests that the methyl group blocks one side of the phosphate, consistent with the observation that nonspecific phospholipase C enzymes which are able to hydrolyze PI, albeit poorly, are unable to hydrolyze diheptanoylphosphatidyl-2-O-methylinositol.

  11. Phospholipase C activation during elicitation of the oxidative burst in cultured plant cells.

    PubMed

    Legendre, L; Yueh, Y G; Crain, R; Haddock, N; Heinstein, P F; Low, P S

    1993-11-25

    Although phospholipase C hydrolysis of polyphosphoinositides constitutes one of the major second messenger pathways in animal cells, its participation in signal transduction in higher plants has not been established. To determine whether activation of phosphatidylinositol-directed phospholipase C might be involved in signaling the elicitor-induced oxidative burst in plants, suspension-cultured soybean cells were treated with two stimulants of the H2O2 burst and examined for polyphosphoinositide turnover. Both polygalacturonic acid elicitor and the G protein activator, mastoparan, promoted a transient increase in inositol 1,4,5-trisphosphate (IP3) content that exceeded basal IP3 levels (0.9 +/- 0.4 pmol of IP3/10(6) cells, n = 28) by 2.6- and 7-fold, respectively. In each case, intracellular IP3 content reached a maximum at 1 min post-stimulation and declined to near basal levels during the subsequent 5-10 min. Neomycin sulfate, an inhibitor of polyphosphoinositide hydrolysis, blocked the IP3 transient, and Mas-17, an inactive analogue of mastoparan, induced no change in IP3. Thin layer chromatography of lipid extracts of the soybean cells corroborated the above results by revealing a rapid decrease in phosphatidyl-inositol monophosphate and phosphatidylinositol 4,5-bisphosphate following polygalacturonic acid elicitor and mastoparan (but not Mas-17) stimulation. Since the rise in IP3 preceded H2O2 production and since neomycin sulfate inhibited the appearance of both, we hypothesize that phospholipase C activation might constitute one pathway by which elicitors trigger the soybean oxidative burst.

  12. High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

    PubMed Central

    Truan, Daphné; Vasil, Adriana; Stonehouse, Martin; Vasil, Michael L.; Pohl, Ehmke

    2013-01-01

    The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95 kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced μ-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4 Å, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75 Å. PMID:23201280

  13. Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C.

    PubMed

    Low, M G; Finean, J B

    1978-04-20

    The release of plasma membrane ecto-enzymes by a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was investigated. There was no effect on L-leucyl-beta-naphthylamidase, alkaline phosphodeisterase I and Ca2+- or MG2+-ATPase, but substantial proportions of the alkaline phosphatase and 5-nucleotidase were released. There was no simultaneous release of phospholipid and the solubilized enzymes were not exluded from Sepharose 6-B. It was therefore concluded that release was not a secondary consequence of membrane vesiculation but occurred as a result of the disruption of specific interactions involving the phosphatidylinositol molecule.

  14. Serotonin 5-HT(2A) receptor activation induces 2-arachidonoylglycerol release through a phospholipase c-dependent mechanism.

    PubMed

    Parrish, Jason C; Nichols, David E

    2006-11-01

    To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors. PMID:17010161

  15. Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens.

    PubMed

    Fach, P; Guillou, J P

    1993-01-01

    A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10(2) bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans. The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.

  16. Phosphatidylinositol-specific phospholipase C activity of chromaffin granule-binding proteins

    SciTech Connect

    Creutz, C.E.; Dowling, L.G.; Kyger, E.M.; Franson, R.C.

    1985-06-25

    Using (U-/sup 14/C)phosphatidylinositol as substrate, Ca/sup 2 +/-dependent phospholipase C activity was detected in a group of bovine adrenal medullary proteins that bind to chromaffin granule membranes in the presence of Ca/sup 2 +/. The activity was maximal at neutral pH and represented an 80- to 240-fold enrichment of adrenal medullary cytosol phospholipase C activity measured at pH 7.3. The stimulation of activity by Ca/sup 2 +/ was complex; no activity was present in the absence of Ca/sup 2 +/, 25% activation occurred at 1 microM Ca/sup 2 +/, and full activation at 5 mM Ca/sup 2 +/. The enzyme bound to chromaffin granule membranes in the presence of 2 mM Ca/sup 2 +/ but was released at 40 microM Ca/sup 2 +/, suggesting that intrinsic enzyme activity may be regulated by (Ca/sup 2 +/) at 1 microM, but additional activation at higher concentrations of Ca/sup 2 +/ is seen in vitro as a result of Ca/sup 2 +/-dependent binding of the active enzyme to substrate-containing membranes. This enzyme may generate diacylglycerol and phosphorylated inositol to act as intracellular messengers in the vicinity of the chromaffin granule membrane during the process of exocytosis.

  17. Substance P receptor desensitization requires receptor activation but not phospholipase C

    SciTech Connect

    Sugiya, Hiroshi; Putney, J.W. Jr. )

    1988-08-01

    Previous studies have shown that exposure of parotid acinar cells to substance P at 37{degree}C results in activation of phospholipase C, formation of ({sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to {approximately}20% of control values, IP{sub 3} formation was completely inhibited, but desensitization was unaffected. When cells were treated with antimycin in the absence of glucose, cellular ATP was decreased to {approximately}5% of control values, and both IP{sub 3} formation and desensitization were blocked. A series of substance P-related peptides increased the formation of ({sup 3}H)IP{sub 3} and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, (D-Pro{sup 2}, D-Try{sup 7,9})-substance P, inhibited substance P-induced IP{sub 3} formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP{sub 3} formation requires agonist activation of a P-type substance P receptor, and that one or more cellular ATP-dependent processes are required for this reaction. However, activation of phospholipase C and the generation of inositol phosphates does not seem to be a prerequisite for desensitization.

  18. Saccharomyces cerevisiae phospholipase C regulates transcription of Msn2p-dependent stress-responsive genes.

    PubMed

    Demczuk, Agnieszka; Guha, Nilanjan; Nguyen, Peter H; Desai, Parima; Chang, Jennifer; Guzinska, Katarzyna; Rollins, Janet; Ghosh, Chandra C; Goodwin, Leslie; Vancura, Ales

    2008-06-01

    Phosphatidylinositol phosphates are involved in signal transduction, cytoskeletal organization, and membrane trafficking. Inositol polyphosphates, produced from phosphatidylinositol phosphates by the phospholipase C-dependent pathway, regulate chromatin remodeling. We used genome-wide expression analysis to further investigate the roles of Plc1p (phosphoinositide-specific phospholipase C in Saccharomyces cerevisiae) and inositol polyphosphates in transcriptional regulation. Plc1p contributes to the regulation of approximately 2% of yeast genes in cells grown in rich medium. Most of these genes are induced by nutrient limitation and other environmental stresses and are derepressed in plc1 Delta cells. Surprisingly, genes regulated by Plc1p do not correlate with gene sets regulated by Swi/Snf or RSC chromatin remodeling complexes but show correlation with genes controlled by Msn2p. Our results suggest that the increased expression of stress-responsive genes in plc1 Delta cells is mediated by decreased cyclic AMP synthesis and protein kinase A (PKA)-mediated phosphorylation of Msn2p and increased binding of Msn2p to stress-responsive promoters. Accordingly, plc1 Delta cells display other phenotypes characteristic of cells with decreased PKA activity. Our results are consistent with a model in which Plc1p acts together with the membrane receptor Gpr1p and associated G(alpha) protein Gpa2p in a pathway separate from Ras1p/Ras2p and converging on PKA.

  19. Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes.

    PubMed

    Allan, D; Low, M G; Finean, J B; Michell, R H

    1975-12-01

    When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis. Only phosphatidylcholine and sphingomyelin were attacked. Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate. Up to 12% of the cell phospholipid could be converted into phosphatidate in this way. Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack. Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles. This change appeared to be due to ceramide and de and diacylglycerol accumulation rather than to increased phosphatidate content of the cells.

  20. An autoinhibitory helix in the C-terminal region of phospholipase C-[beta] mediates G[alpaha subscript q] activation

    SciTech Connect

    Lyon, Angeline M.; Tesmer, Valerie M.; Dhamsania, Vishan D.; Thal, David M.; Gutierrez, Joanne; Chowdhury, Shoaib; Suddala, Krishna C.; Northup, John K.; Tesmer, John J.G.

    2012-03-16

    The enzyme phospholipase C-{beta} (PLC{beta}) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the G{sub q} family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLC{beta} (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLC{beta}3 considerably increase basal activity and diminish stimulation by G{alpha}{sub q}. G{alpha}{sub q} binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLC{beta}.

  1. Synthesis of substrates for periodate-coupled assay of phospholipases C and sphingomyelinases.

    PubMed

    Larsen, Kira Løw; Andersen, Rokhsana J; Brask, Jesper

    2016-09-01

    A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high KM and low kcat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations. PMID:27444331

  2. Regulation of Phosphatidylinositol-specific Phospholipase C at the Nuclear Envelope in Cardiac Myocytes

    PubMed Central

    Smrcka, Alan V.

    2014-01-01

    Phosphatidylinositol 4,5 bisphosphate hydrolysis at the plasma membrane by phospholipase C is one of the major hormone regulated intracellular signaling systems. The system generates the diffusible second messenger IP3 and the membrane bound messenger diacylglycerol. Spatial regulation of this system has been thought to be through specific subcellular distributions of the IP3 receptor or PKC. As is becoming increasingly apparent, receptor-stimulated signaling systems are also found at intracellular membranes. As discussed in this issue, GPCRs have been identified at the nuclear envelope implying intracellular localization of the signaling systems that respond to GPCRs. Here we discuss the evidence for the existence of PLC signals that regulate nuclear processes, as well as the evidence for nuclear and nuclear envelope localization of PLC signaling components, and their implications for cardiac physiology and disease. PMID:25658460

  3. Endogenous glycosylphosphatidylinositol-specific phospholipase C releases renal dipeptidase from kidney proximal tubules in vitro.

    PubMed

    Park, S W; Choi, K; Kim, I C; Lee, H H; Hooper, N M; Park, H S

    2001-01-15

    Spontaneous enzymic release of renal dipeptidase (RDPase; EC 3.4.13.19), a glycosylphosphatidylinositol (GPI)-linked ectoenzyme, was observed in vitro during incubation of porcine proximal tubules at 37 degrees C. Triton X-114 phase separation of the released RDPase showed that the majority of the enzyme activity partitioned into the aqueous phase, indicating its hydrophilic nature. Immunoblot analyses using an antibody against the cross-reacting determinant (CRD) inositol 1,2-cyclic monophosphate, the epitope formed by phospholipase C (PLC) cleavage of the GPI anchor on a protein, detected the released RDPase. Reprobing the immunoblot with an anti-RDPase serum showed the RDPase band co-migrating with the CRD band. The release of RDPase from the proximal tubules was a Ca(2+)-dependent process and had a pH optimum of 9.0. These results indicate that RDPase is released from the proximal tubules by the action of a distinct endogenous GPI-specific PLC.

  4. Regulation of Transient Receptor Potential channels by the phospholipase C pathway

    PubMed Central

    Rohacs, Tibor

    2013-01-01

    Transient Receptor Potential (TRP) channels were discovered while analyzing visual mutants in drosophila. The protein encoded by the transient receptor potential (trp) gene is a Ca2+ permeable cation channel activated downstream of the phospholipase C (PLC) pathway. While searching for homologues in other organisms, a surprisingly large number of mammalian TRP channels were cloned. The regulation of TRP channels is quite diverse, but many of them are either activated downstream of the PLC pathway, or modulated by it. This review will summarize the current knowledge on regulation of TRP channels by the PLC pathway, with special focus on TRPC-s, which can be considered as effectors of the PLC pathway, and the heat and capsaicin sensitive TRPV1, which is modulated by the PLC pathway in a complex manner. PMID:23916247

  5. Requirement for a phospholipase C in odor response: overlap between olfaction and vision in Drosophila.

    PubMed Central

    Riesgo-Escovar, J; Raha, D; Carlson, J R

    1995-01-01

    A central problem in sensory system biology is the identification of the signal transduction pathways used in different sensory modalities. Genetic analysis of transduction mutants provides a means of studying in vivo the contributions of different pathways. This report shows that odorant response in one olfactory organ of Drosophila melanogaster depends on the norpA phospholipase C (EC 3.1.4.3) gene, providing evidence for use of the inositol 1,4,5-trisphosphate (IP3) signal transduction pathway. Since the norpA gene is also essential to phototransduction, this work demonstrates overlap in the genetic and molecular underpinnings of vision and olfaction. Genetic and molecular data also indicate that some olfactory information flows through a pathway which does not depend on norpA. Images Fig. 1 Fig. 5 PMID:7708738

  6. High-level production of Bacillus cereus phospholipase C in Corynebacterium glutamicum.

    PubMed

    Ravasi, Pablo; Braia, Mauricio; Eberhardt, Florencia; Elena, Claudia; Cerminati, Sebastián; Peirú, Salvador; Castelli, Maria Eugenia; Menzella, Hugo G

    2015-12-20

    Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme. PMID:26519562

  7. Angiotensin II stimulates phospholipases C and A/sub 2/ in cultured rat mesangial cells

    SciTech Connect

    Schlondorff, D.; DeCandido, S.; Satriano, J.A.

    1987-07-01

    Angiotensin II stimulates prostaglandin (PG) E/sub 2/ formation in mesangial cells cultured from rat renal glomeruli. The interactions between angiotensin II and PGE/sub 2/ are important in modulating glomerular function. The authors examined the mechanism for stimulation of PGE/sub 2/ production in mesangial cells using the putative diacylglycerol-lipase inhibitor RHC 80267 and trifluoperazine (TFP), an agent interfering with Ca/sup 2 +/-CaM-mediated processes. Although RHC 80267 inhibited diacylglycerol-lipase activity in mesangial cells, it did not influence PGE/sub 2/ production in response to either angiotensin II or A23187. TFP also decreased /sup 14/C release in response to either angiotensin II of A23187. In contrast, TFP (50 ..mu..M) inhibited basal PGE/sub 2/ production and stimulation by angiotensin II and A23187. TFP also decreased /sup 14/C release in response to angiotensin from cells prelabeled with (/sup 14/C)arachidonic acid, which was associated with inhibition of /sup 14/C loss from phosphatidylinositol. In cells prelabeled with /sup 32/P, orthophosphate angiotensin II caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. TFP enhanced formation of (/sup 3/H)inositol trisphosphate both under basal- and angiotensin II-stimulated conditions. Thus TFP did not inhibit phospholipase C activation by angiotensin. Angiotensin II caused marked increases in (/sup 32/P)lysophospholipids, indicating activation of also phospholipase A/sub 2/. Taken together, these results are consistent with stimulation of both phospholipase C and A/sub 2/ by angiotensin, the latter step responsible for the release of arachidonic acid and PGE/sub 2/ formation.

  8. Phospholipase C-delta1 expression is linked to proliferation, DNA synthesis, and cyclin E levels.

    PubMed

    Stallings, Jonathan D; Zeng, Yue X; Narvaez, Francisco; Rebecchi, Mario J

    2008-05-16

    We previously reported that phospholipase C-delta1 (PLC-delta1) accumulates in the nucleus at the G1/S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate ( Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060-22069 ). Here, using small interfering RNA (siRNA) that specifically targets rat PLC-delta1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-delta1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3H]Thymidine labeling and fluorescence-activated cell sorting analysis indicated that the rates of G1/S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G1/S boundary were slower to reach full G2/M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G1/S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G1/S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-delta1, resistant to these siRNA, suppressed expression of cyclin E at the G1/S boundary despite treatment of cultures with rat-specific siRNA. Although whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-delta1 led to a significant rise in the nuclear levels of this phospholipid at the G1/S boundary. These results support a role for PLC-delta1 and nuclear phospholipid metabolism in regulating cell cycle progression.

  9. In vivo detection of phospholipase C by enzyme-activated near-infrared probes.

    PubMed

    Mawn, Theresa M; Popov, Anatoliy V; Beardsley, Nancy J; Stefflova, Klara; Milkevitch, Matthew; Zheng, Gang; Delikatny, E James

    2011-12-21

    In this article, the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported, and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Because of the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC(50) of 34 ± 8 μM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor/muscle ratio. Tumor fluorescence enhancement was inhibited with the administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment.

  10. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2016-05-20

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site.

  11. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2016-05-20

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

  12. A Cell-Permeable Phospholipase C[gamma]1-Binding Peptide Transduces Neurons and Impairs Long-Term Spatial Memory

    ERIC Educational Resources Information Center

    Blum, Sonja; Dash, Pramod K.

    2004-01-01

    Growth factor-mediated signaling has emerged as an essential component of memory formation. In this study, we used a phospholipase C gamma 1 (PLC[gamma]1) binding, cell-penetrating peptide to sequester PLC[gamma]1 away from its target, the phosphotyrosine residues within the activated growth factor receptor. Peptides appear to transduce neurons…

  13. Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria.

    PubMed Central

    Johansen, K A; Gill, R E; Vasil, M L

    1996-01-01

    Resurgence of mycobacterial infections in the United States has led to an intense effort to identify potential virulence determinants in the genus Mycobacterium, particularly ones that would be associated with the more virulent species (e.g., Mycobacterium tuberculosis). Thin-layer chromatography (TLC) using radiolabeled phosphatidylcholine and sphingomyelin as substrates indicated that cell extracts of M. tuberculosis contain both phospholipase C (PLC) and phospholipase D (PLD) activities. In contrast, only PLD activity was detected in cell extracts of M. smegmatis. Neither activity was detected in cell-free culture supernatants from these organisms. We and others recently identified two open reading frames in M. tuberculosis with the potential to encode proteins which are highly homologous to the nonhemolytic (PlcN) and hemolytic (PlcH) phospholipase C enzymes of Pseudomonas aeruginosa. In contrast to the plc genes in P. aeruginosa, which are considerably distal to each other (min 34 and 64 on the chromosome), the mycobacterial genes, designated mpcA and mpcB, are tandemly arranged in the same relative orientation and separated by only 191 bp. Both the mpcA and the mpcB genes were individually cloned in M. smegmatis, and PLC activity was expressed from each gene in this organism. Hybridization experiments using the mpcA and the mpcB genes as probes under conditions of moderate stringency identified sequences homologous to these genes in M. bovis, M. bovis BCG, and M. marinum but not in several other Mycobacterium species, including M. smegmatis, M. avium, and M. intracellulare. TLC analysis using radiolabeled substrates indicated that M. bovis and M. marinum cell extracts contain PLC and PLD activities, but only PLD activity was detected in M. bovis BCG cell extracts. Sphingomyelinase activity was also detected in whole-cell extracts of M. tuberculosis, M. marinum, M. bovis, and M. bovis BCG, but this activity was not detected in extracts of M. smegmatis

  14. GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

    SciTech Connect

    Oberdisse, E.; Lapetina, E.G.

    1987-05-14

    Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.

  15. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C.

    PubMed

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically. PMID:26658739

  16. Sodium and potassium regulate endothelial phospholipase C-γ and Bmx.

    PubMed

    Ying, Wei-Zhong; Aaron, Kristal J; Sanders, Paul W

    2014-07-01

    The amount of Na(+) and K(+) in the diet promotes significant changes in endothelial cell function. In the present study, a series of in vitro and in vivo experiments determined the role of Na(+) and K(+) in the regulation of two pleckstrin homology domain-containing intracellular signaling molecules, phospholipase C (PLC)-γ1 and epithelial and endothelial tyrosine kinase/bone marrow tyrosine kinase on chromosome X (Bmx), and agonist-generated Ca(2+) signaling in the endothelium. Extracellular K(+) concentration regulated the levels of activated PLC-γ1, Bmx, and carbachol-stimulated intracellular Ca(2+) mobilization in human endothelial cells. Additional experiments confirmed that high-conductance Ca(2+)-activated K(+) channels and phosphatidylinositol 3-kinase mediated these effects. The content of Na(+) and K(+) in the diet also regulated Bmx levels in endothelial cells and activated PLC-γ1 levels in rats in vivo. The effects of dietary K(+) on Bmx were more pronounced in rats fed a high-salt diet compared with rats fed a low-salt diet. These experiments elucidated an endothelial cell signaling mechanism regulated by electrolytes, further demonstrating an integral relationship between endothelial cell function and dietary Na(+) and K(+) content. PMID:24785188

  17. PI(3,4,5)P3 Potentiates Phospholipase C-β Activity

    PubMed Central

    Zhang, Yong; Kwon, Sun Hyung; Vogel, Walter K.; Filtz, Theresa M.

    2010-01-01

    Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we had shown that PLC-β1 and PLC-β3 bound immobilized PIP3. In this study, PIP3 was found to potentiate Ca2+-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 minutes agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 seconds agonist-stimulated IP3 accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 seconds oxytocin-stimulated IP3 accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP3 in directly potentiating PLC-β activity. When co-expressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-β is not important for p110CAAX-induced membrane association. PMID:19519170

  18. Sodium and potassium regulate endothelial phospholipase C-γ and Bmx.

    PubMed

    Ying, Wei-Zhong; Aaron, Kristal J; Sanders, Paul W

    2014-07-01

    The amount of Na(+) and K(+) in the diet promotes significant changes in endothelial cell function. In the present study, a series of in vitro and in vivo experiments determined the role of Na(+) and K(+) in the regulation of two pleckstrin homology domain-containing intracellular signaling molecules, phospholipase C (PLC)-γ1 and epithelial and endothelial tyrosine kinase/bone marrow tyrosine kinase on chromosome X (Bmx), and agonist-generated Ca(2+) signaling in the endothelium. Extracellular K(+) concentration regulated the levels of activated PLC-γ1, Bmx, and carbachol-stimulated intracellular Ca(2+) mobilization in human endothelial cells. Additional experiments confirmed that high-conductance Ca(2+)-activated K(+) channels and phosphatidylinositol 3-kinase mediated these effects. The content of Na(+) and K(+) in the diet also regulated Bmx levels in endothelial cells and activated PLC-γ1 levels in rats in vivo. The effects of dietary K(+) on Bmx were more pronounced in rats fed a high-salt diet compared with rats fed a low-salt diet. These experiments elucidated an endothelial cell signaling mechanism regulated by electrolytes, further demonstrating an integral relationship between endothelial cell function and dietary Na(+) and K(+) content.

  19. Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis

    PubMed Central

    Le Chevalier, Fabien; Cascioferro, Alessandro; Frigui, Wafa; Pawlik, Alexandre; Boritsch, Eva C.; Bottai, Daria; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2015-01-01

    Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis. PMID:26603639

  20. Mechanosensitivity of human osteosarcoma cells and phospholipase C {beta}2 expression

    SciTech Connect

    Hoberg, M. . E-mail: Maik.Hoberg@med.uni-tuebingen.de; Gratz, H.-H.; Noll, M.; Jones, D.B.

    2005-07-22

    Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 {mu}strains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC {beta}1, {beta}3, {gamma}1, {gamma}2, and {delta}1 in all cells, but PLC {beta}2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC {beta}2 in mechanotransduction. Inducible antisense expression for 24 h inhibited the translation of PLC {beta}1 in U-2/OS cells by 35% and PLC {beta}2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC {beta}2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.

  1. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C.

    PubMed

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.

  2. Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development

    SciTech Connect

    Sone, Yoshie; Ito, Masahiko; Shirakawa, Hideki; Shikano, Tomohide; Takeuchi, Hiroyuki; Kinoshita, Katsuyuki; Miyazaki, Shunichi . E-mail: shunm@research.twmu.ac.jp

    2005-05-13

    Phospholipase C-zeta (PLC{zeta}), a strong candidate of the egg-activating sperm factor, causes intracellular Ca{sup 2+} oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLC{zeta}. Changes in the localization of expressed PLC{zeta} were investigated by tagging with a fluorescent protein. PLC{zeta} began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLC{zeta} in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLC{zeta} was recognized in every embryo up to blastocyst. Thus, PLC{zeta} exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca{sup 2+} oscillations in early embryogenesis.

  3. Relationship between phospholipase C zeta immunoreactivity and DNA fragmentation and oxidation in human sperm

    PubMed Central

    Park, Ju Hee; Kim, Seul Ki; Kim, Jayeon; Kim, Ji Hee; Chang, Jae Hoon; Kim, Seok Hyun

    2015-01-01

    Objective The study aimed to evaluate the feasibility and reproducibility of measuring phospholipase C zeta (PLCζ) using immunostaining in human sperm and to investigate the relationship between PLCζ immunoreactivity and DNA fragmentation and oxidation in human sperm. Methods Semen samples were obtained from participants (n=44) and processed by the conventional swim-up method. Sperm concentration, motility, normal form by strict morphology, DNA fragmentation index assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling method and immunofluorescent expression for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and PLCζ were assessed. Results When duplicate PLCζ tests were performed on two sperm samples from each of the 44 participants, similar results were obtained (74.1±9.4% vs. 75.4±9.7%). Two measurements of PLCζ were found to be highly correlated with each other (r=0.759, P<0.001). Immunoreactivity of PLCζ was not associated with donor's age, sperm concentration, motility, and the percentage of normal form as well as DNA fragmentation index. However, immunoreactivity of PLCζ showed a significant negative relationship with 8-OHdG immunoreactivity (r=-0.404, P=0.009). Conclusion Measurement of PLCζ by immunostaining is feasible and reproducible. Lower expression of PLCζ in human sperm may be associated with higher sperm DNA oxidation status. PMID:26023673

  4. Melanopsin-Expressing Amphioxus Photoreceptors Transduce Light via a Phospholipase C Signaling Cascade

    PubMed Central

    Angueyra, Juan Manuel; Pulido, Camila; Malagón, Gerardo; Nasi, Enrico; Gomez, Maria del Pilar

    2012-01-01

    Melanopsin, the receptor molecule that underlies light sensitivity in mammalian ‘circadian’ receptors, is homologous to invertebrate rhodopsins and has been proposed to operate via a similar signaling pathway. Its downstream effectors, however, remain elusive. Melanopsin also expresses in two distinct light-sensitive cell types in the neural tube of amphioxus. This organism is the most basal extant chordate and can help outline the evolutionary history of different photoreceptor lineages and their transduction mechanisms; moreover, isolated amphioxus photoreceptors offer unique advantages, because they are unambiguously identifiable and amenable to single-cell physiological assays. In the present study whole-cell patch clamp recording, pharmacological manipulations, and immunodetection were utilized to investigate light transduction in amphioxus photoreceptors. A Gq was identified and selectively localized to the photosensitive microvillar membrane, while the pivotal role of phospholipase C was established pharmacologically. The photocurrent was profoundly depressed by IP3 receptor antagonists, highlighting the importance of IP3 receptors in light signaling. By contrast, surrogates of diacylglycerol (DAG), as well as poly-unsaturated fatty acids failed to activate a membrane conductance or to alter the light response. The results strengthen the notion that calcium released from the ER via IP3-sensitive channels may fulfill a key role in conveying - directly or indirectly - the melanopsin-initiated light signal to the photoconductance; moreover, they challenge the dogma that microvillar photoreceptors and phoshoinositide-based light transduction are a prerogative of invertebrate eyes. PMID:22235344

  5. Identification of phospholipase C zeta in normospermic and teratospermic domestic cat sperm.

    PubMed

    Villaverde, Ana Izabel S Balbin; Fioratti, Eduardo G; Fissore, Rafael A; He, Changli; Lee, Hoi Chang; Souza, Fabiana F; Landim-Alvarenga, Fernanda C; Lopes, Maria Denise

    2013-10-15

    In mammalian species, oocyte activation is initiated by oscillations in the intracellular concentration of free calcium ([Ca(2+)]i), which are also essential to allow embryonic development. To date, evidence supporting the hypothesis that a sperm factor is responsible for initiating oocyte activation has been presented in various mammalian species. Among the possible candidates to be the active sperm factor is the novel sperm-specific phospholipase C ζ (PLCζ), which besides its testis-specific expression is capable of initiating [Ca(2+)]i oscillations. In this study, we investigated the presence of PLCζ in the sperm of the domestic cat and whether normospermic and teratospermic cats differ in their PLCζ expression. Immunoblotting with anti-PLCζ antibodies confirmed the presence of an immunoreactive band of ∼70 kDa in whole sperm lysates of domestic cat as well as in both soluble and "insoluble" fractions from this sperm. Additional immunoreactive bands, probably C- and N-terminal truncated versions of PLCζ, were also visualized in the soluble sperm fractions. Interestingly, immunoreactivity of PLCζ was detectable in teratospermic sperm, although with slightly less intensity than in normospermic sperm. In conclusion, domestic cat sperm express PLCζ in both cytosolic and high-pH fractions, which is consistent with data in other mammals. Sperm from teratospermic cats also express PLCζ, albeit at reduced concentrations, which may affect the fertility of these males.

  6. Phospholipase C-β1 Hypofunction in the Pathogenesis of Schizophrenia

    PubMed Central

    Kim, Seong-Wook; Cho, Taesup; Lee, Sukchan

    2015-01-01

    Schizophrenia is a mental disorder that is characterized by various abnormal symptoms. Previous studies indicate decreased expression of phospholipase C-β1 (PLC-β1) in the brains of patients with schizophrenia. PLC-β1-null (PLC-β1−/−) mice exhibit multiple endophenotypes of schizophrenia. Furthermore, a study of PLC-β1 knockdown in the medial prefrontal cortex of mice has shown a specific behavioral deficit, impaired working memory. These results support the notion that disruption of PLC-β1-linked signaling in the brain is strongly involved in the pathogenesis of schizophrenia. In this review, we broadly investigate recent studies regarding schizophrenia-related behaviors as well as their various clinical and biological correlates in PLC-β1−/− and knockdown mouse models. This will provide a better understanding of the pathological relevance of the altered expression of PLC-β1 in the brains of patients with schizophrenia. Evidence accumulated will shed light on future in-depth studies, possibly in human subjects. PMID:26635636

  7. Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as an effector.

    PubMed Central

    Park, E S; Won, J H; Han, K J; Suh, P G; Ryu, S H; Lee, H S; Yun, H Y; Kwon, N S; Baek, K J

    1998-01-01

    Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified. Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghalpha is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem. J. 315, 739-744]. To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor-Ghalpha-PLC-delta1 complex. PLC-delta1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghalpha (GTP-bound Ghalpha). Furthermore the stimulated PLC-delta1 activity resulting from activation of Ghalpha via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP. Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-delta1 in the three-component preparation by anti-Gh7alpha antibody resulted in the PLC-delta1 being tightly coupled to activated Ghalpha on stimulation of the oxytocin receptor. These results indicate that PLC-delta1 is the effector for Ghalpha-mediated oxytocin receptor signalling. PMID:9512491

  8. Reduced fertilization after ICSI and abnormal phospholipase C zeta presence in spermatozoa from the wobbler mouse.

    PubMed

    Heytens, Elke; Schmitt-John, Thomas; Moser, Jakob M; Jensen, Nanna Mandøe; Soleimani, Reza; Young, Claire; Coward, Kevin; Parrington, John; De Sutter, Petra

    2010-12-01

    Failed fertilization after intracytoplasmic sperm injection (ICSI) can be due to a reduced oocyte-activation capacity caused by reduced concentrations and abnormal localization of the oocyte-activation factor phospholipase C (PLC) zeta. Patients with this condition can be helped to conceive by artificial activation of oocytes after ICSI with calcium ionophore (assisted oocyte activation; AOA). However some concern still exists about this approach. Mouse models could help to identify potential oocyte-activation strategies and evaluate their safety. In this study, the fertilizing capacity of wobbler sperm cells was tested and the efficiency of AOA with two exposures to ionomycin to restore fertilization and embryo development was studied. The quality of the obtained blastocysts was assessed and embryo transfer was performed to evaluate post-implantation development. The presence of PLCzeta in the spermatozoa and testis of the wobbler mouse was evaluated by PLCzeta immunostaining and quantitative reverse-transcription polymerase chain reaction. Sperm cells from wobbler mice had reduced fertilizing capacity and abnormalities in PLCzeta localization, but not in its expression. Artificially activating the oocytes restored fertilization and embryo development. Therefore, the wobbler mouse can be a model for failed fertilization after ICSI to study PLCzeta dynamics and aid in optimization of the AOA method.

  9. Phospholipase C-related catalytically inactive protein (PRIP) controls KIF5B-mediated insulin secretion

    PubMed Central

    Asano, Satoshi; Nemoto, Tomomi; Kitayama, Tomoya; Harada, Kae; Zhang, Jun; Harada, Kana; Tanida, Isei; Hirata, Masato; Kanematsu, Takashi

    2014-01-01

    ABSTRACT We previously reported that phospholipase C-related catalytically inactive protein (PRIP)-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6) cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrin-labeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA-receptor-associated protein (GABARAP), a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms. PMID:24812354

  10. Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis.

    PubMed

    Le Chevalier, Fabien; Cascioferro, Alessandro; Frigui, Wafa; Pawlik, Alexandre; Boritsch, Eva C; Bottai, Daria; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2015-01-01

    Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis.

  11. Genetic evidence for a role of phospholipase C at the budding yeast kinetochore.

    PubMed

    DeLillo, N; Romero, C; Lin, H; Vancura, A

    2003-05-01

    Chromosome segregation during mitosis requires kinetochores, specialized organelles that mediate chromosome attachment to spindle microtubules. We have shown previously that in budding yeast, Plc1p (phosphoinositide-specific phospholipase C) localizes to centromeric loci, associates with the kinetochore proteins Ndc10p and Cep3p, and affects the function of kinetochores. Deletion of PLC1 results in nocodazole sensitivity, mitotic delay, and a higher frequency of chromosome loss. We report here that despite the nocodazole sensitivity of plc1Delta cells, Plc1p is not required for the spindle checkpoint. However, plc1Delta cells require a functional BUB1/BUB3-dependent spindle checkpoint for viability. PLC1 displays strong genetic interactions with genes encoding components of the inner kinetochore, including NDC10, SKP1, MIF2, CEP1, CEP3, and CTF13. Furthermore, plc1Delta cells display alterations in chromatin structure in the core centromere. Chromatin immunoprecipitation experiments indicate that Plc1p localizes to centromeric loci independently of microtubules, and accumulates at the centromeres during G(2)/M stage of cell cycle. These results are consistent with the view that Plc1p affects kinetochore function, possibly by modulating the structure of centromeric chromatin.

  12. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C

    PubMed Central

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically. PMID:26658739

  13. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    SciTech Connect

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).

  14. Distinction in vitro between rat liver phosphatidate phosphatase and phospholipase C

    SciTech Connect

    Lamb, R.; Foster, K.; McGuffin, M.

    1986-03-05

    Hepatocellular membranes (1000 x g) were incubated with sn-(1,3-/sup 14/C) glycerol-3-P, ATP, Ca/sup 2 +/, NaF and palmitate to form labeled, membrane-associated phosphatidate(PA). Membranes incubated with 2mM oleate or 5mM bromobenzene showed rapid (5-10 min) and significant (2-6 fold) increases in the dephosphorylation of PA. However, oleate and bromobenzene activated the dephosphorylation of PA by phosphatidate phosphatase (PAP) and phospholipase C (PLC), respectively. This conclusion is supported by the observation that the PAP stimulated by oleate is: 1) Mg/sup 2 +/-dependent; 2) inhibited by Ca/sup 2 +/ and NaF; 3) specific for PA; 4) associated with a rise in liver cell triacylglycerol (TG) formation. Bromobenzene, however, activated a PLC that is: 1) stimulated by various metals; 2) enhanced by NaF; 3) is associated with a rise in the degradation of membrane phospholipids and liver cell injury. These results suggest that under the appropriate conditions in vitro the dephosphorylation of PA can be used to assess chemical-dependent changes in PAP and/or PLC activity.

  15. Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity.

    PubMed

    Siqueira, Flávia F; Almeida, Marcelle O; Barroca, Tatiana M; Horta, Carolina C R; Carmo, Anderson O; Silva, Rodrigo O S; Pires, Prhiscylla S; Lobato, Francisco C F; Kalapothakis, Evanguedes

    2012-10-12

    Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.

  16. Role of Inositol Phosphosphingolipid Phospholipase C1, the Yeast Homolog of Neutral Sphingomyelinases in DNA Damage Response and Diseases

    PubMed Central

    Tripathi, Kaushlendra

    2015-01-01

    Sphingolipids play a very crucial role in many diseases and are well-known as signaling mediators in many pathways. Sphingolipids are produced during the de novo process in the ER (endoplasmic reticulum) from the nonsphingolipid precursor and comprise both structural and bioactive lipids. Ceramide is the central core of the sphingolipid pathway, and its production has been observed following various treatments that can induce several different cellular effects including growth arrest, DNA damage, apoptosis, differentiation, and senescence. Ceramides are generally produced through the sphingomyelin hydrolysis and catalyzed by the enzyme sphingomyelinase (SMase) in mammals. Presently, there are many known SMases and they are categorized into three groups acid SMases (aSMases), alkaline SMases (alk-SMASES), and neutral SMases (nSMases). The yeast homolog of mammalians neutral SMases is inositol phosphosphingolipid phospholipase C. Yeasts generally have inositol phosphosphingolipids instead of sphingomyelin, which may act as a homolog of mammalian sphingomyelin. In this review, we shall explain the structure and function of inositol phosphosphingolipid phospholipase C1, its localization inside the cells, mechanisms, and its roles in various cell responses during replication stresses and diseases. This review will also give a new basis for our understanding for the mechanisms and nature of the inositol phosphosphingolipid phospholipase C1/nSMase. PMID:26346287

  17. Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: Involvement of a phosphatidylcholine-hydrolyzing phospholipase C

    SciTech Connect

    Nishijima, J.; Wright, T.M.; Hoffman, R.D.; Liao, F.; Symer, D.E.; Shin, H.S. )

    1989-04-04

    The authors have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using ({sup 14}C)glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis.

  18. Galpha12/13- and rho-dependent activation of phospholipase C-epsilon by lysophosphatidic acid and thrombin receptors.

    PubMed

    Hains, Melinda D; Wing, Michele R; Maddileti, Savitri; Siderovski, David P; Harden, T Kendall

    2006-06-01

    Because phospholipase C epsilon (PLC-epsilon) is activated by Galpha(12/13) and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC-epsilon. Expression of the LPA(1) or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC-epsilon. Both LPA(1) and PAR1 receptor-mediated activation of PLC-epsilon was inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for Galpha(12/13) but not by expression of the RGS domain of GRK2, which inhibits Galpha(q) signaling. In contrast, activation of the G(q)-coupled M1 muscarinic or P2Y(2) purinergic receptor was neither enhanced by coexpression with PLC-epsilon nor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPA- or SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLC-epsilon but completely prevented the PLC-epsilon-dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC-epsilon-dependent stimulatory effects of the LPA(1), LPA(2), LPA(3), or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y(2) receptor. Moreover, PLC-epsilon-dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Ras- but not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via

  19. Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-beta.

    PubMed Central

    Filtz, T M; Cunningham, M L; Stanig, K J; Paterson, A; Harden, T K

    1999-01-01

    The potential role of protein kinase C (PKC)-promoted phosphorylation has been examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [32P]Pi-labelled turkey erythrocytes with either the P2Y1 receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA resulted in a marked increase in incorporation of 32P into the G-protein-activated phospholipase C PLC-betaT. Purified PLC-betaT also was phosphorylated by PKC in vitro to a stoichiometry (mean+/-S. E.M.) of 1.06+/-0.2 mol of phosphate/mol of PLC-betaT. Phosphorylation by PKC was isoenzyme-specific because, under identical conditions, mammalian PLC-beta2 also was phosphorylated to a stoichiometry near unity, whereas mammalian PLC-beta1 was not phosphorylated by PKC. The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-betaT with turkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylation resulted in a decrease in basal activity, AlF4(-)-stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5'-[gamma-thio]triphosphate in the reconstituted membranes. The decreases in enzyme activities were proportional to the extent of PKC-promoted phosphorylation. Catalytic activity assessed by using mixed detergent/phospholipid micelles also was decreased by up to 60% by phosphorylation. The effect of phosphorylation on Gqalpha-stimulated PLC-betaT in reconstitution experiments with purified proteins was not greater than that observed on basal activity alone. Taken together, these results illustrate that PKC phosphorylates PLC-betaT in vivo and to a physiologically relevant stoichiometry in vitro. Phosphorylation is accompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-protein-regulated activity. PMID:10024500

  20. Phospholipase C Epsilon (PLCε) Induced TRPC6 Activation: A Common but Redundant Mechanism in Primary Podocytes

    PubMed Central

    Kalwa, Hermann; Storch, Ursula; Demleitner, Jana; Fiedler, Susanne; Mayer, Tim; Kannler, Martina; Fahlbusch, Meike; Barth, Holger; Smrcka, Alan; Hildebrandt, Friedhelm; Gudermann, Thomas; Dietrich, Alexander

    2016-01-01

    In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca2+ concentration [Ca2+]i. Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG-sensitive classical transient receptor channels 3, 6, and 7 (TRPC3/6/7), initiating Ca2+ influx from the extracellular space. Patients with focal segmental glomerulosclerosis (FSGS) express gain-of-function mutants of TRPC6, while others carry loss-of-function mutants of PLCε, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLCβ and PLCγ isozymes was extensively studied, the role of PLCε in TRPC6 activation remains elusive. TRPC6 was co-immunoprecipitated with PLCε in a heterologous overexpression system in HEK293 cells as well as in freshly isolated murine podocytes. Receptor-operated TRPC6 currents in HEK293 cells expressing TRPC6 were reduced by a specific PLCε siRNA and by a PLCε loss-of-function mutant isolated from a patient with FSGS. PLCε-induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking Gαq/11 proteins. Further analysis of the signal transduction pathway revealed a Gα12/13 Rho-GEF activation which induced Rho-mediated PLCε stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLCε. PLCε-/- podocytes however, were undistinguishable from WT podocytes in their angiotensin II-induced formation of actin stress fibers and their GTPγS-induced TRPC6 activation, pointing to a redundant role of PLCε-mediated TRPC6 activation at least in podocytes. PMID:25521631

  1. The sperm phospholipase C-ζ and Ca2+ signalling at fertilization in mammals.

    PubMed

    Swann, Karl; Lai, F Anthony

    2016-02-01

    A series of intracellular oscillations in the free cytosolic Ca(2+) concentration is responsible for activating mammalian eggs at fertilization, thus initiating embryo development. It has been proposed that the sperm causes these Ca(2+) oscillations after membrane fusion by delivering a soluble protein into the egg cytoplasm. We previously identified sperm-specific phospholipase C (PLC)-ζ as a protein that can trigger the same pattern of Ca(2+) oscillations in eggs seen at fertilization. PLCζ appears to be the elusive sperm factor mediating egg activation in mammals. It has potential therapeutic use in infertility treatments to improve the rate of egg activation and early embryo development after intra-cytoplasmic sperm injection. A stable form of recombinant human PLCζ could be a prototype for use in such in vitro fertilization (IVF) treatments. We do not yet understand exactly how PLCζ causes inositol 1,4,5-trisphosphate (InsP3) production in eggs. Sperm PLCζ is distinct among mammalian PI-specific PLCs in that it is far more potent in triggering Ca(2+) oscillations in eggs than other PLCs, but it lacks a PH domain that would otherwise be considered essential for binding to the phosphatidylinositol 4,5-bisphosphate (PIP2) substrate. PLCζ is also unusual in that it does not appear to interact with or hydrolyse plasma membrane PIP2. We consider how other regions of PLCζ may mediate its binding to PIP2 in eggs and how interaction of PLCζ with egg-specific factors could enable the hydrolysis of internal sources of PIP2.

  2. Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein

    PubMed Central

    Liu, Pengcheng; Peng, Hong-Juan; Zhu, Jinsong

    2015-01-01

    Juvenile hormone (JH) is a key regulator of a wide diversity of developmental and physiological events in insects. Although the intracellular JH receptor methoprene-tolerant protein (MET) functions in the nucleus as a transcriptional activator for specific JH-regulated genes, some JH responses are mediated by signaling pathways that are initiated by proteins associated with plasma membrane. It is unknown whether the JH-regulated gene expression depends on the membrane-mediated signal transduction. In Aedes aegypti mosquitoes, we found that JH activated the phospholipase C (PLC) pathway and quickly increased the levels of inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation and autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). When abdomens from newly emerged mosquitoes were cultured in vitro, the JH-activated gene expression was repressed substantially if specific inhibitors of PLC or CaMKII were added to the medium together with JH. In newly emerged female mosquitoes, RNAi-mediated depletion of PLC or CaMKII considerably reduced the expression of JH-responsive genes, including the Krüppel homolog 1 gene (AaKr-h1) and the early trypsin gene (AaET). JH-induced loading of MET to the promoters of AaKr-h1 and AaET was weakened drastically when either PLC or CaMKII was inactivated in the cultured tissues. Therefore, the results suggest that the membrane-initiated signaling pathway modifies the DNA-binding activity of MET via phosphorylation and thus facilitates the genomic responses to JH. In summary, this study reveals an interplay of genomic and nongenomic signaling mechanisms of JH. PMID:25825754

  3. Growth hormone activates phospholipase C in proximal tubular basolateral membranes from canine kidney

    SciTech Connect

    Rogers, S.A.; Hammerman, M.R. )

    1989-08-01

    To delineate pathways for signal transduction by growth hormone (GH) in proximal tubule, the authors incubated basolateral membranes isolated from canine kidney with human growth hormone (hGH) or human prolactin (hPrl) and measured levels of inositol trisphosphate (InsP{sub 3}) in suspensions and of diacylglycerol extractable from the membranes. Incubation with hGH, but not hPrl, increased levels of InsP{sub 3} and diacylglycerol in a concentration-dependent manner. Half-maximal effects occurred between 0.1 and 1 nM hGH. Increased levels of InsP{sub 3} were measured after as little as 5 sec of incubation with 1 nM hGH, and increase was maximal after 15 sec. Increases were no longer detectable after 60 sec because of dephosphorylation of InsP{sub 3} in membrane suspensions. hGH did not affect rates of dephosphorylation. hGH-stimulated increases in InsP{sub 3} were detectable in membranes suspended in 0, 0.1, and 0.2 {mu}M calcium but not in 0.3 or 1.0 {mu}M calcium. {sup 125}I-labeled hGH-receptor complexes with M{sub r} values of 66,000 and 140,000 were identified in isolated basolateral membranes. The findings establish that GH activates phospholipase C in isolated canine renal proximal tubular basolateral membranes, potentially after binding to a specific receptor. This process could mediate signal transmission by GH across the plasma membrane of the proximal tubular cell and elsewhere.

  4. Dopamine D1 Receptor Signaling: Does GαQ–Phospholipase C Actually Play a Role?

    PubMed Central

    Lee, Sang-Min; Yang, Yang

    2014-01-01

    Despite numerous studies showing therapeutic potential, no central dopamine D1 receptor ligand has ever been approved, because of potential limitations, such as hypotension, seizures, and tolerance. Functional selectivity has been widely recognized as providing a potential mechanism to develop novel therapeutics from existing targets, and a highly biased, functionally selective D1 ligand might overcome some of the past limitations. SKF-83959 [6-chloro-3-methyl-1-(m-tolyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine-7,8-diol] is reported to be a highly biased D1 ligand, having full agonism at D1-mediated activation of phospholipase C (PLC) signaling (via GαQ) and antagonism at D1-mediated adenylate cyclase signaling (via GαOLF/S). For this reason, numerous studies have used this compound to elucidate the physiologic role of D1-PLC signaling, including a novel molecular mechanism (GαQ-PLC activation via D1-D2 heterodimers). There is, however, contradictory literature that suggests that SKF-83959 is actually a partial agonist at both D1-mediated adenylate cyclase and β-arrestin recruitment. Moreover, the D1-mediated PLC stimulation has also been questioned. This Minireview examines 30 years of relevant literature and proposes that the data strongly favor alternate hypotheses: first, that SKF-83959 is a typical D1 partial agonist; and second, that the reported activation of PLC by SKF-83959 and related benzazepines likely is due to off-target effects, not actions at D1 receptors. If these hypotheses are supported by future studies, it would suggest that caution should be used regarding the role of PLC and downstream pathways in D1 signaling. PMID:25052835

  5. Osmotic activation of phospholipase C triggers structural adaptation in osmosensitive rat supraoptic neurons.

    PubMed

    Shah, Love; Bansal, Vimal; Rye, Peter L; Mumtaz, Naima; Taherian, Amir; Fisher, Thomas E

    2014-10-01

    The magnocellular neurosecretory cells of the hypothalamus (MNCs) synthesize and secrete vasopressin or oxytocin. A stretch-inactivated cation current mediated by TRPV1 channels rapidly transduces increases in external osmolality into a depolarization of the MNCs leading to an increase in action potential firing and thus hormone release. Prolonged increases in external osmolality, however, trigger a reversible structural and functional adaptation that may enable the MNCs to sustain high levels of hormone release. One poorly understood aspect of this adaptation is somatic hypertrophy. We demonstrate that hypertrophy can be evoked in acutely isolated rat MNCs by exposure to hypertonic solutions lasting tens of minutes. Osmotically evoked hypertrophy requires activation of the stretch-inactivated cation channel, action potential firing, and the influx of Ca(2+). Hypertrophy is prevented by pretreatment with a cell-permeant inhibitor of exocytotic fusion and is associated with an increase in total membrane capacitance. Recovery is disrupted by an inhibitor of dynamin function, suggesting that it requires endocytosis. We also demonstrate that hypertonic solutions cause a decrease in phosphatidylinositol 4,5-bisphosphate in the plasma membranes of MNCs that is prevented by an inhibitor of phospholipase C (PLC). Inhibitors of PLC or protein kinase C (PKC) prevent osmotically evoked hypertrophy, and treatment with a PKC-activating phorbol ester can elicit hypertrophy in the absence of changes in osmolality. These studies suggest that increases in osmolality cause fusion of internal membranes with the plasma membrane of the MNCs and that this process is mediated by activity-dependent activation of PLC and PKC.

  6. Macroscopic consequences of the action of phospholipase C on giant unilamellar liposomes.

    PubMed Central

    Holopainen, Juha M; Angelova, Miglena I; Söderlund, Tim; Kinnunen, Paavo K J

    2002-01-01

    Macroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10-100 microm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution of the microscope. The presence of N-palmitoyl-sphingomyelin (mole fraction X = 0.25) in the GUVs did not affect the outcome of the PC-PLC reaction. In addition to hydrolysis, PC-PLC induced adhesion of vicinal vesicles. When multilamellar SOPC vesicles were used only a minor decrease in their diameter was evident suggesting that PC-PLC can exert its hydrolytic activity only in the outer monolayer. A series of stepwise shrinkages was observed also for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) GUVs above their main phase transition temperature, T(m), i.e., when the bilayer is in the liquid crystalline state. However, this process was not observed for DMPC GUVs in the gel state, below T(m). These results are supported by the enhanced activity of PC-PLC upon exceeding T(m) of DMPC large unilamellar vesicles (diameter approximately 0.1 microm) used as a substrate. Studies on SOPC monolayers revealed that PC-PLC can exert its hydrolytic activity only at surface pressures below approximately 30 mN/m. Accordingly, the lack of changes in the gel state DMPC GUVs could be explained by the equilibrium lateral pressure in these vesicles exceeding this critical value. PMID:12124275

  7. Signal-dependent Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate without Activation of Phospholipase C

    PubMed Central

    Lev, Shaya; Katz, Ben; Tzarfaty, Vered; Minke, Baruch

    2012-01-01

    In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P2 in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P2 was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P2 is not an inhibitor of TRPL channel activation. PI(4,5)P2 hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P2 levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P2 is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating. PMID:22065576

  8. Expression of FLR1 transporter requires phospholipase C and is repressed by Mediator.

    PubMed

    Romero, Carlos; Desai, Parima; DeLillo, Nicholas; Vancura, Ales

    2006-03-01

    In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in benomyl sensitivity, alterations in chromatin structure of centromeres, mitotic delay, and a higher frequency of chromosome loss. Here we intended to utilize benomyl sensitivity as a phenotype that would allow us to identify genes that are important for kinetochore function and are downstream of Plc1p. However, our screen identified SIN4, encoding a component of the Mediator complex of RNA polymerase II. Deletion of SIN4 gene (sin4Delta) does not suppress benomyl sensitivity of plc1Delta cells by improving the function of kinetochores. Instead, benomyl sensitivity of plc1Delta cells is caused by a defect in expression of FLR1, and the suppression of benomyl sensitivity in plc1Delta sin4Delta cells occurs by derepression of FLR1 transcription. FLR1 encodes a plasma membrane transporter that mediates resistance to benomyl. Several other mutations in the Mediator complex also result in significant derepression of FLR1 and greatly increased resistance to benomyl. Thus, benomyl sensitivity is not a phenotype exclusively associated with mitotic spindle defect. These results demonstrate that in addition to promoter-specific transcription factors that are components of the pleiotropic drug resistance network, expression of the membrane transporters can be regulated by Plc1p, a component of a signal transduction pathway, and by Mediator, a general transcription factor. The results thus suggest another layer of complexity in regulation of pleiotropic drug resistance.

  9. Phosphatidylinositol-specific phospholipase C, a possible virulence factor of Staphylococcus aureus.

    PubMed Central

    Marques, M B; Weller, P F; Parsonnet, J; Ransil, B J; Nicholson-Weller, A

    1989-01-01

    Phosphatidylinositol-specific phospholipase C (PIPLC), an enzyme that can specifically release phosphatidylinositol-linked proteins from host cells, is one of the extracellular enzymes produced by Staphylococcus aureus. To investigate whether PIPLC might be a virulence factor, we assessed PIPLC production by S. aureus strains that had been isolated from healthy carriers and from infected patients with or without toxic shock syndrome. Although none of five vaginal isolates from healthy women was a PIPLC producer, only 10 of 32 selected pathogenic strains that caused significant infections or toxic shock syndrome elaborated PIPLC enzyme activity. Seven of 24 toxic-shock-associated strains, compared with 3 of 8 non-toxic-shock-associated strains, were positive for PIPLC. The majority of strains that produced PIPLC were negative for toxic shock syndrome toxin 1 (P less than 0.05); this association between PIPLC production and strains negative for toxic shock syndrome toxin 1 was even stronger among strains isolated only from patients with toxic shock syndrome (P less than 0.01). Among all 32 pathogenic isolates, PIPLC-producing S. aureus strains were isolated from four of four patients developing adult respiratory distress syndrome and four of five patients with disseminated intravascular coagulation, suggesting a significant association between PIPLC production and adult respiratory distress syndrome and/or disseminated intravascular coagulation (P less than 0.002). On the basis of these results, we propose that PIPLC is a virulence factor of S. aureus and is implicated in the development of adult respiratory distress syndrome and disseminated intravascular coagulation. PMID:2808668

  10. Thromboxane A2-mediated shape change: independent of Gq-phospholipase C--Ca2+ pathway in rabbit platelets.

    PubMed Central

    Ohkubo, S.; Nakahata, N.; Ohizumi, Y.

    1996-01-01

    an unknown mechanism. 8. U46619-induced shape change was unaffected by W-7 (30 microM), a calmodulin antagonist or ML-7 (30 microM), a myosin light-chain kinase inhibitor, indicating that an increase in [Ca2+]i might not be involved in the shape change. In fact, U46619 (10 nM) could cause shape change without affecting [Ca2+]i level, determined by simultaneous recordings. 9. [3H]-SQ29548 and [3H]-U46619 bound to platelets at a single site with a Kd value of 14.88 nM and Bmax of 106.1 fmol/10(8) platelets and a Kd value of 129.8 nM and Bmax of 170.4 fmol/10(8) platelets, respectively. The inhibitory constant Ki value for U46619 as an inhibitor of 3H-ligand binding was similar to the EC50 value of U46619 in GTPase activity, phosphoinositide hydrolysis and Ca2+ mobilization, but significantly different (P < 0.001 by Student's t test) from the effect on shape change. 10. Neither U46619 nor ONO NT-126 affected the adenosine 3',5'-cyclic monophosphate (cyclic AMP) level in the presence or absence of external Ca2+ and/or isobutyl methylxanthine. 11. The results indicate that TXA2 receptor stimulation causes phospholipase C activation and increase in [Ca2+]i via a G protein of the Gq/11 family leading to aggregation in the presence of external Ca2+, and that shape change induced by TXA2 receptor stimulation might occur without involvement of the Gq-phospholipase C-Ca2+ pathway. PMID:8882602

  11. Oocyte activation and phospholipase C zeta (PLCζ): diagnostic and therapeutic implications for assisted reproductive technology

    PubMed Central

    2012-01-01

    Infertility affects one in seven couples globally and has recently been classified as a disease by the World Health Organisation (WHO). While in-vitro fertilisation (IVF) offers effective treatment for many infertile couples, cases exhibiting severe male infertility (19–57%) often remain difficult, if not impossible to treat. In such cases, intracytoplasmic sperm injection (ICSI), a technique in which a single sperm is microinjected into the oocyte, is implemented. However, 1–5% of ICSI cycles still fail to fertilise, affecting over 1000 couples per year in the UK alone. Pregnancy and delivery rates for IVF and ICSI rarely exceed 30% and 23% respectively. It is therefore imperative that Assisted Reproductive Technology (ART) protocols are constantly modified by associated research programmes, in order to provide patients with the best chances of conception. Prior to fertilisation, mature oocytes are arrested in the metaphase stage of the second meiotic division (MII), which must be alleviated to allow the cell cycle, and subsequent embryogenesis, to proceed. Alleviation occurs through a series of concurrent events, collectively termed ‘oocyte activation’. In mammals, oocytes are activated by a series of intracellular calcium (Ca2+) oscillations following gamete fusion. Recent evidence implicates a sperm-specific phospholipase C, PLCzeta (PLCζ), introduced into the oocyte following membrane fusion as the factor responsible. This review summarises our current understanding of oocyte activation failure in human males, and describes recent advances in our knowledge linking certain cases of male infertility with defects in PLCζ expression and activity. Systematic literature searches were performed using PubMed and the ISI-Web of Knowledge. Databases compiled by the United Nations and World Health Organisation databases (UNWHO), and the Human Fertilization and Embryology Authority (HFEA) were also scrutinised. It is clear that PLCζ plays a fundamental role in

  12. The effects of mastoparan on the carrot cell plasma membrane polyphosphoinositide phospholipase C.

    PubMed Central

    Cho, M H; Tan, Z; Erneux, C; Shears, S B; Boss, W F

    1995-01-01

    When [3H]inositol-labeled carrot (Daucus carota L.) cells were treated with 10 or 25 microM wasp venom peptide mastoparan or the active analog Mas-7 there was a rapid loss of more than 70% of [3H]phosphatidylinositol-4-monophosphate (PIP) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) and a 3- and 4-fold increase in [3H]inositol-1,4-P2 and [3H]inositol-1,4,5-P3, respectively. The identity of [3H]inositol-1,4,5-P3 was confirmed by phosphorylation with inositol-1,4,5-P3 3-kinase and co-migration with inositol-1,3,4,5-P4. The changes in phosphoinositides were evident within 1 min. The loss of [3H]PIP was evident only when cells were treated with the higher concentrations (10 and 25 microM) of mastoparan or Mas-7. At 1 microM Mas-7, [3H]PIP increased. The inactive mastoparan analog Mas-17 had little or no effect on [3H]PIP or [3H]PIP2 hydrolysis in vivo. Neomycin (100 microM) inhibited the uptake of Mas-7 and thereby inhibited the Mas-7-stimulated hydrolysis of [3H]PIP and [3H]PIP2. Plasma membranes isolated from mastoparan-treated cells had increased PIP-phospholipase C (PLC) activity. However, when Mas-7 was added to isolated plasma membranes from control cells, it had no effect on PIP-PLC activity at low concentrations and inhibited PIP-PLC at concentrations greater than 10 microM. In addition, guanosine-5'-O-(3-thiotriphosphate) had no effect on the PIP-PLC activity when added to plasma membranes isolated from either the Mas-7-treated or control cells. The fact that Mas-7 did not stimulate PIP-PLC activity in vitro indicated that the Mas-7-induced increase in PIP-PLC in vivo required a factor that was lost from the membrane during isolation. PMID:7716245

  13. Mu-opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium-channel opening.

    PubMed

    Smart, D; Smith, G; Lambert, D G

    1995-01-15

    We have recently reported that, in SH-SY5Y cells, mu-opioid receptor occupancy activates phospholipase C via a pertussis toxin-sensitive G-protein. In the present study we have further characterized the mechanisms involved in this process. Fentanyl (0.1 microM) caused a monophasic increase in inositol 1,4,5-trisphosphate mass formation, with a peak (20.5 +/- 3.6 pmol/mg of protein) at 15 s. Incubation in Ca(2+)-free buffer abolished this response, while Ca2+ replacement 1 min later restored the stimulation of inositol 1,4,5-trisphosphate formation (20.1 +/- 0.6 pmol/mg of protein). In addition, nifedipine (1 nM-0.1 mM), an L-type Ca(2+)-channel antagonist, caused a dose-dependent inhibition of inositol 1,4,5-trisphosphate formation, with an IC50 of 60.3 +/- 1.1 nM. Elevation of endogenous beta/gamma subunits by selective activation of delta-opioid and alpha 2 adrenoceptors failed to stimulate phospholipase C. Fentanyl also caused a dose-dependent (EC50 of 16.2 +/- 1.0 nM), additive enhancement of carbachol-induced inositol 1,4,5-trisphosphate formation. In summary, we have demonstrated that in SH-SY5Y cells activation of the mu-opioid receptor allows Ca2+ influx to activate phospholipase C. However, the possible role of this mechanism in the process of analgesia remains to be elucidated. PMID:7832776

  14. Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C.

    PubMed

    Shukla, S D; Coleman, R; Finean, J B; Michell, R H

    1980-04-01

    When isolated hepatocytes are incubated with phosphatidylinositol-specific phospholipase C, three cell-surface enzymes show markedly different behaviour. Most of the alkaline phosphatase is released at very low values of phosphatidylinositol hydrolysis, whereas further phosphatidylinositol hydrolysis releases only a maximum of about one-third of the 5'-nucleotidase. Alkaline phosphodiesterase I is not released. If cells containing phosphatidyl[3H]inositol are similarly treated, then the released [3H]inositol is in the form of inositol phosphate: no evidence has been obtained for any covalent association between released [3H]inositol and alkaline phosphatase.

  15. Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity.

    PubMed

    Kravchuk, Alexander V; Zhao, Li; Bruzik, Karol S; Tsai, Ming-Daw

    2003-03-01

    Eukaryotic phosphatidylinositol-specific phospholipase Cs (PI-PLCs) utilize calcium as a cofactor during catalysis, whereas prokaryotic PI-PLCs use a spatially conserved guanidinium group from Arg69. In this study, we aimed to construct a metal-dependent mutant of a bacterial PI-PLC and characterize the catalytic role of the metal ion, seeking an enhanced understanding of the functional differences between these two positively charged moieties. The following results indicate that a bona fide catalytic metal binding site was created by the single arginine-to-aspartate mutation at position 69: (1) The R69D mutant was activated by Ca(2+), and the activation was specific for R69D, not for other mutants at this position. (2) Titration of R69D with Ca(2+), monitored by (15)N-(1)H HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca(2+) to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme. (3) Upon Ca(2+) activation, the thio effect of the S(P)-isomer of the phosphorothioate analogue (k(O)/k(Sp) = 4.4 x 10(5)) approached a value similar to that of the WT enzyme, suggesting a structural and functional similarity between the two positively charged moieties (Arg69 and Asp69-Ca(2+)). The R(P)-thio effect (k(O)/k(Rp) = 9.4) is smaller than that of the WT enzyme by a factor of 5. Consequently, R69D-Ca(2+) displays higher stereoselectivity (k(Rp)/k(Sp) = 47,000) than WT (k(Rp)/k(Sp) = 7600). (4) Results from additional mutagenesis analyses suggest that the Ca(2+) binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. Our studies provide new insight into the mechanism of metal-dependent and metal-independent PI-PLCs.

  16. Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase

    PubMed Central

    An, Sungwon; Irwin, Robert P.; Allen, Charles N.; Tsai, Connie

    2011-01-01

    Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities. PMID:21389307

  17. Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase C(alpha), src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.

    PubMed

    Canet-Aviles, Rosa-Maria; Anderton, Mark; Hooper, Nigel M; Turner, Anthony J; Vaughan, Peter F T

    2002-06-15

    The signalling pathways by which muscarine and epidermal growth factor (EGF) regulate the secretion of the alpha-secretase cleavage product (sAPPalpha) of the amyloid precursor protein (APP) were examined in the human neuroblastoma SH-SY5Y. Using specific inhibitors it was found that over 80% of sAPPalpha secretion, enhanced by muscarine, occurred via the extracellular signal-regulated kinase (ERK1/2) member of the mitogen-activated protein kinase (MAPK) family and was dependent on protein kinase Calpha (PKCalpha) and a member of the Src family of non-receptor tyrosine kinases (Src-TK). In contrast the stimulation of sAPPalpha secretion by EGF was not affected by inhibitors of PKC nor Src-TK but was dependent on ERK1/2. In addition muscarine-enhanced sAPPalpha secretion and ERK1/2 activation were inhibited 60 and 80%, respectively, by micromolar concentrations of the phosphatidylinositol 3 kinase (PI-3K) inhibitor wortmannin. In comparison wortmannin decreased EGF stimulation of sAPPalpha secretion and ERK 1/2 activation by approximately 40%. Unexpectedly, U73122, an inhibitor of phosphoinositide-specific phospholipase C, did not inhibit muscarine enhancement of sAPPalpha secretion. These data are discussed in relation to a pathway for the enhancement of sAPPalpha secretion by muscarine which involves the activation of a Src-TK by G-protein beta/gamma-subunits leading to activation of PKCalpha, and ERK1/2 by a mechanism not involving phospholipase C. PMID:12191495

  18. A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2

    PubMed Central

    1996-01-01

    Defects in the gene encoding Bruton's tyrosine kinase (Btk) result in a disease called X-linked agammaglobulinemia, in which there is a profound decrease of mature B cells due to a block in B cell development. Recent studies have shown that Btk is tyrosine phosphorylated and activated upon B cell antigen receptor (BCR) stimulation. To elucidate the functions of this kinase, we examined BCR signaling of DT40 B cells deficient in Btk. Tyrosine phosphorylation of phospholipase C (PLC)-gamma 2 upon receptor stimulation was significantly reduced in the mutant cells, leading to the loss of both BCR-coupled phosphatidylinositol hydrolysis and calcium mobilization. Pleckstrin homology and Src-homology 2 domains of Btk were required for PLC-gamma 2 activation. Since Syk is also required for the BCR-induced PLC-gamma 2 activation, our findings indicate that PLC-gamma 2 activation is regulated by Btk and Syk through their concerted actions. PMID:8691147

  19. Aluminum ions alter the function of non-specific phospholipase C through the changes in plasma membrane physical properties.

    PubMed

    Pejchar, Přemysl; Martinec, Jan

    2015-01-01

    The first indication of the aluminum (Al) toxicity in plants growing in acidic soils is the cessation of root growth, but the detailed mechanism of Al effect is unknown. Here we examined the impact of Al stress on the activity of non-specific phospholipase C (NPC) in the connection with the processes related to the plasma membrane using fluorescently labeled phosphatidylcholine. We observed a rapid and significant decrease of labeled diacylglycerol (DAG), product of NPC activity, in Arabidopsis seedlings treated with AlCl₃. Interestingly, an application of the membrane fluidizer, benzyl alcohol, restored the level of DAG during Al treatment. Our observations suggest that the activity of NPC is affected by Al-induced changes in plasma membrane physical properties. PMID:26024014

  20. 2-aminohydroxamic acid derivatives as inhibitors of Bacillus cereus phosphatidylcholine preferred phospholipase C PC-PLC(Bc).

    PubMed

    González-Bulnes, Patricia; González-Roura, Albert; Canals, Daniel; Delgado, Antonio; Casas, Josefina; Llebaria, Amadeu

    2010-12-15

    Phosphatidylcholine preferring phospholipase C (PC-PLC) is an important enzyme that plays a key role in a variety of cellular events and lipid homoeostases. Bacillus cereus phospholipase C (PC-PLC(Bc)) has antigenic similarity with the elusive mammalian PC-PLC, which has not thus far been isolated and purified. Therefore the discovery of inhibitors of PC-PLC(Bc) is of current interest. Here, we describe the synthesis and biological evaluation of a new type of compounds inhibiting PC-PLC(Bc). These compounds have been designed by evolution of previously described 2-aminohydroxamic acid PC-PLC(Bc) inhibitors that block the enzyme by coordination of the zinc active site atoms present in PC-PLC(Bc) [Gonzalez-Roura, A.; Navarro, I.; Delgado, A.; Llebaria, A.; Casas, J. Angew. Chem. Int. Ed.2004, 43, 862]. The new compounds maintain the zinc coordinating groups and possess an extra trimethylammonium function, linked to the hydroxyamide nitrogen by an alkyl chain, which is expected to mimic the trimethylammonium group of the phosphatidylcholine PC-PLC(Bc) substrates. Some of the compounds described inhibit the enzyme with IC(50)'s in the low micromolar range. Unexpectedly, the most potent inhibitors found are those that possess a trimethylammonium group but have chemically blocked the zinc coordinating functionalities. The results obtained suggest that PC-PLC(Bc) inhibition is not due to the interaction of compounds with the phospholipase catalytic zinc atoms, but rather results from the inhibitor cationic group recognition by the PC-PLC(Bc) amino acids involved in choline lipid binding.

  1. Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: evaluation of the roles of phospholipases C and D.

    PubMed

    Booz, G W; Taher, M M; Baker, K M; Singer, H A

    1994-12-21

    Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In 32P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar1]angiotensin II was shown to rapidly induce formation of 32P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as 32P- or 3H-phosphatidylethanol was produced when cells labeled with [32P]H3PO4 or 1-O-[1,2- 3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar1]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca2+, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar1]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar1]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar1]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar1]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such as de novo synthesis, may contribute to [Sar1]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar1]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Neuropeptide Y reduces the expression of PLCB2, PLCD1 and selected PLC genes in cultured human endothelial cells.

    PubMed

    Lo Vasco, V R; Leopizzi, M; Puggioni, C; Della Rocca, C; Businaro, R

    2014-09-01

    Endothelial cells (EC) are the first elements exposed to mediators circulating in the bloodstream, and react to stimulation with finely tuned responses mediated by different signal transduction pathways, leading the endothelium to adapt. Neuropeptide Y (NPY), the most abundant peptide in heart and brain, is mainly involved in the neuroendocrine regulation of the stress response. The regulatory roles of NPY depend on many factors, including its enzymatic processing, receptor subtypes and related signal transduction systems, including the phosphoinositide (PI) pathway and related phospholipase C (PI-PLC) family of enzymes. The panel of expression of PI-PLC enzymes differs comparing quiescent versus differently stimulated human EC. Growing evidences indicate that the regulation of the expression of PLC genes, which codify for PI-PLC enzymes, might act as an additional mechanism of control of the PI signal transduction pathway. NPY was described to potentiate the activation of PI-PLC enzymes in different cell types, including EC. In the present experiments, we stimulated human umbilical vein EC using different doses of NPY in order to investigate a possible role upon the expression PLC genes. NPY reduced the overall transcription of PLC genes, excepting for PLCE. The most significant effects were observed for PLCB2 and PLCD1, both isoforms recruited by means of G-proteins and G-protein-coupled receptors. NPY behavior was comparable with other PI-PLC interacting molecules that, beside the stimulation of phospholipase activity, also affect the upcoming enzymes' production acting upon gene expression. That might represent a mode to regulate the activity of PI-PLC enzymes after activation.

  3. Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: A sup 31 P NMR study

    SciTech Connect

    Shashidhar, M.S.; Kuppe, A. ); Volwerk, J.J.; Griffith, O.H.

    1990-09-04

    The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by {sup 31}P NMR. {sup 31}P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are {minus}0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. The authors also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by {sup 31}P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B.cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. They propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.

  4. Nonspecific Phospholipase C NPC4 Promotes Responses to Abscisic Acid and Tolerance to Hyperosmotic Stress in Arabidopsis[W

    PubMed Central

    Peters, Carlotta; Li, Maoyin; Narasimhan, Rama; Roth, Mary; Welti, Ruth; Wang, Xuemin

    2010-01-01

    Diacyglycerol (DAG) is an important class of cellular lipid messengers, but its function in plants remains elusive. Here, we show that knockout of the Arabidopsis thaliana nonspecific phospholipase C (NPC4) results in a decrease in DAG levels and compromises plant response to abscisic acid (ABA) and hyperosmotic stresses. NPC4 hydrolyzes various phospholipids in a calcium-independent manner, producing DAG and a phosphorylated head group. NPC4 knockout (KO) plants display decreased ABA sensitivity in seed germination, root elongation, and stomatal movement and had decreased tolerance to high salinity and water deficiency. Overexpression of NPC4 renders plants more sensitive to ABA and more tolerant to hyperosmotic stress than wild-type plants. Addition of a short-chain DAG or a short-chain phosphatidic acid (PA) restores the ABA response of NPC4-KO to that of the wild type, but the addition of DAG together with a DAG kinase inhibitor does not result in a wild-type phenotype. These data suggest that NPC4-produced DAG is converted to PA and that NPC4 and its derived lipids positively modulate ABA response and promote plant tolerance to drought and salt stresses. PMID:20699393

  5. Calcineurin phosphatase and phospholipase C are required for developmental and pathological functions in the citrus fungal pathogen Alternaria alternata.

    PubMed

    Tsai, Hsieh-Chin; Chung, Kuang-Ren

    2014-07-01

    Excessive Ca(2+) or compounds interfering with phosphoinositide cycling have been found to inhibit the growth of the tangerine pathotype of Alternaria alternata, suggesting a crucial role of Ca(2+) homeostasis in this pathotype. The roles of PLC1, a phospholipase C-coding gene and CAL1, a calcineurin phosphatase-coding gene were investigated. Targeted gene disruption showed that both PLC1 and CAL1 were required for vegetative growth, conidial formation and pathogenesis in citrus. Fungal strains lacking PLC1 or CAL1 exhibited extremely slow growth and induced small lesions on calamondin leaves. Δplc1 mutants produced fewer conidia, which germinated at slower rates than wild-type. Δcal1 mutants produced abnormal hyphae and failed to produce any mature conidia, but instead produced highly melanized bulbous hyphae with distinct septae. Fluorescence microscopy using Fluo-3 dye as a Ca(2+) indicator revealed that the Δplc1 mutant hyphae emitted stronger cytosolic fluorescence, and the Δcal1 mutant hyphae emitted less cytosolic fluorescence, than those of wild-type. Infection assessed on detached calamondin leaves revealed that application of CaCl2 or neomycin 24 h prior to inoculation provided protection against Alt. alternata. These data indicate that a dynamic equilibrium of cellular Ca(2+) is critical for developmental and pathological processes of Alt. alternata.

  6. Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice.

    PubMed

    Bakala N'Goma, Jean Claude; Le Moigne, Vincent; Soismier, Nathalie; Laencina, Laura; Le Chevalier, Fabien; Roux, Anne-Laure; Poncin, Isabelle; Serveau-Avesque, Carole; Rottman, Martin; Gaillard, Jean-Louis; Etienne, Gilles; Brosch, Roland; Herrmann, Jean-Louis; Canaan, Stéphane; Girard-Misguich, Fabienne

    2015-02-01

    Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

  7. Fluorescent phosphatidylinositol 4,5-bisphosphate derivatives with modified 6-hydroxy group as novel substrates for phospholipase C.

    PubMed

    Wang, Xiaoyang; Barrett, Matthew; Sondek, John; Harden, T Kendall; Zhang, Qisheng

    2012-07-01

    The capacity to monitor spatiotemporal activity of phospholipase C (PLC) isozymes with a PLC-selective sensor would dramatically enhance understanding of the physiological function and disease relevance of these signaling proteins. Previous structural and biochemical studies defined critical roles for several of the functional groups of the endogenous substrate of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP(2)), indicating that these sites cannot be readily modified without compromising interactions with the lipase active site. However, the role of the 6-hydroxy group of PIP(2) for interaction and hydrolysis by PLC has not been explored, possibly due to challenges in synthesizing 6-hydroxy derivatives. Here, we describe an efficient route for the synthesis of novel, fluorescent PIP(2) derivatives modified at the 6-hydroxy group. Two of these derivatives were used in assays of PLC activity in which the fluorescent PIP(2) substrates were separated from their diacylglycerol products and reaction rates quantified by fluorescence. Both PIP(2) analogues effectively function as substrates of PLC-δ1, and the K(M) and V(max) values obtained with one of these are similar to those observed with native PIP(2) substrate. These results indicate that the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for further development of PLC-selective sensors.

  8. Function of the p55 tumor necrosis factor receptor "death domain" mediated by phosphatidylcholine-specific phospholipase C

    PubMed Central

    1996-01-01

    Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammation that has been implicated in the pathogenesis of devastating clinical syndromes including septic shock. We have investigated the role of a TNF-responsive phosphatidylcholine-specific phospholipase C (PC-PLC) for the cytotoxic and proinflammatory activity of TNF. We show here that the cytotoxicity signaled for by the so-called "death domain" of the p55 TNF receptor is associated with the activation of PC-PLC. The xanthogenate tricyclodecan-9-yl (D609), a specific and selective inhibitor of PC-PLC, blocked the cytotoxic action of TNF on L929 and Wehi164 cells. In vivo, D609 prevented both adhesion molecule expression in the pulmonary vasculature and the accompanying leukocyte infiltration in TNF-treated mice. More strikingly, D609 protects BALB/c mice from lethal shock induced either by TNF, lipopolysaccharide, or staphylococcal enterotoxin B. Together these findings imply PC-PLC as an important mediator of the pathogenic action of TNF, suggesting that PC-PLC may serve as a novel target for anti-inflammatory TNF antagonists. PMID:8760826

  9. Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm?

    PubMed Central

    Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Yelumalai, Suseela; Mounce, Ginny; da Silva, Sarah J. Martins; Child, Tim; Coward, Kevin

    2016-01-01

    Socio-economic factors have led to an increasing trend for couples to delay parenthood. However, advancing age exerts detrimental effects upon gametes which can have serious consequences upon embryo viability. While such effects are well documented for the oocyte, relatively little is known with regard to the sperm. One fundamental role of sperm is to activate the oocyte at fertilisation, a process initiated by phospholipase C zeta (PLCζ), a sperm-specific protein. While PLCζ deficiency can lead to oocyte activation deficiency and infertility, it is currently unknown whether the expression or function of PLCζ is compromised by advancing male age. Here, we evaluate sperm motility and the proportion of sperm expressing PLCζ in 71 males (22–54 years; 44 fertile controls and 27 infertile patients), along with total levels and localisation patterns of PLCζ within the sperm head. Three different statistical approaches were deployed with male age considered both as a categorical and a continuous factor. While progressive motility was negatively correlated with male age, all three statistical models concurred that no PLCζ–related parameter was associated with male age, suggesting that advancing male age is unlikely to cause problems in terms of the sperm’s fundamental ability to activate an oocyte. PMID:27270687

  10. Clostridium perfringens Phospholipase C Induced ROS Production and Cytotoxicity Require PKC, MEK1 and NFκB Activation

    PubMed Central

    Monturiol-Gross, Laura; Flores-Díaz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis. PMID:24466113

  11. Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

    PubMed

    Monturiol-Gross, Laura; Flores-Díaz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis. PMID:24466113

  12. Diacylglycerol-Rich Domain Formation in Giant Stearoyl-Oleoyl Phosphatidylcholine Vesicles Driven by Phospholipase C Activity

    PubMed Central

    Riske, Karin A.; Döbereiner, Hans-Günther

    2003-01-01

    We have studied the effect of phospholipase C from Bacillus cereus and Clostridium perfringens (α-toxin) on giant stearoyl-oleoyl phosphatidylcholine (SOPC) vesicles. Enzyme activity leads to a binary mixture of SOPC and the diacylglycerol SOG, which phase separates into a SOPC-rich bilayer phase and a SOG-rich isotropic bulk-like domain embedded within the membrane, as seen directly by phase contrast microscopy. After prolonged enzymatic attack, all bilayer membranes are transformed into an isotropic pure SOG phase as characterized by fluorescence microscopy, differential scanning calorimetry, fluorescence anisotropy measurements, and small angle x-ray scattering. These domains may have biological relevance, serving as storage compartments for hydrophobic molecules and/or catalyzing cellular signaling events at their boundaries. Furthermore, in the early stages of asymmetric enzymatic attack to the external monolayer of giant vesicles, we observe a transient coupling of the second-messenger diacylglycerol to membrane spontaneous curvature, which decreases due to enzyme activity, before domain formation and final vesicle collapse occurs. PMID:14507699

  13. Mutation of the phospholipase C-gamma1-binding site of LAT affects both positive and negative thymocyte selection.

    PubMed

    Sommers, Connie L; Lee, Jan; Steiner, Kevin L; Gurson, Jordan M; Depersis, Corinne L; El-Khoury, Dalal; Fuller, Claudette L; Shores, Elizabeth W; Love, Paul E; Samelson, Lawrence E

    2005-04-01

    Linker for activation of T cells (LAT) is a scaffolding adaptor protein that is critical for T cell development and function. A mutation of LAT (Y136F) that disrupts phospholipase C-gamma1 activation and subsequent calcium influx causes a partial block in T cell development and leads to a severe lymphoproliferative disease in homozygous knock-in mice. One possible contribution to the fatal disease of LAT Y136F knock-in mice could be from autoreactive T cells generated in these mice because of altered thymocyte selection. To examine the impact of the LAT Y136F mutation on thymocyte positive and negative selection, we bred this mutation onto the HY T cell receptor (TCR) transgenic, recombination activating gene-2 knockout background. Female mice with this genotype showed a severe defect in positive selection, whereas male mice exhibited a phenotype resembling positive selection (i.e., development and survival of CD8(hi) HY TCR-specific T cells) instead of negative selection. These results support the hypothesis that in non-TCR transgenic, LAT Y136F knock-in mice, altered thymocyte selection leads to the survival and proliferation of autoreactive T cells that would otherwise be negatively selected in the thymus.

  14. Angiogenin activates phospholipase C and elicits a rapid incorporation of fatty acid into cholesterol esters in vascular smooth muscle cells

    SciTech Connect

    Moore, F.; Riordan, J.F. )

    1990-01-09

    Angiogenin activates the phosphoinositide-specific phospholipase C (PLC) in cultured rat aortic smooth muscle cells to yield a transient (30 s) peak of 1,2-diacylglycerol (DG) and inositol trisphosphate. Within 1 min, the DG level falls below that of the control and remains so for at least 20 min. A transient increase in monoacylglycerol indicates that depletion of DG may be the consequence of hydrolysis by DG lipase. In addition to these changes in second messengers, a rapid increase in incorporating of radiolabeled tracer into cellular cholesterol esters is observed. Stimulated cholesterol ester labeling is inhibited by preincubation with either the DG lipase inhibitor RHC 80267 or the acyl coenzyme A:cholesterol acyltransferase inhibitor Sandoz 58035. Cells prelabeled with ({sup 3}H)arachidonate show a sustained increase in labeling of cholesterol esters following exposure to angiogenin. In contrast, cells prelabeled with ({sup 3}H)oleate show only a transient elevation that returns to the basal level by 5 min. This suggests initial cholesterol esterification by oleate followed by arachidonate that is released by stimulation of the PLC/DG lipase pathway.

  15. Non-specific phospholipase C1 affects silicon distribution and mechanical strength in stem nodes of rice.

    PubMed

    Cao, Huasheng; Zhuo, Lin; Su, Yuan; Sun, Linxiao; Wang, Xuemin

    2016-05-01

    Silicon, the second abundant element in the crust, is beneficial for plant growth, mechanical strength, and stress responses. Here we show that manipulation of the non-specific phospholipase C1, NPC1, alters silicon content in nodes and husks of rice (Oryza sativa). Silicon content in NPC1-overexpressing (OE) plants was decreased in nodes but increased in husks compared to wild-type, whereas RNAi suppression of NPC1 resulted in the opposite changes to those of NPC1-OE plants. NPC1 from rice hydrolyzed phospholipids and galactolipids to generate diacylglycerol that can be phosphorylated to phosphatidic acid. Phosphatidic acid interacts with Lsi6, a silicon transporter that is expressed at the highest level in nodes. In addition, the node cells of NPC1-OE plants have lower contents of cellulose and hemicellulose, and thinner sclerenchyma and vascular bundle fibre cells than wild-type plants; whereas NPC1-RNAi plants displayed the opposite changes. These data indicate that NPC1 modulates silicon distribution and secondary cell wall deposition in nodes and grains, affecting mechanical strength and seed shattering. PMID:26991499

  16. Dephosphorylation of the adaptor LAT and phospholipase C-γ by SHP-1 inhibits natural killer cell cytotoxicity.

    PubMed

    Matalon, Omri; Fried, Sophia; Ben-Shmuel, Aviad; Pauker, Maor H; Joseph, Noah; Keizer, Danielle; Piterburg, Marina; Barda-Saad, Mira

    2016-01-01

    Natural killer (NK) cells discriminate between healthy cells and virally infected or transformed self-cells by tuning activating and inhibitory signals received through cell surface receptors. Inhibitory receptors inhibit NK cell function by recruiting and activating the tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) to the plasma membrane. However, to date, the guanine nucleotide exchange factor VAV1 is the only direct SHP-1 substrate identified in NK cells. We reveal that the adaptor protein linker for activation of T cells (LAT) as well as phospholipase C-γ1 (PLC-γ1) and PLC-γ2 are SHP-1 substrates. Dephosphorylation of Tyr(132) in LAT by SHP-1 in NK cells abrogated the recruitment of PLC-γ1 and PLC-γ2 to the immunological synapse between the NK cell and a cancer cell target, which reduced NK cell degranulation and target cell killing. Furthermore, the ubiquitylation of LAT by the E3 ubiquitin ligases c-Cbl and Cbl-b, which was induced by LAT phosphorylation, led to the degradation of LAT in response to the engagement of inhibitory receptors on NK cells, which abrogated NK cell cytotoxicity. Knockdown of the Cbl proteins blocked LAT ubiquitylation, which promoted NK cell function. Expression of a ubiquitylation-resistant mutant LAT blocked inhibitory receptor signaling, enabling cells to become activated. Together, these data identify previously uncharacterized SHP-1 substrates and inhibitory mechanisms that determine the response of NK cells.

  17. Phospholipase C-gamma1 is required for subculture-induced terminal differentiation of normal human oral keratinocytes.

    PubMed

    Oh, Ju-Eun; Kook, Joong-Ki; Park, Kyung-Hee; Lee, Gene; Seo, Byoung-Moo; Min, Byung-Moo

    2003-04-01

    Serial subculture of primary normal human oral keratinocytes (NHOKs) to the post-mitotic stage induces terminal differentiation, which is in part linked to elevated levels of phospholipase C (PLC)-gamma1. Therefore, PLC-gamma1 may be involved in the signal transduction system that leads to the calcium regulation of subculture-induced keratinocyte differentiation. To test this hypothesis, the expression of PLC-gamma1 in primary NHOKs was blocked by transfecting cells with the antisense PLC-gamma1 cDNA construct. These cells demonstrated dramatic reductions in PLC-gamma1 protein and in the differentiation markers involucrin and transglutaminase following calcium exposure and an increase (15-20%) in in vitro life span versus empty vector-transfected cells. In addition, we established the ability of antisense PLC-gamma1 to block the serial subculture-induced rise in intracellular calcium. Similar observations were made following treatment with the specific PLC inhibitor U73122. These results indicate that the terminal differentiation of NHOKs by serial subculture is associated with PLC-gamma1, which mediates calcium regulation by mobilizing intracellular calcium.

  18. How far does phospholipase C activity depend on the cell calcium concentration? A study in intact cells.

    PubMed Central

    Renard, D; Poggioli, J; Berthon, B; Claret, M

    1987-01-01

    The dependence of phospholipase C activity on the cytosolic Ca2+ concentration ([Ca2+]i) was studied in intact liver cells treated with the Ca2+-mobilizing hormone vasopressin, or not so treated. Phospholipase C (PLC) activity was estimated from the formation of [3H]inositol trisphosphate (InsP3) and the degradation of [3H]phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The [Ca2+]i of the cells was clamped from 29 to 1130 nM by quin2 loading. This wide concentration range was obtained by loading the hepatocytes with a high concentration of the Ca2+ indicator in low-Ca2+ medium or by using the Ca2+ ionophore ionomycin in medium containing Ca2+. In resting cells, in which [Ca2+]i was 193 nM, treatment with 0.1 microM-vasopressin which stimulates liver PLC maximally, tripled InsP3 content and raised [Ca2+]i to 2 microM within 15 s. Lowering [Ca2+]i partially decreased cell InsP3 content as well as the ability of vasopressin to stimulate InsP3 formation maximally. At 29 nM, the lowest Ca2+ concentration obtained in isolated liver cells, basal InsP3 content was 64% of that measured in control cells. Addition of vasopressin no longer affected [Ca2+]i, but significantly increased InsP3 by 200%, although less than in the controls (300%). The maintenance of the greater part of the PLC response at constant [Ca2+]i indicated that, in the liver, InsP3 formation does not result from an increase in [Ca2+]i. The effects of lowering [Ca2+]i were reversible. When low cell [Ca2+]i was restored to a normal value, resting InsP3 content and the ability of vasopressin to stimulate InsP3 formation maximally by 300% were also restored. Raising [Ca2+]i from 193 to 1130 nM had little effect on the InsP3 content or the vasopressin-mediated increase in InsP3. In agreement with the stimulation of PLC activity by vasopressin, cell [3H]PtdInsP2 and total PtdInsP2 were degraded by application of this hormone for 15 s. In contrast, when [Ca2+]i was lowered to 29 nM, basal [3H]PtdInsP2 and total

  19. ISC1-encoded inositol phosphosphingolipid phospholipase C is involved in Na+/Li+ halotolerance of Saccharomyces cerevisiae.

    PubMed

    Betz, Christian; Zajonc, Dirk; Moll, Matthias; Schweizer, Eckhart

    2002-08-01

    In Saccharomyces cerevisiae, toxic concentrations of Na+ orLi+ ions induce the expression of the cation-extrusion ATPase gene, ENA1. Several well-studied signal transduction pathways are known correlating high salinity to the transcriptional activation of ENA1. Nevertheless, information on the actual sensing mechanism initiating these pathways is limited. Here, we report that the ISC1-encoded phosphosphingolipid-specific phospholipase C appears to be involved in stimulation of ENA1 expression and, consequently, in mediating Na+ and Li+ tolerance in yeast. Deletion of ISC1 distinctly decreased cellular Na+ and Li+ tolerance as growth of the Deltaisc1::HIS5 mutant, DZY1, was severely impaired by 0.5 m NaCl or 0.01 m LiCl. In contrast,K+ tolerance and general osmostress regulation wereunaffected. Isc1Delta mutant growth with 0.9 m KCl and glycerol accumulation in the presence of 0.9 m NaCl or 1.5 m sorbitol were comparable to that of the wild-type. ENA1-lacZ reporter studies suggested that the increased salt sensitivity of the isc1Delta mutant is related to a significant reduction of Na+/Li+-stimulated ENA1 expression. Correspondingly, Ena1p-dependent extrusion of Na+/Li+ ions was less efficient in the isc1Delta mutant than in wild-type cells. Itis suggested that ISC1-dependent hydrolysis of an unidentified yeast inositol phosphosphingolipid represents an early event in one of the salt-induced signalling pathways of ENA1 transcriptional activation. PMID:12180980

  20. Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling.

    PubMed

    Kim, Jung Kuk; Choi, Jung Woong; Lim, Seyoung; Kwon, Ohman; Seo, Jeong Kon; Ryu, Sung Ho; Suh, Pann-Ghill

    2011-06-01

    Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-η1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling.

  1. Calcium fluxes cause nuclear shrinkage and the translocation of phospholipase C-delta1 into the nucleus.

    PubMed

    Okada, Masashi; Taguchi, Katsutoshi; Maekawa, Shohei; Fukami, Kiyoko; Yagisawa, Hitoshi

    2010-03-26

    Phospholipase C-delta1 (PLCdelta1) is the most fundamental form of the eukaryotic PLC and thought to play important roles in the regulation of cells. We previously reported that PLCdelta1 shuttles between the cytoplasm and nucleus, and an influx of Ca2+ triggers the nuclear import of PLCdelta1 via Ca2+-dependent interaction with importin beta1, although the physiological meaning of this is unclear. Here we have examined the distribution of PLCdelta1 using primary cultures of rat hippocampal neurons. Treatment of 7DIV neurons with ionomycin or thapsigargin caused the nuclear localization of PLCdelta1 as has been observed in other cell lines. Similar results were obtained with neurons treated with glutamate, suggesting that the nuclear localization of PLCdelta1 plays some roles in excitotoxicity associated with ischemic stress. Generally, cells undergoing ischemic or hypoxic cell death show nuclear shrinkage. We confirmed that a massive influx of Ca2+ caused similar results. Furthermore, overexpression of GFP-PLCdelta1 facilitated ionomycin-induced nuclear shrinkage in embryonic fibroblasts derived from PLCdelta1 gene-knockout mice (PLCdelta1KO-MEF). By contrast, an E341A mutant that cannot bind with importin beta1 and be imported into the nucleus by ionomycin and also lacks enzymatic activity did not cause nuclear shrinkage in PLCdelta1KO-MEF. Nuclear translocation and the PLC activity of PLCdelta1, therefore, may regulate the nuclear shape by controlling the nuclear scaffold during stress-induced cell death caused by high levels of Ca2+.

  2. Selectivity of phospholipase C phosphorylation by the epidermal growth factor receptor, the insulin receptor, and their cytoplasmic domains.

    PubMed Central

    Nishibe, S; Wahl, M I; Wedegaertner, P B; Kim, J W; Rhee, S G; Carpenter, G; Kim, J J

    1990-01-01

    Phosphatidylinositol-specific phospholipase C isozyme gamma (PLC-gamma, Mr 145,000) is an excellent substrate for the epidermal growth factor (EGF) receptor both in vivo and in vitro. PLC-beta-1, another PLC isozyme, is a poor substrate for the EGF receptor. We examined the relative phosphorylation of PLC-gamma and PLC-beta-1 by the 170-kDa native EGF receptor molecule, the 66-kDa cytoplasmic kinase domain of the EGF receptor (Arg647-Ala1186), the alpha 2 beta 2 native insulin receptor, and the 48-kDa cytoplasmic kinase domain of the insulin receptor beta subunit (Gly947-Ser1343). Similar to the intact EGF receptor, the cytoplasmic kinase domain of the EGF receptor preferentially phosphorylated PLC-gamma. High-performance liquid chromatographic comparison of tryptic phosphopeptides from PLC-gamma phosphorylated by both forms of the EGF receptor kinase indicated similar patterns of multiple tyrosine phosphorylations. These results imply that substrate selectivity, at least in terms of PLC isozymes, is independent of the extracellular ligand-binding and membrane anchor domains of the EGF receptor. In comparison, neither the intact insulin receptor nor the beta-chain kinase domain was able to phosphorylate PLC-gamma to a significant extent. Also, insulin failed to stimulate the phosphorylation of PLC-gamma in NIH 3T3/HIR cells, which overexpress the human insulin receptor. Thus PLC-gamma is not a phosphorylation substrate for the insulin receptor in vitro or in the intact cell. Images PMID:2153302

  3. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

    PubMed Central

    Wong, Raymond; Fabian, Lacramioara; Forer, Arthur; Brill, Julie A

    2007-01-01

    Background Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring. PMID:17509155

  4. Intercellular Odontoblast Communication via ATP Mediated by Pannexin-1 Channel and Phospholipase C-coupled Receptor Activation

    PubMed Central

    Sato, Masaki; Furuya, Tadashi; Kimura, Maki; Kojima, Yuki; Tazaki, Masakazu; Sato, Toru; Shibukawa, Yoshiyuki

    2015-01-01

    Extracellular ATP released via pannexin-1 channels, in response to the activation of mechanosensitive-TRP channels during odontoblast mechanical stimulation, mediates intercellular communication among odontoblasts in dental pulp slice preparation dissected from rat incisor. Recently, odontoblast cell lines, such as mouse odontoblast lineage cells, have been widely used to investigate physiological/pathological cellular functions. To clarify whether the odontoblast cell lines also communicate with each other by diffusible chemical substance(s), we investigated the chemical intercellular communication among cells from mouse odontoblast cell lines following mechanical stimulation. A single cell was stimulated using a glass pipette filled with standard extracellular solution. We measured intracellular free Ca2+ concentration ([Ca2+]i) by fura-2 in stimulated cells, as well as in cells located nearby. Direct mechanical stimulation to a single odontoblast increased [Ca2+]i, which showed sensitivity to capsazepine. In addition, we observed increases in [Ca2+]i not only in the mechanically stimulated odontoblast, but also in nearby odontoblasts. We could observe mechanical stimulation-induced increase in [Ca2+]i in a stimulated human embryo kidney (HEK) 293 cell, but not in nearby HEK293 cells. The increase in [Ca2+]i in nearby odontoblasts, but not in the stimulated odontoblast, was inhibited by adenosine triphosphate (ATP) release channel (pannexin-1) inhibitor in a concentration- and spatial-dependent manner. Moreover, in the presence of phospholipase C (PLC) inhibitor, the increase in [Ca2+]i in nearby odontoblasts, following mechanical stimulation of a single odontoblast, was abolished. We could record some inward currents evoked from odontoblasts near the stimulated odontoblast, but the currents were observed in only 4.8% of the recorded odontoblasts. The results of this study showed that ATP is released via pannexin-1, from a mechanically stimulated odontoblast

  5. Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

    PubMed

    Mensa-Wilmot, K; Hereld, D; Englund, P T

    1990-02-01

    The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.

  6. CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C-gamma(1) upon B cell activation

    PubMed Central

    1996-01-01

    Cross-linking B cell antigen receptor (BCR) elicits early signal transduction events, including activation of protein tyrosine kinases, phosphorylation of receptor components, activation of phospholipase C- gamma (PLC-gamma), and increases in intracellular free Ca2+. In this article, we report that cross-linking the BCR led to a rapid translocation of cytosolic protein tyrosine phosphatase (PTP) 1C to the particulate fraction, where it became associated with a 140-150-kD tyrosyl-phosphorylated protein. Western blotting analysis identified this 140-150-kD protein to be CD22. The association of PTP-1C with CD22 was mediated by the NH2-terminal Src homology 2 (SH2) domain of PTP-1C. Complexes of either CD22/PTP-1C/Syk/PLC-gamma(1) could be isolated from B cells stimulated by BCR engagement or a mixture of hydrogen peroxidase and sodium orthovanadate, respectively. The binding of PLC- gamma(1) and Syk to tyrosyl-phosphorylated CD22 was mediated by the NH2- terminal SH2 domain of PLC-gamma(1) and the COOH-terminal SH2 domain of Syk, respectively. These observations suggest that tyrosyl- phosphorylated CD22 may downmodulate the activity of this complex by dephosphorylation of CD22, Syk, and/or PLC-gamma(1). Transient expression of CD22 and a null mutant of PTP-1C (PTP-1CM) in COS cells resulted in an increase in tyrosyl phosphorylation of CD22 and its interaction with PTP-1CM. By contrast, CD22 was not tyrosyl phosphorylated or associated with PTP-1CM in the presence of wild-type PTP-1C. These results suggest that tyrosyl-phosphorylated CD22 may be a substrate for PTP-1C regulates tyrosyl phosphorylation of CD22. PMID:8627166

  7. GTP-dependent hydrolysis of phosphatidylinositol-4,5-bisphosphate by a soluble phospholipase C from adult human epidermis

    SciTech Connect

    Fisher, G.J.; Baldassare, J.J.; Voorhees, J.J.

    1987-05-01

    The effects of tumor promoting phorbol esters, which activate protein kinase (C (PK-C), on epidermis suggest that PK-C is important in the regulation of epidermal growth and differentiation. Since in vivo PK-C is activated by the products of phospholipase C (PL-C)-catalyzed hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP/sub 2/), they have investigated the properties of this reaction. Soluble PL-C from adult human epidermis was incubated with sonicated lipid vesicles containing (/sup 3/H-inositol)PIP/sub 2/, for 10 minutes at 37/sup 0/C. Water soluble reaction products were extracted, chromatographed and quantitated. In the presence of physiological concentrations of Ca/sup + +/ magnesium and GTP PIP/sub 2/, but not phosphatidylinositol, was hydrolyzed by PL-C (11.7 nmol/min/mg). Addition of GTP or GTP..gamma..S stimulated activity greater than 15 fold. Half maximal and maximal activity were observed at 10 ..mu..M and 100 ..mu..M GTP..gamma..S, respectively. ATP was unable to substitute for GTP, and GDP/S inhibited PIP/sub 2/ hydrolysis in a dose dependent manner. Activity was sensitive to pH, and exhibited a sharp optimum at pH 6.5. In addition, the PL-C preparation specifically bound (/sup 35/S)GTP S. These data demonstrate that adult human epidermis contains PL-C activity that specifically hydrolyzes PIP/sub 2/ and suggest the involvement of a GTP-binding regulatory protein in this reaction.

  8. Methylmercury-induced toxicity is mediated by enhanced intracellular calcium through activation of phosphatidylcholine-specific phospholipase C

    SciTech Connect

    Kang, Mi Sun; Jeong, Ju Yeon; Seo, Ji Heui; Jeon, Hyung Jun; Jung, Kwang Mook; Chin, Mi-Reyoung; Moon, Chang-Kiu; Bonventre, Joseph V.; Jung, Sung Yun; Kim, Dae Kyong . E-mail: proteinlab@hanmail.net

    2006-10-15

    Methylmercury (MeHg) is a ubiquitous environmental toxicant to which humans can be exposed by ingestion of contaminated food. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of free intracellular Ca{sup 2+} levels ([Ca{sup 2+}]{sub i}). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity in MDCK cells. D609, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner with concomitant inhibition of the diacylglycerol (DAG) generation and the phosphatidylcholine (PC)-breakdown. MeHg activated the group IV cytosolic phospholipase A{sub 2} (cPLA{sub 2}) and acidic form of sphingomyelinase (A-SMase) downstream of PC-PLC, but these enzymes as well as protein kinase C (PKC) were not linked to the toxicity by MeHg. Furthermore, MeHg produced ROS, which did not affect the toxicity. Addition of EGTA to culture media resulted in partial decrease of [Ca{sup 2+}]{sub i} and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca{sup 2+} in the culture media, D609 completely prevented cell death with parallel decrease in [Ca{sup 2+}]{sub i}. Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca{sup 2+}]{sub i} through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA{sub 2}, A-SMase, and PKC, or to the generation of ROS.

  9. Phospholipase C epsilon 1 (PLCE1) Haplotypes are Associated with Increased Risk of Gastric Cancer in Kashmir Valley

    PubMed Central

    Malik, Manzoor A.; Srivastava, Priya; Zargar, Showkat A.; Mittal, Balraj

    2014-01-01

    Background/Aim: Phospholipase C epsilon 1 (PLCE1) plays a crucial role in carcinogenesis and progression of several types of cancers. A single nucleotide polymorphism (SNP, rs2274223) in PLCE1 has been identified as a novel susceptibility locus. The aim of the present study was to investigate the role of three potentially functional SNPs (rs2274223A > G, rs3765524C > T, and rs7922612C > T) of PLCE1 in gastric cancer patients from Kashmir Valley. Patients and Methods: The study was conducted in 108 GC cases and 195 healthy controls from Kashmir Valley. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism method. Data were statistically analyzed using χ2 test and logistic regression models. A P value of less than 0.05 was regarded as statistically significant. Results: The frequency of PLCE1 A2274223C3765524T7922612, G2274223C3765524T7922612, and G2274223T3765524C7922612 haplotypes were higher in patients compared with controls, conferred high risk for GC [odds ratio (OR) =6.29; P = 0.001; Pcorr = 0.003], (OR = 3.23; P = 0.011; Pcorr = 0.033), and (OR = 5.14; P = 0.011; Pcorr = 0.033), respectively. Smoking and salted tea are independent risk factors for GC, but we did not find any significant modulation of cancer risk by PLCE1 variants with smoking or excessive consumption of salted tea. Conclusion: These results suggest that variation in PLCE1 may be associated with GC risk in Kashmir Valley. PMID:25434319

  10. Phospholipase C interacts with Sgd1p and is required for expression of GPD1 and osmoresistance in Saccharomyces cerevisiae.

    PubMed

    Lin, H; Nguyen, P; Vancura, A

    2002-05-01

    The Saccharomyces cerevisiae PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C. Cells deleted for PLC1 ( plc1Delta) are viable, but display several phenotypes, including osmotic, temperature, and nocodazole sensitivity. We have used a two-hybrid screen to identify Plc1p-interacting proteins. One of the interacting proteins found was Sgd1p, a recently identified, essential, nuclear protein. The SGD1 gene was originally cloned by complementation of an osmostress-sensitive mutant. The Plc1p-Sgd1p interaction was confirmed biochemically by affinity chromatography. SGD1 interacts genetically with both PLC1 and HOG1 (which encodes an osmosensing mitogen-activated protein kinase). Overexpression of Sgd1p suppresses the temperature sensitivity of cells bearing the plc1-4 allele, and the double mutant strain plc1Delta sgd1-1 displays enhanced temperature and nocodazole sensitivity. The plc1Delta hog1Delta strain displays increased osmosensitivity, and has a synthetic defect in glycerol synthesis and the expression of GPD1 (which encodes the enzyme glycerol 3-phosphate dehydrogenase that is involved in glycerol biosynthesis), suggesting that Plc1p and Hog1p function in independent pathways. The hog1Delta sgd1-1 double mutant displays enhanced osmosensitivity relative to that of either single mutant. The triple mutant plc1Delta hog1Delta sgd1-1 is inviable, while the plc1Delta hog1Delta sgd1-2 strain grows extremely slowly and is more osmosensitive than the plc1Delta hog1Delta or hog1Delta sgd1-2 strain. These results are consistent with a model in which Plc1p and Hog1p function in parallel pathways affecting osmoregulation, and signals from both these pathways converge, at least partly, on Sgd1p.

  11. Interaction of apolipoprotein A-I with lecithin-cholesterol vesicles in the presence of phospholipase C.

    PubMed

    Gudheti, Manasa V; Gonzalez, Yamaira I; Lee, Sum P; Wrenn, Steven P

    2003-12-30

    Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.

  12. Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma

    PubMed Central

    Ferri, Renata; Mercurio, Laura; Canevari, Silvana; Podo, Franca; Miotti, Silvia; Iorio, Egidio

    2015-01-01

    Purpose The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them. Results Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells). Conclusions These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential. PMID:26402860

  13. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    SciTech Connect

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung; Rebecchi, Mario

    2012-10-01

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

  14. Loss of src, topoisomerase 1 and phospholipase C genes in 20q deletions in myeloid leukemia by FISH

    SciTech Connect

    Rao, P.N.; Hayworth, R.; Carroll, A.J. |

    1994-09-01

    Deletion of 20q is one of the more consistent abnormalities in myeloid leukemia (ML). This has led to the suggestion of a loss of(a) tumor-suppressor gene(s) that could provide a proliferative advantage to myeloid cells. Using in situ hybridization mapping, the deleted region was determined to be between 20q11.2 to q12, flanked proximally by ribophorin and distally by D20S17. We have been constructing a genetic map of the 20q12-q13.1 region and the MODY locus. We further defined the del(20q) by studying 5 patients with ML and cytogenetically identifiable deletions of 20q, using multicolor FISH, and scored for the presence or absence of the following probes: oncogenes hck and src, phospholipase C (plc1), adenosine deaminase (ada), DNA topoisomerase 1 (top1), D20S17, and D20S16. hck mapped to proximal 20q11.2 and was present in the normal and deleted chromosome 20. D20S16 was observed in all but one patient suggesting that it is also not part of the critical region of deletion. src, ada, top1, plc1, and 20S17 were deleted in all patients. The loss of plc1 and top1 in ML is reported for the first time and is important given their function in cell growth and replication. top1 relaxes supercoiled DNA and is important for RNA transcription, DNA replication and repair. A lack of DNA repair caused by the inactivation of top1 may lead to genomic instability and thus neoplasia and apoptosis. plc1 catalyzes inositol phospholipids to second messenger molecules diacylglycerol and inositol triphosphate which directly affect cell division and growth. If there is a loss or alteration of plc1, it is speculated that the leukemic cells instead of undergoing apoptosis, may follow an alternative pathway that provides proliferative signals causing the progression to an aggressive acute myeloid leukemia. Further studies regarding the role of plc1 in apoptosis and ML may lead to promising results.

  15. Metabotropic glutamate receptor/phospholipase C pathway: a vulnerable target to Creutzfeldt-Jakob disease in the cerebral cortex.

    PubMed

    Rodríguez, A; Freixes, M; Dalfó, E; Martín, M; Puig, B; Ferrer, I

    2005-01-01

    Glutamate is the main excitatory neurotransmitter in the cerebral cortex. Altered glutamatergic transmission has been suggested as having a central role in many neurodegenerative diseases. Metabotropic glutamate receptors (mGluRs) are coupled to intracellular signal transduction via G proteins, and they mediate slower responses than ionotropic glutamate receptors. Group I mGluRs are positively coupled to phospholipase C beta1 (PLCbeta1). Creutzfeldt-Jakob disease (CJD) is a human transmissible spongiform encephalopathy associated with a dysfunction in the membrane glycoprotein PrP which is converted into an abnormal isoform, with a predominant beta-sheet structure, that is pathogenic and partially resistant to protease digestion. Proteins associated with the signal transduction of group I mGluRs were examined in the frontal cortex (area 8) of 12 cases with sCJD and four age-matched controls, by means of gel electrophoresis and Western blotting of total homogenates. Densitometric analysis of the bands demonstrated decreased expression levels of PLCbeta1 and PLCgamma, a non-related phospholipase which is a substrate of tyrosine kinase, in CJD cases when compared with controls. Novel protein kinase C delta (nPKCdelta) has also been found to be significantly decreased in CJD cases. However, no modifications in mGluR1 cPKCalpha expression levels are found in CJD when compared with controls. No modifications in PLCbeta1 solubility in PBS-, deoxycholate- and sodium dodecylsulphate-soluble fractions have been observed in CJD when compared with controls. Finally, no interactions between PLCbeta1 and PrP, as revealed by immunoprecipitation assays, have been found in CJD and controls. The present results show, for the first time, reduced expression levels of phospholipases, particularly PLCbeta1, which may interfere with group I mGluR signaling in the cerebral cortex in CJD. These abnormalities are not the result of abnormal PLC solubility or interactions with PrP. Selective

  16. Phospholipase C gamma mediates endogenous brain-derived neurotrophic factor-regulated calcitonin gene-related peptide expression in colitis-induced visceral pain

    PubMed Central

    Hashmi, Fiza; Liu, Miao; Shen, Shanwei

    2016-01-01

    Background Visceral hypersensitivity is a complex pathophysiological paradigm with unclear mechanisms. Primary afferent neuronal plasticity marked by alterations in neuroactive compounds such as calcitonin gene-related peptide is suggested to underlie the heightened sensory responses. Signal transduction that leads to calcitonin gene-related peptide expression thereby sensory neuroplasticity during colitis remains to be elucidated. Results In a rat model with colitis induced by 2,4,6-trinitrobenzene sulfonic acid, we found that endogenously elevated brain-derived neurotrophic factor elicited an up-regulation of calcitonin gene-related peptide in the lumbar L1 dorsal root ganglia. At seven days of colitis, neutralization of brain-derived neurotrophic factor with a specific brain-derived neurotrophic factor antibody reversed calcitonin gene-related peptide up-regulation in the dorsal root ganglia. Colitis-induced calcitonin gene-related peptide transcription was also inhibited by brain-derived neurotrophic factor antibody treatment. Signal transduction studies with dorsal root ganglia explants showed that brain-derived neurotrophic factor-induced calcitonin gene-related peptide expression was mediated by the phospholipase C gamma, but not the phosphatidylinositol 3-kinase/Akt or the mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway. Application of PLC inhibitor U73122 in vivo confirmed that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide up-regulation in the dorsal root ganglia was regulated by the phospholipase C gamma pathway. In contrast, suppression of the phosphatidylinositol 3-kinase activity in vivo had no effect on colitis-induced calcitonin gene-related peptide expression. During colitis, calcitonin gene-related peptide also co-expressed with phospholipase C gamma but not with p-Akt. Calcitonin gene-related peptide up-regulation during colitis correlated to the activation

  17. BDNF attenuates hippocampal LTD via activation of phospholipase C: implications for a vertical shift in the frequency-response curve of synaptic plasticity.

    PubMed

    Ikegaya, Yuji; Ishizaka, Yoko; Matsuki, Norio

    2002-07-01

    Recent evidence shows that neurotrophins are not only involved in neuronal survival and differentiation during development but also in modulating synaptic strength in the mature brain. To understand how neurotrophins alter this synaptic modification, we have investigated the effect of brain-derived neurotrophic factor (BDNF) on long-term depression (LTD) at Schaffer collateral-CA1 synapses in rat hippocampal slices. The slices treated with BDNF for 5 min showed significantly less LTD in response to a 1-Hz tetanus compared with controls but displayed normal LTD when the afferents were tetanized at 10 Hz. Because BDNF enhanced long-term potentiation (LTP) induced by a 30-Hz tetanus, the synaptic modification threshold (theta(m)) as defined in the 'BCM' theory of Bienenstock Cooper & Monroe [Bienenstock et al. (1982), J. Neurosci., 2, 32-48] was not shifted. BNDF is likely to alter the capability of the plastic changes in synaptic efficacy, i.e. to produce an upward shift in the BCM curve. The suppressive effect of BDNF on LTD was prevented by either the tyrosine kinase (Trk) receptor inhibitor K252a or the phospholipase C inhibitor U73122. Thus, TrkB activation may attenuate LTD through phospholipase C signalling pathway.

  18. A phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5: purification and characterization of conditions for optimal activity with an artificial substrate.

    PubMed

    Baine, W B

    1988-02-01

    Phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5 was purified from buffered yeast extract culture supernate by ion-exchange chromatography followed by fractionation by manganous chloride and ammonium sulphate precipitation steps. Enzyme activity was assayed by hydrolysis of p-nitrophenylphosphorylcholine and confirmed by release of radioactivity from tritiated L-alpha-dipalmitoylphosphatidylcholine labelled in the methyl groups of choline. After SDS-PAGE, the purified preparation yielded a single band upon Coomassie-blue staining. This protein migrated with an apparent Mr of 50,000-54,000. Phospholipase C activity was maximal at pH greater than or equal to 8.4 and was enhanced in the presence of sorbitol and of several nonionic detergents but was eliminated by SDS. EDTA, Cu2+, Fe2+ and Zn2+ inhibited enzyme activity, whereas Ba2+, Ca2+, Co2+, Mg2+ and Mn2+ restored activity to EDTA-treated material. No haemolytic activity was demonstrated with the purified enzyme. PMID:3171547

  19. Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response.

    PubMed

    Kanehara, Kazue; Yu, Chao-Yuan; Cho, Yueh; Cheong, Wei-Fun; Torta, Federico; Shui, Guanghou; Wenk, Markus R; Nakamura, Yuki

    2015-09-01

    Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response. PMID:26401841

  20. The effects of acute exposure to ethanol on neurotensin and guanine nucleotide-stimulation of phospholipase C activity in intact NIE-115 neuroblastoma cells

    SciTech Connect

    Smith, T.L. )

    1990-01-01

    Both ethanol and neurotensin produce sedation and hypothermia. When administered in combination the behavioral effects of these two substances are potentiated. In order to better understand the biochemical nature of this interaction, the direct effects of ethanol on neurotensin receptors and an associated signal transduction process were determined in NIE-115 neuroblastoma cells. Ethanol in physiologically relevant concentrations significantly reduced neurotensin stimulated ({sup 3}H)inositol phosphate production while having no effect on the specific binding of ({sup 3}H)neurotensin. In addition, ethanol up to 200 mM had no effect on GTPYS mediated ({sup 3}H)inositol phosphate production. The results indicate that acute exposure ethanol partially disrupts the normal coupling of activated neurotensin receptors to the guanine nucleotide binding protein associated with phospholipase C.

  1. Full-Length Gαq–Phospholipase C-β3 Structure Reveals Interfaces of the C-terminal Coiled-Coil Domain

    PubMed Central

    Lyon, Angeline M.; Dutta, Somnath; Boguth, Cassandra A.; Skiniotis, Georgios; Tesmer, John J. G.

    2013-01-01

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM 3D reconstructions of human full-length PLCβ3 in complex with murine Gαq. The distal CTD forms an extended, monomeric helical bundle consisting of three anti-parallel segments with structural similarity to membrane-binding bin–amphiphysin–Rvs (BAR) domains. Sequence conservation of the distal CTD identifies putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests the distal CTD plays roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis. PMID:23377541

  2. Phospholipase C{gamma}1 stimulates transcriptional activation of the matrix metalloproteinase-3 gene via the protein kinase C/Raf/ERK cascade

    SciTech Connect

    Shin, Soon Young; Choi, Ha Young; Ahn, Bong-Hyun; Son, Sang Wook; Lee, Young Han . E-mail: younghan@hanyang.ac.kr

    2007-02-16

    The phospholipid hydrolase phospholipase C{gamma}1 (PLC{gamma}1) plays a major role in regulation of cell proliferation, development, and cell motility. Overexpression of PLC{gamma}1 is associated with tumor development, and it is overexpressed in some tumors. Matrix metalloproteinase-3 (MMP-3) is a protein involved in tumor invasion and metastasis. Here, we demonstrate that overexpression of PLC{gamma}1 stimulates MMP-3 expression at the transcriptional level via the PKC-mediated Raf/MEK1/ERK signaling cascade. We propose that modulation of PLC{gamma}1 activity might be of value in controlling the activity of MMPs, which are important regulators of invasion and metastasis in malignant tumors.

  3. Full-length Gαq-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain

    SciTech Connect

    Lyon, Angeline M.; Dutta, Somnath; Boguth, Cassandra A.; Skiniotis, Georgios; Tesmer, John J.G.

    2014-08-21

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

  4. Full-length Gα(q)-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain.

    PubMed

    Lyon, Angeline M; Dutta, Somnath; Boguth, Cassandra A; Skiniotis, Georgios; Tesmer, John J G

    2013-03-01

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis. PMID:23377541

  5. Long-term depression induced by postsynaptic group II metabotropic glutamate receptors linked to phospholipase C and intracellular calcium rises in rat prefrontal cortex.

    PubMed

    Otani, S; Daniel, H; Takita, M; Crépel, F

    2002-05-01

    We have previously shown (Otani et al., 1999b) that bath application of (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV), the agonist of group II metabotropic glutamate receptors (mGluRs), induces postsynaptic Ca2+-dependent long-term depression (LTD) of layer I-II to layer V pyramidal neuron glutamatergic synapses of rat medial prefrontal cortex. In the present study, we examined detailed mechanisms of this DCG IV-induced LTD. First, the group II mGluR antagonist (RS)-alpha-methylserine-O-phosphate monophenyl ester blocked DCG IV-induced LTD, and another group II agonist (2S,3S,4S)-CCG/(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine-induced LTD, suggesting that LTD is indeed mediated by the activation of group II mGluRs. Second, DCG IV-induced LTD was blocked by the NMDA receptor antagonist AP-5, whereas DCG IV did not potentiate NMDA receptor-mediated synaptic responses. Interruption of single test stimuli during DCG IV application blocked DCG IV-induced LTD. These results suggest that small NMDA receptor-mediated responses evoked by single synaptic stimuli contribute to DCG IV-induced LTD. Third, DCG IV-induced LTD was blocked or reduced by the following drugs: phospholipase C inhibitor U-73122 (bath-applied or postsynaptically injected), postsynaptically injected IP3 receptor blocker heparin, phospholipase D-linked mGluR blocker PCCG-13, PKC inhibitor RO318220, postsynaptically injected PKC inhibitor PKC(19-36), and PKA inhibitor KT-5720. Fourth, fluorescent Ca2+ analysis techniques revealed that DCG IV increases Ca2+ concentration in prefrontal layer V pyramidal neurons. These Ca2+ rises and the LTD were both blocked by postsynaptic heparin in the same cells. Taken together, these results suggest that postsynaptic group II mGluRs, linked to phospholipase C and probably also phospholipase D, induce LTD through postsynaptic PKC activation and IP3 receptor-mediated postsynaptic increases of Ca2+ concentration.

  6. Transfected adenosine A1 receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A2 activity in CHO cells.

    PubMed

    Dickenson, J M; Hill, S J

    1997-02-19

    ]inositol phosphates and the release of [3H]arachidonic acid through pertussis-toxin-insensitive G-proteins. Experiments using PMA suggest that protein kinase C differentially regulates thrombin receptor activation of phospholipase C and phospholipase A2. Co-activation of the transfected human adenosine A1 receptor augments thrombin-stimulated phospholipase C and phospholipase A2 activity. Finally, the augmentation of phospholipase A2 activity by the adenosine A1 receptor is inhibited by selective protein kinase C inhibitors, suggesting the involvement of protein kinase C. PMID:9083789

  7. A chimeric protein composed of the binding domains of Clostridium perfringens phospholipase C and Trueperella pyogenes pyolysin induces partial immunoprotection in a mouse model.

    PubMed

    Hu, Yunhao; Zhang, Wenlong; Bao, Jun; Wu, Yuhong; Yan, Minghui; Xiao, Ya; Yang, Lingxiao; Zhang, Yue; Wang, Junwei

    2016-08-01

    Trueperella pyogenes and Clostridium perfringens are two kinds of conditional pathogens frequently associated with wound infections and succeeding lethal complications in various economic livestock. Pyolysin (PLO) and phospholipase C (PLC) are the key virulence factors of these two pathogens, respectively. In our study, a chimeric protein called rPC-PD4, which is composed of the binding regions of PLO and PLC, was synthesized. The toxicity of rPC-PD4 was evaluated. Results revealed that rPC-PD4 is a safe chimeric molecule that can be used to develop vaccines. Immunizing BALB/c mice with rPC-PD4 induced high titers of serum antibodies that could efficiently neutralize the hemolytic activity of recombinant PLO and PLC. After the challenge with T. pyogenes or C. perfringens was performed through the intraperitoneal route, we observed that rPC-PD4 immunization could provide partial immunoprotection and reduce lung, intestine, and liver tissue damage to mice. This work demonstrated the efficacy of the rationally designed rPC-PD4 chimeric protein as a potential vaccine candidate against C. perfringens and T. pyogenes. PMID:27473983

  8. Activity-dependent regulation of synapse and dendritic spine morphology in developing barrel cortex requires phospholipase C-beta1 signalling.

    PubMed

    Spires, Tara L; Molnár, Zoltán; Kind, Peter C; Cordery, Patricia M; Upton, A Louise; Blakemore, Colin; Hannan, Anthony J

    2005-04-01

    The phospholipase C-beta1 (PLC-beta1) signalling pathway, activated via metabotropic glutamate receptors (mGluRs), is implicated in activity-dependent development of the cerebral cortex, as both PLC-beta1 and mGluR5 knockout mice exhibit disrupted barrel formation in somatosensory cortex. To characterize the effects of this signalling system on development of synaptic circuitry in barrel cortex, we have examined neuronal ultrastructure, synapse formation and dendritic spine morphology in PLC-beta1 knockout mice. Qualitative ultrastructure of neurons and synapse density in layers 2-4 of barrel cortex were unchanged in PLC-beta1 knockout mice during development [postnatal day (P) 5] and in mature cortex (P19-21). We found a decrease in the proportion of synapses with symmetric morphology at P5 that was gone by P19-21, indicating a transient imbalance in excitatory and inhibitory circuitry. We also investigated dendritic spines by back-labelling layer 5 pyramidal neurons with carbocyanine. We observed normal dendritic spine densities on apical dendrites as they passed through layer 4 of barrel cortex, but spine morphology was altered in PLC-beta1 knockout mice at P9. These observations indicate that the PLC-beta1 signalling pathway plays a role in the development of normal cortical circuitry. Interrupting this regulation leads to changes in synapse and dendritic spine morphology, possibly altering post-synaptic integration of signal.

  9. Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels.

    PubMed

    Sakai, Hiromichi; Kado, Sayaka; Taketomi, Akinobu; Sakane, Fumio

    2014-09-19

    Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels. PMID:25112873

  10. Overexpression of kidney phosphatidylinositol 4-kinasebeta and phospholipase C(gamma1) proteins in two rodent models of polycystic kidney disease.

    PubMed

    Cuozzo, F P; Mishra, S; Jiang, J; Aukema, H M

    2002-05-21

    Our studies of renal phosphoinositide levels and metabolism in the pcy mouse with polycystic kidney disease (PKD) suggest that phosphatidylinositol kinase (PtdInsK) and phospholipase C (PLC) are elevated in this renal disorder. Therefore, the steady-state levels of select isoforms of these enzymes were examined in renal cytosolic and particulate (detergent-soluble) fractions in male and female normal and CD1-pcy/pcy (pcy) mice at 60, 120 and 180 days of age, and in male and female normal and diseased (Han:SPRD-cy) rats at 28 and 70 days of age. Disease-related increases in phosphatidylinositol 4-kinasebeta (PtdIns4Kbeta) and PLC(gamma1) levels were present in both models. PtdIns4Kbeta levels were higher by as much as 233% in pcy mice and by 95% in diseased Han:SPRD-cy rats compared to normals of the same age and gender. Steady-state levels of PLC(gamma1) were as much as 74% and 35% higher in pcy mice and diseased Han:SPRD-cy rats, respectively, compared to their controls. The consistency of these alterations in two accepted models of PKD indicates the importance of the phosphoinositide signalling pathway in the evolution of this disorder, and represents a potential site for therapeutic intervention. PMID:12009430

  11. Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation

    PubMed Central

    Liu, Ta-Ming; Woyach, Jennifer A.; Zhong, Yiming; Lozanski, Arletta; Lozanski, Gerard; Dong, Shuai; Strattan, Ethan; Lehman, Amy; Zhang, Xiaoli; Jones, Jeffrey A.; Flynn, Joseph; Andritsos, Leslie A.; Maddocks, Kami; Jaglowski, Samantha M.; Blum, Kristie A.; Byrd, John C.; Dubovsky, Jason A.

    2015-01-01

    Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib’s capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2R665W confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance. PMID:25972157

  12. Compartmentation of the cerebellar cortex of hummingbirds (Aves: Trochilidae) revealed by the expression of zebrin II and phospholipase C beta 4.

    PubMed

    Iwaniuk, Andrew N; Marzban, Hassan; Pakan, Janelle M P; Watanabe, Masahiko; Hawkes, Richard; Wylie, Douglas R W

    2009-01-01

    The parasagittal organization of the mammalian cerebellar cortex into zones has been well characterized by immunohistochemical, hodological and physiological studies in recent years. The pattern of these parasagittal bands across the cerebellum is highly conserved across mammals, but whether a similar conservation of immunohistochemically defined parasagittal bands occurs within birds has remained uncertain. Here, we examine the compartmentation of the cerebellar cortex of a group of birds with unique cerebellar morphology-hummingbirds (Trochilidae). Immunohistochemical techniques were used to characterize the expression of zebrin II (aldolase C) and phospholipase C beta 4 (PLC beta 4) in the cerebellar cortex of two hummingbird species. A series of zebrin II immunopositive/immunonegative parasagittal stripes was apparent across most folia representing three major transverse zones: an anterior zone with a central stripe flanked by three lateral stripes on either side; a central zone of high/low immunopositive stripes; and a posterior zone with a central stripe flanked by four to six lateral stripes on either side. In addition, both folia I and X were uniformly immunopositive. The pattern of PLC beta 4 immunoreactivity was largely complementary-PLC beta 4 positive stripes were zebrin II negative and vice versa. The similarity of zebrin II expression between the hummingbirds and the pigeon indicates that the neurochemical compartmentation of the cerebellar cortex in birds is highly conserved, but species differences in the number and width of stripes do occur.

  13. Phospholipase C Signaling via the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Is Essential for Normal Bone Responses to PTH

    PubMed Central

    Guo, Jun; Liu, Minlin; Yang, Dehong; Bouxsein, Mary L.; Thomas, Clare C.; Schipani, Ernestina; Bringhurst, F. Richard; Kronenberg, Henry M.

    2010-01-01

    We have previously shown that differentiation of hypertrophic chondrocytes is delayed in mice expressing a mutated PTH/PTHrP receptor (PTHR) (called DSEL here) that stimulates adenylyl cyclase normally but fails to activate phospholipase C (PLC). To better understand the role of PLC signaling via the PTHR in skeletal and mineral homeostasis, we examined these mice fed a normal or calcium-deficient diet. On a standard diet, DSEL mice displayed a modest decrease in bone mass. Remarkably, when fed a low-calcium diet or infused with PTH, DSEL mice exhibited strikingly curtailed peritrabecular stromal cell responses and attenuated new bone formation when compared with Wt mice. Attenuated in vitro colony formation was also observed in bone marrow cells derived from DSEL mice fed a low-calcium diet. Furthermore, PTH stimulated proliferation and increased mRNAs encoding cyclin D1 in primary osteoblasts derived from Wt but not from DSEL mice. Our data indicate that PLC signaling through the PTHR is required for skeletal homeostasis. PMID:20501677

  14. Cbl Suppresses B Cell Receptor–Mediated Phospholipase C (Plc)-γ2 Activation by Regulating B Cell Linker Protein–Plc-γ2 Binding

    PubMed Central

    Yasuda, Tomoharu; Maeda, Akito; Kurosaki, Mari; Tezuka, Tohru; Hironaka, Katsunori; Yamamoto, Tadashi; Kurosaki, Tomohiro

    2000-01-01

    Accumulating evidence indicates that the Cbl protein plays a negative role in immune receptor signaling; however, the mode of Cbl action in B cell receptor (BCR) signaling still remains unclear. DT40 B cells deficient in Cbl showed enhanced BCR-mediated phospholipase C (PLC)-γ2 activation, thereby leading to increased apoptosis. A possible explanation for the involvement of Cbl in PLC-γ2 activation was provided by findings that Cbl interacts via its Src homology 2 (SH2) domain with B cell linker protein (BLNK) after BCR ligation. BLNK is a critical adaptor molecule for PLC-γ2 tyrosine phosphorylation through its binding to the PLC-γ2 SH2 domains. As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-γ2 to BLNK and the subsequent PLC-γ2 tyrosine phosphorylation were inhibited. Thus, our data suggest that Cbl negatively regulates the PLC-γ2 pathway by inhibiting the association of PLC-γ2 with BLNK. PMID:10684856

  15. Propolis Reduces Phosphatidylcholine-Specific Phospholipase C Activity and Increases Annexin a7 Level in Oxidized-LDL-Stimulated Human Umbilical Vein Endothelial Cells

    PubMed Central

    Xuan, Hongzhuan; Li, Zhen; Wang, Jiying; Fu, Chongluo; Yuan, Jianlong; Hu, Fuliang

    2014-01-01

    To understand the mechanisms underlying the regulating dyslipidemia action of Chinese propolis and Brazilian green propolis, we investigated their effects on phosphatidylcholine-specific phospholipase C (PC-PLC) activity and annexin a7 (ANXA7) level which play crucial roles in the control of the progress of atherosclerosis. Furthermore, active oxygen species (ROS) levels, nuclear factor-KappaB p65 (NF-κB p65), and mitochondrial membrane potential (MMP) were also investigated in oxidized-LDL- (ox-LDL-) stimulated human umbilical vein endothelial cells (HUVECs). Our data indicated that the treatment of both types of propolis 12.5 μg/mL significantly increased cell viability and attenuated apoptosis rate, increased ANXA7 level, and decreased PC-PLC activity. Both types of propolis also inhibited ROS generation as well as the subsequent MMP collapse, and NF-κB p65 activation induced by ox-LDL in HUVECs. Our results also indicated that Chinese propolis and Brazilian green propolis had similar biological activities and prevented ox-LDL induced cellular dysfunction in HUVECs. PMID:24864152

  16. The Cryptococcal Enzyme Inositol Phosphosphingolipid-Phospholipase C Confers Resistance to the Antifungal Effects of Macrophages and Promotes Fungal Dissemination to the Central Nervous System†

    PubMed Central

    Shea, John M.; Kechichian, Talar B.; Luberto, Chiara; Del Poeta, Maurizio

    2006-01-01

    In recent years, sphingolipids have emerged as critical molecules in the regulation of microbial pathogenesis. In fungi, the synthesis of complex sphingolipids is important for the regulation of pathogenicity, but the role of sphingolipid degradation in fungal virulence is not known. Here, we isolated and characterized the inositol phosphosphingolipid-phospholipase C1 (ISC1) gene from the fungal pathogen Cryptococcus neoformans and showed that it encodes an enzyme that metabolizes fungal inositol sphingolipids. Isc1 protects C. neoformans from acidic, oxidative, and nitrosative stresses, which are encountered by the fungus in the phagolysosomes of activated macrophages, through a Pma1-dependent mechanism(s). In an immunocompetent mouse model, the C. neoformans Δisc1 mutant strain is almost exclusively found extracellularly and in a hyperencapsulated form, and its dissemination to the brain is remarkably reduced compared to that of control strains. Interestingly, the dissemination of the C. neoformans Δisc1 strain to the brain is promptly restored in these mice when alveolar macrophages are pharmacologically depleted or when infecting an immunodeficient mouse in which macrophages are not efficiently activated. These studies suggest that Isc1 plays a key role in protecting C. neoformans from the intracellular environment of macrophages, whose activation is important for preventing fungal dissemination of the Δisc1 strain to the central nervous system and the development of meningoencephalitis. PMID:16988277

  17. Mutation of the phospholipase C-γ1–binding site of LAT affects both positive and negative thymocyte selection

    PubMed Central

    Sommers, Connie L.; Lee, Jan; Steiner, Kevin L.; Gurson, Jordan M.; DePersis, Corinne L.; El-Khoury, Dalal; Fuller, Claudette L.; Shores, Elizabeth W.; Love, Paul E.; Samelson, Lawrence E.

    2005-01-01

    Linker for activation of T cells (LAT) is a scaffolding adaptor protein that is critical for T cell development and function. A mutation of LAT (Y136F) that disrupts phospholipase C-γ1 activation and subsequent calcium influx causes a partial block in T cell development and leads to a severe lymphoproliferative disease in homozygous knock-in mice. One possible contribution to the fatal disease of LAT Y136F knock-in mice could be from autoreactive T cells generated in these mice because of altered thymocyte selection. To examine the impact of the LAT Y136F mutation on thymocyte positive and negative selection, we bred this mutation onto the HY T cell receptor (TCR) transgenic, recombination activating gene-2 knockout background. Female mice with this genotype showed a severe defect in positive selection, whereas male mice exhibited a phenotype resembling positive selection (i.e., development and survival of CD8hi HY TCR-specific T cells) instead of negative selection. These results support the hypothesis that in non-TCR transgenic, LAT Y136F knock-in mice, altered thymocyte selection leads to the survival and proliferation of autoreactive T cells that would otherwise be negatively selected in the thymus. PMID:15795236

  18. Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels.

    PubMed

    Sakai, Hiromichi; Kado, Sayaka; Taketomi, Akinobu; Sakane, Fumio

    2014-09-19

    Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.

  19. Glucocorticoid exerts its non-genomic effect on IPSC by activation of a phospholipase C-dependent pathway in prefrontal cortex of rats.

    PubMed

    Teng, Zenghui; Zhang, Mingyue; Zhao, Minggao; Zhang, Weiqi

    2013-07-01

    In response to stressor, the brain activates a comprehensive stress system. Among others, this stress system causes release of glucocorticoids that also feed back to the brain. Glucocorticoids affect brain function by activation of both delayed, genomic and rapid, non-genomic mechanisms in rodents. Here we report that application of the potent glucocorticoid receptor agonist dexamethasone (DEX) caused a rapid increase of spontaneous and miniature inhibitory postsynaptic currents (IPSCs) and elicited intermittent burst activities through a non-genomic pathway, involving membrane-located receptors. The onset of the rapid effect in prefrontal cortex (PFC, <15 min) was much slower than in hippocampus (<5 min). The intermittent burst activities were abolished in the presence of TTX. Furthermore, the nitric oxide (NO) pathway was present and endogenously activated in PFC. Part of the rapid DEX effect in PFC remained after blocking NO-sensitive guanylyl cyclase that was due to activation of a phospholipase C-diacylglycerol-dependent signalling pathway. Thus, our data demonstrated that glucocorticoids could rapidly enhance IPSCs and evoke burst activities by activation of at least two different signalling pathways in hippocampus and PFC of rats.

  20. The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.

    PubMed

    de Munnik, Sabrina M; van der Lee, Rosan; Velders, Daniëlle M; van Offenbeek, Jody; Smits-de Vries, Laura; Leurs, Rob; Smit, Martine J; Vischer, Henry F

    2016-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce β-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74. PMID:26931381

  1. Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

    PubMed

    Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

    2008-12-25

    Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

  2. Binding of phosphoinositide-specific phospholipase C-zeta (PLC-zeta) to phospholipid membranes: potential role of an unstructured cluster of basic residues.

    PubMed

    Nomikos, Michail; Mulgrew-Nesbitt, Anna; Pallavi, Payal; Mihalyne, Gyongyi; Zaitseva, Irina; Swann, Karl; Lai, F Anthony; Murray, Diana; McLaughlin, Stuart

    2007-06-01

    Phospholipase C-zeta (PLC-zeta) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta1, but lacks the PH domain that anchors PLC-delta1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-delta1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5x10(3) M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge=+7) within the linker region, PLC-zeta-(374-385), binds to PC/PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.

  3. L-Theanine Improves Immunity by Altering TH2/TH1 Cytokine Balance, Brain Neurotransmitters, and Expression of Phospholipase C in Rat Hearts

    PubMed Central

    Li, Chengjian; Tong, Haiou; Yan, Qiongxian; Tang, Shaoxun; Han, Xuefeng; Xiao, Wenjun; Tan, Zhiliang

    2016-01-01

    Background This study aimed to investigate the regulatory effects of L-theanine on secretion of immune cytokines, hormones, and neurotransmitters, and mRNA expression of phospholipase C (PLC) in rats, and to explore its regulatory mechanism in immune function. Material/Methods Sixty-four Sprague-Dawley rats received daily intragastric infusion of different doses of L-theanine solution [0, 50 (LT), 200 (MT), and 400 (HT) mg/kg BW]. Cytokines, immunoglobulins, and hormones in the serum, neurotransmitters, and mRNA expression of PLC in the relevant tissues were assayed. Results L-theanine administration increased the splenic organ index and decreased the contents of ILs-4/6/10 and the ratio of IL-4/IFN-γ in the serum. High-dose L-theanine administration increased the levels of dopamine and 5-hydroxytryptamine in the pituitary and hippocampus, resulting in decrease in corticosterone level in the serum. L-theanine administration decreased the mRNA expressions of PLC isomers in the liver and PLC-γ1 and PLC-δ1 in the spleen. Interestingly, mRNA expressions of PLC-βf1 in the spleen and PLC isomers mRNA in the heart were up-regulated by L-theanine administration. Conclusions Administration of 400 mg/kg BWL-theanine improved immune function of the rats by increasing the splenic weight, altering the Th2/Th1 cytokine balance, decreasing the corticosterone level in the serum, elevating dopamine and 5-hydroxytryptamine in the brain, and regulating the mRNA expression of PLC isomers in the heart. PMID:26922362

  4. Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy

    PubMed Central

    Podo, Franca; Paris, Luisa; Cecchetti, Serena; Spadaro, Francesca; Abalsamo, Laura; Ramoni, Carlo; Ricci, Alessandro; Pisanu, Maria Elena; Sardanelli, Francesco; Canese, Rossella; Iorio, Egidio

    2016-01-01

    Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation, and cell proliferation. Recent investigations showed that PC-PLC activation could account for 20–50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC coimmunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and antiproliferative effects. This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be preclinically and clinically monitored by metabolic imaging methods. PMID:27532027

  5. Contrasting role of phospholipase C-{gamma}1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors

    SciTech Connect

    Liao Hongjun; Santos, Josue de los; Carpenter, Graham . E-mail: graham.carpenter@vanderbilt.edu

    2006-04-01

    While significant progress has been achieved in identifying the signal transduction elements that operate downstream of activated receptor tyrosine kinases, it remains unclear how different receptors utilize these signaling elements to achieve a common response. This study compares the capacity of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to elicit the induction of immediate early gene (IEG) mRNAs in the presence or absence of phospholipase C-{gamma}1 (PLC-{gamma}1). The results show that while PDGF induction of nearly all IEG mRNAs is abrogated in plcg1 null cells, EGF induction of the same genes is variable in the null cells and exhibits three distinct responses. Five IEG mRNAs (Nup475, Cyr61, TF, Gly, TS7) are completely inducible by EGF in the presence or absence of PLC-{gamma}1, while three others (JE, KC, FIC) exhibit a stringent requirement for the presence of PLC-{gamma}1. The third type of response is exhibited by c-fos and COX-2. While these mRNAs are completely induced by EGF in the absence of PLC-{gamma}1, the time course of their accumulation is significantly delayed. No IEG was identified as completely inducible by EGF and PDGF in the absence of PLC-{gamma}1. Electrophoretic mobility shift assays (EMSA) demonstrate that PLC-{gamma}1 is necessary for nuclear extracts from PDGF-treated cells, but not EGF-treated cells, to interact with probes for AP-1 or NF-{kappa}B.

  6. Genomic organization and complete cDNA sequence of the human phosphoinositide-specific phospholipase C {beta}3 gene (PLCB3)

    SciTech Connect

    Lagercrantz, J.; Carson, E.; Phelan, C.

    1995-04-10

    We have characterized the complete cDNA sequence, genomic structure, and expression of the human phosphoinositide-specific phospholipase C {beta}3 (PLC {beta}3) gene (gene symbol PLCB3). PLC {beta}3 plays an important role in initiating receptor-mediated signal transduction. Activation of PLC takes place in many cells as a response to stimulation by hormones, growth factors, neurotransmitters, and other ligands. The partial cDNA sequence of PLC {beta}3, previously published, was extended with 876 bp in the 5{prime} direction, giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids. This was in accordance with expression analysis by Northern blotting that revealed a single 4.4-kb transcript in all tissues tested. Genomic data were obtained by sequencing plasmid subclones of a cosmid that contained the whole gene. The size of the complete transcription unit was estimated to be on the order of 15 kb. The gene contains 31 exons, with all splice donor and acceptor sites conforming to the GT/AG rule. No exon exceeds 571 bp in length, and the shortest exon spans only 36 bp. More than half of the introns are smaller than 200 bp, with the smallest being only 79 bp long. The transcription initiation site was determined to be within an 8-bp cluster 328-321 bp upstream of the translation initiation site. The 5{prime} flanking region is highly GC rich, with multiple CpG doublets, and contains multiple binding sites for Sp1. Lacking typical transcriptional regulatory sequences such as TATA and CAAT boxes, the putative promoter region conforms to the group of housekeeping promoters. 28 refs., 4 figs., 1 tab.

  7. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

    SciTech Connect

    Zeng, Ke-Wu; Li, Jun; Dong, Xin; Wang, Ying-Hong; Ma, Zhi-Zhong; Jiang, Yong; Jin, Hong-Wei; Tu, Peng-Fei

    2013-11-15

    Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators. Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.

  8. Knockdown of phospholipase C-β1 in the medial prefrontal cortex of male mice impairs working memory among multiple schizophrenia endophenotypes

    PubMed Central

    Kim, Seong-Wook; Seo, Misun; Kim, Duk-Soo; Kang, Moonkyung; Kim, Yeon-Soo; Koh, Hae-Young; Shin, Hee-Sup

    2015-01-01

    Background Decreased expression of phospholipase C-β1 (PLC-β1) has been observed in the brains of patients with schizophrenia, but, to our knowledge, no studies have shown a possible association between this altered PLC-β1 expression and the pathogenesis of schizophrenia. Although PLC-β1-null (PLC-β1−/−) mice exhibit multiple endophenotypes of schizophrenia, it remains unclear how regional decreases in PLC-β1 expression in the brain contribute to specific behavioural defects. Methods We selectively knocked down PLC-β1 in the medial prefrontal cortex (mPFC) using a small hairpin RNA strategy in mice. Results Silencing PLC-β1 in the mPFC resulted in working memory deficits, as assayed using the delayed non-match-to-sample T-maze task. Notably, however, other schizophrenia- related behaviours observed in PLC-β1−/− mice, including phenotypes related to locomotor activity, sociability and sensorimotor gating, were normal in PLC-β1 knockdown mice. Limitations Phenotypes of PLC-β1 knockdown mice, such as locomotion, anxiety and sensorimotor gating, have already been published in our previous studies. Further, the neural mechanisms underlying the working memory deficit in mice may be different from those in human schizophrenia. Conclusion These results indicate that PLC-β1 signalling in the mPFC is required for working memory. Importantly, these results support the notion that the decrease in PLC-β1 expression in the brains of patients with schizophrenia is a pathogenically relevant molecular marker of the disorder. PMID:25268789

  9. G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

    PubMed Central

    Vaziri, C; Downes, C P

    1992-01-01

    Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5'-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected. PMID:1352448

  10. Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

    SciTech Connect

    Henriksen, E.J.; Rodnick, K.J.; Holloszy, J.O. )

    1989-12-25

    It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin. In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle.

  11. CD45 cross-linking regulates phospholipase C activation and tyrosine phosphorylation of specific substrates in CD3/Ti-stimulated T cells.

    PubMed

    Ledbetter, J A; Schieven, G L; Uckun, F M; Imboden, J B

    1991-03-01

    In lymphocytes, CD45 regulates the increase in cytoplasmic calcium concentration that occurs after receptor cross-linking. Here we show that T cell receptor complex (CD3/Ti)-mediated inositol phosphate production was inhibited by CD45 ligation in Jurkat cells. CD3/Ti signaling in normal T cells was also inhibited by CD45 ligation, but coupling of CD4 with CD3/Ti gave augmented calcium signals that were entirely resistant to the inhibitory effect of CD45. In contrast, CD3-induced T cell proliferation was suppressed by immobilized CD45 mAb even in the presence of CD4 mAb. The effect of CD45 and CD4 ligation on tyrosine phosphorylation during T cell activation was directly examined by immunoblotting with anti-phosphotyrosine. Using immobilized mAb, CD45 ligation suppressed the tyrosine phosphorylation of specific substrates induced by CD3/Ti stimulation, including almost complete suppression of 150-, 36-, and 35-kDa proteins and partial suppression of 76- and 80-kDa proteins. Other tyrosine-phosphorylated proteins induced by CD3/Ti stimulation, including 135- and 21-kDa proteins, were not suppressed by simultaneous ligation of CD3/Ti and CD45. Simultaneous ligation of CD3 and CD4 enhanced tyrosine phosphorylation of all substrates, but did not overcome the CD45-mediated suppression of tyrosine phosphorylation of the 35- and 36-kDa proteins. The CD45-mediated suppression of phospholipase C activation is therefore modulated by association with CD4 without altering the specific inhibition of tyrosine phosphorylation and T cell proliferation after co-ligation of CD45 and CD3/Ti.

  12. Insulin activates glycerol-3-phosphate acyltransferase (de novo phosphatidic acid synthesis) through a phospholipid-derived mediator. Apparent involvement of Gi alpha and activation of a phospholipase C.

    PubMed

    Vila, M C; Milligan, G; Standaert, M L; Farese, R V

    1990-09-18

    We studied the mechanism whereby insulin activates de novo phosphatidic acid synthesis in BC3H-1 myocytes. Insulin rapidly activated glycerol-3-phosphate acyltransferase (G3PAT) in intact and cell-free preparations of myocytes in a dose-related manner. The apparent Km of the enzyme was decreased by treatment with insulin, whereas the Vmax was unaffected. No activation was found by ACTH, insulin-like growth factor-I, angiotensin II, or phenylephrine, but epidermal growth factor, which, like insulin, is known to activate de novo phosphatidic acid synthesis in intact myocytes, also stimulated G3PAT activity. In homogenates or membrane fractions, the effect of insulin on G3PAT was fully mimicked by nonspecific or phosphatidylinositol (PI)-specific phospholipase C (PLC). An antiserum raised against PI-glycan-PLC completely blocked the effect of insulin on G3PAT. Although the above findings suggested involvement of a PLC in insulin-induced activation of G3PAT, neither diacylglycerol nor protein kinase C activation appeared to be involved. On the other hand, insulin stimulated the release of a cytosolic factor, which activated membrane-associated G3PAT. This cytosolic factor had a molecular weight of less than 5K as determined by Sephadex G-25 chromatography. NaF, a phosphatase inhibitor, blocked the activation of G3PAT by insulin, suggesting involvement of a phosphatase. Insulin-induced activation of G3PAT was also blocked by pretreatment of intact myocytes with pertussis toxin and by prior addition, to homogenates, of an antiserum that recognizes the C-terminal decapeptide of Gi alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Progesterone-Dependent Induction of Phospholipase C-Related Catalytically Inactive Protein 1 (PRIP-1) in Decidualizing Human Endometrial Stromal Cells

    PubMed Central

    Muter, Joanne; Brighton, Paul J.; Lucas, Emma S.; Lacey, Lauren; Shmygol, Anatoly; Quenby, Siobhan; Blanks, Andrew M.

    2016-01-01

    Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca2+ release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors. PMID:27167772

  14. Modulation of Enzymatic Activity and Biological Function of Listeria monocytogenes Broad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with the Bacillus cereus Ortholog

    PubMed Central

    Zückert, Wolfram R.; Marquis, Hélène; Goldfine, Howard

    1998-01-01

    The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens α-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells. PMID:9746585

  15. Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy.

    PubMed

    Podo, Franca; Paris, Luisa; Cecchetti, Serena; Spadaro, Francesca; Abalsamo, Laura; Ramoni, Carlo; Ricci, Alessandro; Pisanu, Maria Elena; Sardanelli, Francesco; Canese, Rossella; Iorio, Egidio

    2016-01-01

    Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation, and cell proliferation. Recent investigations showed that PC-PLC activation could account for 20-50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC coimmunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and antiproliferative effects. This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be preclinically and clinically monitored by metabolic imaging methods. PMID:27532027

  16. Platelet activation by bacterial phospholipase C involves phosphoinositide turnover and phosphorylation of 47,000 dalton but not 20,000 dalton protein

    SciTech Connect

    Huzoor-Akbar; Anwer, K.

    1986-05-01

    This study was conducted to examine the role of phosphoinositides (PIns) and phosphorylation of 47,000 dalton (P47) and 20,000 dalton (P20) proteins in platelet activation by bacterial phospholipase C (PLC). PLC induced serotonin secretion (SS) and platelet aggregation (PA) in a concentration dependent manner. PLC (0.02 U/ml) caused phosphorylation of P47 in a time dependent manner (27% at 0.5 min to 378% at 7 min). PLC did not induce more than 15% phosphorylation of P20 by 7 min. Aspirin (500 ..mu..M) blocked phosphorylation of P20 but did not inhibit SS, PA or phosphorylation of P47. PLC (0.04 U/ml) decreased radioactivity (cpm) in /sup 32/P labeled phosphatidylinositol (PI), PI-4,5-bis-PO4 (PIP2) and PI-4-PO4 (PIP) by 20%, 12% and 7.5% respectively at 15 sec. The level of PI but not that of PIP2 returned to base line in 3 min. PIP level increased above control values within one min. PLC increased phosphatidic acid level (75% at 0.5 min. to 1545% at 3 min). In other experiments PLC produced diacylglycerol (DAG) in a time and concentration dependent manner. However, no DAG was detectable in the first 60 sec. These data suggest that: (a) PIns turnover and phosphorylation of P47 but not that of P20 is involved in platelet activation by PLC; and (b) DAG production from outer membrane phospholipids is not a prerequisite for platelet activation by PLC.

  17. Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-delta 1.

    PubMed

    Yagisawa, H; Hirata, M; Kanematsu, T; Watanabe, Y; Ozaki, S; Sakuma, K; Tanaka, H; Yabuta, N; Kamata, H; Hirata, H

    1994-08-01

    It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the delta 1 isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-delta 1 in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(1,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding. The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-delta 1 consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-delta 1 which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-delta 1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4

  18. Abnormal metabotropic glutamate receptor expression and signaling in the cerebral cortex in diffuse Lewy body disease is associated with irregular alpha-synuclein/phospholipase C (PLCbeta1) interactions.

    PubMed

    Dalfó, E; Albasanz, J L; Martin, M; Ferrer, I

    2004-10-01

    Diffuse Lewy body disease (DLBD) is a degenerative disease of the nervous system, involving the brain stem, diencephalic nuclei and cerebral cortex, associated with abnormal a-synuclein aggregation and widespread formation of Lewy bodies and Lewy neurites. DLBD presents as pure forms (DLBDp) or in association with Alzheimer disease (AD) in the common forms (DLBDc). Several neurotransmitter abnormalities have been reported including those of the nigrostriatal and mesocorticolimbic dopaminergic system, and central noradrenergic, serotoninergic and cholinergic pathways. The present work examines metabotropic glutamate receptor (mGluR) expression and signaling in the frontal cortex of DLBDp and DLBDc cases in comparison with age-matched controls. Abnormal L-[3H]glutamate specific binding to group I and II mGluRs, and abnormal mGluR1 levels have been found in DLBD. This is associated with reduced expression levels of phospholipase C beta1 (PLCbeta1), the effector of group I mGluRs following protein G activation upon glutamate binding. Additional modification in the solubility of PLCbeta1 and reduced PLCbeta1 activity in pure and common DLBD further demonstrates for the first time abnormal mGluR signaling in the cerebral cortex in DLBD. In order to look for a possible link between abnormal mGluR signaling and a-synuclein accumulation in DLBD, immunoprecipitation studies have shown alpha-synuclein/PLCbeta1 binding in controls and decreased alpha-synuclein/PLCbeta1 binding in DLBD. This is accompanied by a shift in the distribution of a-synuclein, but not of PLCbeta1, in DLBD when compared with controls. Together, these results support the concept that abnormal a-synuclein in DLBD produces functional effects on cortical glutamatergic synapses, which are associated with reduced alpha-synuclein/PLCbeta1 interactions, and, therefore, that mGluRs are putative pharmacological targets in DLBD. Finally, these results emphasize the emergence of a functional neuropathology that has

  19. The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.

    PubMed Central

    Batty, I H; Downes, C P

    1994-01-01

    Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively

  20. Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2

    SciTech Connect

    Slivka, S.R.; Insel, P.A.

    1987-03-25

    alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2.

  1. Specificity of G alpha q and G alpha 11 gene expression in platelets and erythrocytes. Expressions of cellular differentiation and species differences.

    PubMed Central

    Johnson, G J; Leis, L A; Dunlop, P C

    1996-01-01

    G alpha q and G alpha 11, members of the Gq family of G-proteins, transduce signals from receptors to the beta isoenzymes of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The receptor specificity of these alpha subunits is unknown. G alpha q and G alpha 11 are ubiquitously expressed in tissues; however, there have been conflicting reports of the presence or absence of G alpha 11 protein in haematopoietic cells. Platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors activate PI-PLC via G alpha q, but the role of G alpha 11 is uncertain. To define their roles in platelet activation we studied G alpha q and G alpha 11 gene expression by immunotransfer blotting and by reverse transcription of mRNA followed by PCR (RT-PCR) and direct sequencing. An antiserum specific for mouse G alpha 11 failed to identify G alpha 11 in dog or human platelets or in dog liver, a tissue known to contain G alpha 11. RT-PCR performed with gene-specific primers demonstrated G alpha q mRNA, but not G alpha 11 mRNA, in normal human and mouse platelets and in thromboxane-sensitive and thromboxane-insensitive dog platelets. Studies of mouse and dog liver and human retina confirmed that the cDNA, primers and probes used could amplify and recognize G alpha 11 in other tissues. However, species-specific oligonucleotide primers and probes were essential to demonstrate G alpha 11, but not G alpha q, mRNA. Compared with mouse cDNA, dog and human G alpha 11 cDNA had twice as many nucleotide substitutions (approx. 12% compared with approx. 6%) as G alpha q, G alpha q mRNA was also found in mature erythrocytes but G alpha 11 mRNA was not identified, whereas both G alpha q and G alpha 11 mRNAs were found in bone marrow stem cells. Therefore G alpha 11 gene expression in haematopoietic cells is linked with cellular differentiation. The lack of G alpha 11 indicates that signal transduction from platelet TXA2/PGH2 receptors to PI-PLC occurs via G alpha q, and that G alpha 11 deficiency is

  2. Over-expression of Brassica napus phosphatidylinositol-phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation.

    PubMed

    Georges, Fawzy; DAS, Shankar; Ray, Heather; Bock, Cheryl; Nokhrina, Kateryna; Kolla, Venkat Apparao; Keller, Wilf

    2009-12-01

    Phosphatidylinositol-specific phospholipase C (PtdIns-PLC2) plays a central role in the phosphatidylinositol-specific signal transduction pathway. It catalyses the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to produce two second messengers, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The former is a membrane activator of protein kinase C in mammalian systems, and the latter is a Ca(2+) modulator which induces distinctive oscillating bursts of cytosolic Ca(2+), resulting in regulation of gene expression and activation of proteins. Sustained over-expression of BnPtdIns-PLC2 in transgenic Brassica napus lines brought about an early shift from vegetative to reproductive phases, and shorter maturation periods, accompanied by notable alterations in hormonal distribution patterns in various tissues. The photosynthetic rate increased, while stomata were partly closed. Numerous gene expression changes that included induction of stress-related genes such as glutathione S-transferase, hormone-regulated and regulatory genes, in addition to a number of kinases, calcium-regulated factors and transcription factors, were observed. Other changes included increased phytic acid levels and phytohormone organization patterns. These results suggest the importance of PtdIns-PLC2 as an elicitor of a battery of events that systematically control hormone regulation, and plant growth and development in what may be a preprogrammed mode.

  3. Family.

    ERIC Educational Resources Information Center

    Hurst, Hunter, Ed.; And Others

    1985-01-01

    This document contains the fourth volume of "Today's Delinquent," an annual publication of the National Center for Juvenile Justice. This volume deals with the issue of the family and delinquency. "The Family and Delinquency" (LaMar T. Empey) systematically reviews and weighs the evidence to support prominent theories on the origins of…

  4. Temporal profiling of changes in phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol allows comprehensive analysis of phospholipase C-initiated signalling in single neurons1

    PubMed Central

    Nelson, Carl P; Nahorski, Stefan R; Challiss, R A John

    2008-01-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) fulfils vital signalling roles in an array of cellular processes, yet until recently it has not been possible selectively to visualize real-time changes in PIP2 levels within living cells. Green fluorescent protein (GFP)-labelled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Gαq/11-coupled muscarinic acetylcholine receptors in both SH-SY5Y human neuroblastoma cells and primary rat hippocampal neurons. GFP-Tubby translocation is independent of changes in cytosolic inositol 1,4,5-trisphosphate and instead reports dynamic changes in levels of plasma membrane PIP2. In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCδ1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C12 domain of protein kinase C-γ [eGFP-C1(2); to detect diacylglycerol] allowed a selective and comprehensive analysis of PLC-initiated signalling in living cells. Manipulating intracellular Ca2+ concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca2+ up to 100–200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca2+ concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP2 in hippocampal neurons. PMID:18665913

  5. PDZ domain-containing 1 (PDZK1) protein regulates phospholipase C-β3 (PLC-β3)-specific activation of somatostatin by forming a ternary complex with PLC-β3 and somatostatin receptors.

    PubMed

    Kim, Jung Kuk; Kwon, Ohman; Kim, Jinho; Kim, Eung-Kyun; Park, Hye Kyung; Lee, Ji Eun; Kim, Kyung Lock; Choi, Jung Woong; Lim, Seyoung; Seok, Heon; Lee-Kwon, Whaseon; Choi, Jang Hyun; Kang, Byoung Heon; Kim, Sanguk; Ryu, Sung Ho; Suh, Pann-Ghill

    2012-06-15

    Phospholipase C-β (PLC-β) is a key molecule in G protein-coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-β subtypes have different physiological functions despite their similar structures. Because the PLC-β subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, we identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-β3. To elucidate the functional roles of PDZK1, we next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-β3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-β3, but not PLC-β1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-β3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-β3, but not that of PLC-β1, significantly inhibited SST-induced intracellular Ca(2+) mobilization, which further attenuated subsequent ERK1/2 phosphorylation. Taken together, our results strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-β3 is essential for the specific activation of PLC-β3 and the subsequent physiologic responses by SST.

  6. Evidence that a lipolytic enzyme—hematopoietic-specific phospholipase C-β2—promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes

    PubMed Central

    Adamiak, M; Poniewierska-Baran, A; Borkowska, S; Schneider, G; Abdelbaset-Ismail, A; Suszynska, M; Abdel-Latif, A; Kucia, M; Ratajczak, J; Ratajczak, M Z

    2016-01-01

    Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-β2 (PLC-β2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-β2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs. PMID:26582648

  7. Angiotensin II Reduces Lipoprotein Lipase Expression in Visceral Adipose Tissue via Phospholipase C β4 Depending on Feeding but Increases Lipoprotein Lipase Expression in Subcutaneous Adipose Tissue via c-Src.

    PubMed

    Uchiyama, Tsuyoshi; Tomono, Shoichi; Sato, Koichi; Nakamura, Tetsuya; Kurabayashi, Masahiko; Okajima, Fumikazu

    2015-01-01

    Metabolic syndrome is characterized by visceral adiposity, insulin resistance, high triglyceride (TG)- and low high-density lipoprotein cholesterol-levels, hypertension, and diabetes-all of which often cause cardiovascular and cerebrovascular diseases. It remains unclear, however, why visceral adiposity but not subcutaneous adiposity causes insulin resistance and other pathological situations. Lipoprotein lipase (LPL) catalyzes hydrolysis of TG in plasma lipoproteins. In the present study, we investigated whether the effects of angiotensin II (AngII) on TG metabolism are mediated through an effect on LPL expression. Adipose tissues were divided into visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) for comparison. AngII accelerated LPL expression in SAT but, on the contrary, suppressed its expression in VAT. In both SAT and VAT, AngII signaled through the same type 1 receptor. In SAT, AngII increased LPL expression via c-Src and p38 MAPK signaling. In VAT, however, AngII reduced LPL expression via the Gq class of G proteins and the subsequent phospholipase C β4 (PLCβ4), protein kinase C β1, nuclear factor κB, and inducible nitric oxide synthase signaling pathways. PLCβ4 small interfering RNA experiments showed that PLCβ4 expression is important for the AngII-induced LPL reduction in VAT, in which PLCβ4 expression increases in the evening and falls at night. Interestingly, PLCβ4 expression in VAT decreased with fasting, while AngII did not decrease LPL expression in VAT in a fasting state. In conclusion, AngII reduces LPL expression through PLCβ4, the expression of which is regulated by feeding in VAT, whereas AngII increases LPL expression in SAT. The different effects of AngII on LPL expression and, hence, TG metabolism in VAT and SAT may partly explain their different contributions to the development of metabolic syndrome. PMID:26447765

  8. Phosphatidylcholine-specific phospholipase C and phospholipase D are respectively implicated in mitogen-activated protein kinase and nuclear factor kappaB activation in tumour-necrosis-factor-alpha-treated immature acute-myeloid-leukaemia cells.

    PubMed Central

    Plo, I; Lautier, D; Levade, T; Sekouri, H; Jaffrézou, J P; Laurent, G; Bettaïeb, A

    2000-01-01

    Tumour necrosis factor-alpha (TNFalpha) has been reported to induce potent growth inhibition of committed myeloid progenitor cells, whereas it is a potential growth stimulator of human CD34(+)CD38(-) multipotent haematopoietic cells. The present study was aimed at evaluating the respective role of two phospholipases, phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the response of the CD34(+) CD38(-) KG1a cells to TNFalpha. In these cells TNFalpha triggered phosphoinositide 3-kinase (PI3K)-dependent PC hydrolysis within 4-8 min with concomitant production of both diacylglycerol (DAG) and phosphocholine (P-chol). DAG and P-chol production was accompanied by extracellular-signal-related protein kinase-1 ('ERK-1') activation and DNA-synthesis stimulation. PC-PLC stimulation was followed by PI3K-independent PLD activation with concomitant phosphatidic acid (PA) production followed by PA-derived DAG accumulation and sustained nuclear factor kappaB (NF-kappaB) activation. PLD/NF-kappaB signalling activation played no role in the TNFalpha proliferative effect and conferred no consistent protection of KG1a cells towards antileukaemic agents. Altogether these results suggest that, in KG1a cells, TNFalpha can stimulate in parallel PC-PLC and PLD, whose lipid products activate in turn mitogen-activated protein kinase (MAP kinase) and NF-kappaB signalling respectively. Finally, our study suggests that PC-PLC, but not PLD, plays a role in the TNFalpha proliferative effect in immature myeloid cells. PMID:11023832

  9. Evidence that a lipolytic enzyme--hematopoietic-specific phospholipase C-β2--promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes.

    PubMed

    Adamiak, M; Poniewierska-Baran, A; Borkowska, S; Schneider, G; Abdelbaset-Ismail, A; Suszynska, M; Abdel-Latif, A; Kucia, M; Ratajczak, J; Ratajczak, M Z

    2016-04-01

    Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-β2 (PLC-β2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-β2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs.

  10. Angiotensin II Reduces Lipoprotein Lipase Expression in Visceral Adipose Tissue via Phospholipase C β4 Depending on Feeding but Increases Lipoprotein Lipase Expression in Subcutaneous Adipose Tissue via c-Src

    PubMed Central

    Uchiyama, Tsuyoshi; Tomono, Shoichi; Sato, Koichi; Nakamura, Tetsuya; Kurabayashi, Masahiko; Okajima, Fumikazu

    2015-01-01

    Metabolic syndrome is characterized by visceral adiposity, insulin resistance, high triglyceride (TG)- and low high-density lipoprotein cholesterol-levels, hypertension, and diabetes—all of which often cause cardiovascular and cerebrovascular diseases. It remains unclear, however, why visceral adiposity but not subcutaneous adiposity causes insulin resistance and other pathological situations. Lipoprotein lipase (LPL) catalyzes hydrolysis of TG in plasma lipoproteins. In the present study, we investigated whether the effects of angiotensin II (AngII) on TG metabolism are mediated through an effect on LPL expression. Adipose tissues were divided into visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) for comparison. AngII accelerated LPL expression in SAT but, on the contrary, suppressed its expression in VAT. In both SAT and VAT, AngII signaled through the same type 1 receptor. In SAT, AngII increased LPL expression via c-Src and p38 MAPK signaling. In VAT, however, AngII reduced LPL expression via the Gq class of G proteins and the subsequent phospholipase C β4 (PLCβ4), protein kinase C β1, nuclear factor κB, and inducible nitric oxide synthase signaling pathways. PLCβ4 small interfering RNA experiments showed that PLCβ4 expression is important for the AngII-induced LPL reduction in VAT, in which PLCβ4 expression increases in the evening and falls at night. Interestingly, PLCβ4 expression in VAT decreased with fasting, while AngII did not decrease LPL expression in VAT in a fasting state. In conclusion, AngII reduces LPL expression through PLCβ4, the expression of which is regulated by feeding in VAT, whereas AngII increases LPL expression in SAT. The different effects of AngII on LPL expression and, hence, TG metabolism in VAT and SAT may partly explain their different contributions to the development of metabolic syndrome. PMID:26447765

  11. Phospholipase C mediates (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-, but not lysergic acid diethylamide (LSD)-elicited head bobs in rabbit medial prefrontal cortex.

    PubMed

    Schindler, Emmanuelle A D; Harvey, John A; Aloyo, Vincent J

    2013-01-23

    The phenethylamine and indoleamine classes of hallucinogens demonstrate distinct pharmacological properties, although they share a serotonin(2A) (5-HT(2A)) receptor mechanism of action (MOA). The 5-HT(2A) receptor signals through phosphatidylinositol (PI) hydrolysis, which is initiated upon activation of phospholipase C (PLC). The role of PI hydrolysis in the effects of hallucinogens remains unclear. In order to better understand the role of PI hydrolysis in the MOA of hallucinogens, the PLC inhibitor, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to study the effects of two hallucinogens, the phenethylamine, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and the indoleamine, lysergic acid diethylamide (LSD). PI hydrolysis was quantified through release of [3H]inositol-4-phosphate from living rabbit frontocortical tissue prisms. Head bobs were counted after hallucinogens were infused into the medial prefrontal cortex (mPFC) of rabbits. Both DOI and LSD stimulated PI hydrolysis in frontocortical tissue through activation of PLC. DOI-stimulated PI hydrolysis was blocked by 5-HT(2A/2C) receptor antagonist, ketanserin, whereas the LSD signal was blocked by 5-HT(2B/2C) receptor antagonist, SB206553. When infused into the mPFC, both DOI- and LSD-elicited head bobs. Pretreatment with U73122 blocked DOI-, but not LSD-elicited head bobs. The two hallucinogens investigated were distinct in their activation of the PI hydrolysis signaling pathway. The serotonergic receptors involved with DOI and LSD signals in frontocortical tissue were different. Furthermore, PLC activation in mPFC was necessary for DOI-elicited head bobs, whereas LSD-elicited head bobs were independent of this pathway. These novel findings urge closer investigation into the intracellular mechanism of action of these unique compounds.

  12. The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

    PubMed Central

    Fazioli, F; Kim, U H; Rhee, S G; Molloy, C J; Segatto, O; Di Fiore, P P

    1991-01-01

    The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts. Images PMID:1672440

  13. Membrane associated phospholipase C from bovine brain

    SciTech Connect

    Lee, K.; Ryu, S.H.; Suh, P.; Choi, W.C.; Rhee, S.G.

    1987-05-01

    Cytosolic fractions of bovine brain contain 2 immunologically distinct phosphoinositide-specific phospholipase (PLC), PLC-I and PLC-II, whose MW are 150,000 and 145,000 respectively, under a denaturing condition. Monoclonal antibodies were derived against each form and specific radioimmunoassays were developed. Distribution of PLC-I and PLC-II in cytosolic and particulate fractions was measured using the radioimmunoassay. More than 90% of PLC-II was found in the cytosolic fraction, while the anti-PLC-I antibody cross-reacting protein was distributed nearly equally between the soluble fraction and the 2 M KCl extract of particulate fraction. The PLC enzyme in the particulate fraction was purified to homogeneity, yielding 2 proteins of 140 KDa and 150 KDa when analyzed on SDS-PAGE. Neither of the 2 enzymes cross-reacted with anti-PLC-II antibodies, but both could be immunoblotted by all 4 different anti-PLC-I antibodies. This suggests that the 140 KDa PLC was derived from the 150 KDa form. The 150 Kda form from particulate fraction was indistinguishable from the cytosolic PLC-I when their mixture was analyzed on SDS-PAGE. In addition, the elution profile of tryptic peptides derived from the 150 KDa particulate form was identical to that of cytosolic PLC-I. This result indicates that PLC-I is reversibly associated to membranes.

  14. Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase

    PubMed Central

    1994-01-01

    Interleukin 4 (IL-4) diminishes cytokine activation of human macrophage. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacyglycerol (DAG), an activator of PKC, from membrane phospholipids was investigated to define the proximal events of IL-4R signaling. IL-4 induced a statistically significant time-and dose-dependent generation of DAG. The IL-4-triggered production of DAG was not derived from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in response to IL-4. Experiments were performed using [14C- methyl]choline-labeled U937 cells and monocytes to determine whether IL- 4R activated phospholipase C (PLC), PLD, or PLA2 to use membrane phosphatidylcholine (PC) to form DAG. IL-4 induced a time- and dose- dependent increase of phosphocholine (pchol) with concomitant degradation of membrane PC (p < 0.05 compared with control). The finding that the peak reduction of PC was equivalent to peak production of pchol suggested that IL-4R signaling involved the activation of a PC- specific PLC. Changes in choline (chol) or lyso-PC and glycerolphosphocholine, the respective products of PC cleavage by PLD or PLA2, were not detected in IL-4-treated cells. In contrast, exogenous PLD induced an increase in chol and concomitant loss of membrane PC. Additional investigation suggested that IL-4R signaling does not involve PLD. In cells labeled with L-lyso-3-PC 1-[1- 14C]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with ethanol. Propranolol, an inhibitor of phosphatidate phosphohydrolase, and calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG production in response to FMLP but not to IL-4. In propranolol pretreated cells, PMA but not IL-4 triggered the production of PA and lowered the amount of DAG. Evidence that PLA2 is not

  15. Evidence for a model of integrated inositol phospholipid pools implies an essential role for lipid transport in the maintenance of receptor-mediated phospholipase C activity in 1321N1 cells.

    PubMed Central

    Batty, I H; Currie, R A; Downes, C P

    1998-01-01

    The compartmentation of inositol phospholipids was examined by using a combination of radiolabelling approaches in intact and permeabilized 1321N1 astrocytoma cells. A 'chase' protocol was developed with whole cells in which phosphoinositide (PI) pools were labelled to steady state with [3H]inositol and the cellular [3H]inositol pool was then diluted selectively with non-radioactive inositol. In these cells muscarinic-receptor-stimulated phospholipase C (PLC) hydrolysed [3H]PI at approx. 1-2%/min. However, after the chase procedure the relative specific radioactivity of [3H]Ins(1,3,4)P3, a rapidly metabolized and sensitive marker of PLC activity, decreased only after more than 5 min and over a time course similar to that during which the labelling of each [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 declined by at least 50%. These results demonstrate a large receptor-responsive [3H]PI pool that is accessed by stimulated PLC without apparent metabolic compartmentation, despite its probable distribution between different membrane fractions. Support for this was obtained in intact cells by using an acute [3H]inositol labelling method in which increases in the specific radioactivity of [3H]inositol phosphates stimulated by carbachol occurred only in parallel with similar increases in the labelling of the bulk of cellular [3H]PI. In [3H]inositol-prelabelled cells permeabilized to deplete cytosolic proteins, carbachol and guanosine 5'-[gamma-thio]triphosphate stimulated the endogenous PLC to degrade only approx. 5% of [3H]PI. This was increased to approx. 30% in the presence of exogenous PtdIns transfer protein, which, at a concentration approx. 5-10% of that in 1321N1 cell cytosol, was sufficient to support PLC activity comparable with that observed in response to carbachol in whole cells. These and earlier results in 1321N1 cells suggest a model of integrated PI pools involving an obligatory role for lipid transport. Given the multifunctional capacity of PI in cellular

  16. Null Mutation in PGAP1 Impairing Gpi-Anchor Maturation in Patients with Intellectual Disability and Encephalopathy

    PubMed Central

    Murakami, Yoshiko; Tawamie, Hasan; Maeda, Yusuke; Büttner, Christian; Buchert, Rebecca; Radwan, Farah; Schaffer, Stefanie; Sticht, Heinrich; Aigner, Michael; Reis, André; Kinoshita, Taroh; Jamra, Rami Abou

    2014-01-01

    Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene PGAP1. PGAP1 was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type PGAP1 cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene PGAP1. Our results add PGAP1 to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development. PMID:24784135

  17. Dimer Structure of an Interfacially Impaired Phosphatidylinositol-Specific Pholpholipase C

    SciTech Connect

    Shao,C.; Shi, X.; Wehbi, H.; Zambonelli, C.; Head, J.; Seaton, B.; Roberts, M,.

    2007-01-01

    The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8{angstrom} resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp {yields} Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues.

  18. Physiology and pathology of nuclear phospholipase C β1.

    PubMed

    Cocco, Lucio; Follo, Matilde Y; Faenza, Irene; Fiume, Roberta; Ramazzotti, Giulia; Weber, George; Martelli, Alberto M; Manzoli, Francesco A

    2011-01-01

    The existence and function of inositide signaling in the nucleus is well documented and we know that the existence of the inositide cycle inside the nucleus has a biological role. An autonomous lipid-dependent signaling system, independently regulated from its plasma membrane counterpart, acts in the nucleus and modulates cell cycle progression and differentiation.We and others focused on PLCβ1, which is the most extensively investigated PLC isoform in the nuclear compartment. PLCβ1 is a key player in the regulation of nuclear inositol lipid signaling, and, as discussed above, its function could also be involved in nuclear structure because it hydrolyses PtdIns(4,5)P2, a well accepted regulator of chromatin remodelling. The evidence, in a number of patients with myelodysplastic syndromes, that the mono-allelic deletion of PLCβ1 is associated with an increased risk of developing acute myeloid leukemia paves the way for an entirely new field of investigation. Indeed the genetic defect evidenced, in addition to being a useful prognostic tool, also suggests that altered expression of this enzyme could have a role in the pathogenesis of this disease, by causing an imbalance between proliferation and apoptosis. The epigenetics of PLCβ1 expression in MDS has been reviewed as well.

  19. Major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid

    SciTech Connect

    Nagel, S.D.; Boothroyd, J.C.

    1989-04-05

    P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with (/sup 3/H)palmitic acid and with myo-(2-/sup 3/H)inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological cross-reacting determinant first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with (/sup 35/S)methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purified (/sup 3/H) palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.

  20. Disruption of pioneer growth cone guidance in vivo by removal of glycosyl-phosphatidylinositol-anchored cell surface proteins.

    PubMed

    Chang, W S; Serikawa, K; Allen, K; Bentley, D

    1992-02-01

    Cell surface proteins anchored to membranes via covalently attached glycosyl-phosphatidylinositol (GPI) have been implicated in neuronal adhesion, promotion of neurite outgrowth and directed cell migration. Treatment of grasshopper embryos with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves the GPI anchor, often induced disruptions in the highly stereotyped migrations of peripheral pioneer growth cones and afferent neuron cell bodies. In distal limb regions of embryos treated with PI-PLC at early stages of pioneer axon outgrowth, growth cones lost their proximal orientation toward the central nervous system (CNS) and turned distally. Pioneer growth cones in treated limbs also failed to make a characteristic ventral turn along the trochanter-coxa (Tr-Cx) segment boundary, and instead continued to grow proximally across the boundary. Treatment at an earlier stage of development caused pre-axonogenesis Cx1 neurons to abandon their normal circumferential migration and reorient toward the CNS. None of these abnormal phenotypes were observed in limbs of untreated embryos or embryos exposed to other phospholipases that do not release GPI-anchored proteins. Incubation of embryos with PI-PLC effectively removed immunoreactivity for fasciclin I, a GPI-anchored protein expressed on a subset of neuronal surfaces. These results suggest that cell surface GPI-anchored proteins are involved in pioneer growth cone guidance and in pre-axonogenesis migration of neurons in the grasshopper limb bud in vivo.

  1. Genome-wide prediction and annotation of Burkholderia pseudomallei AraC/XylS family transcription regulator.

    PubMed

    Lim, Boon-San; Chong, Chan-Eng; Zamrod, Zulkeflie; Nathan, Sheila; Mohamed, Rahmah

    2007-01-01

    Many members of the AraC/XylS family transcription regulator have been proven to play a critical role in regulating bacterial virulence factors in response to environmental stress. By using the Hidden Markov Model (HMM) profile built from the alignment of a 99 amino acid conserved domain sequence of 273 AraC/XylS family transcription regulators, we detected a total of 45 AraC/XylS family transcription regulators in the genome of the Gram-negative pathogen, Burkholderia pseudomallei. Further in silico analysis of each detected AraC/XylS family transcription regulatory protein and its neighboring genes allowed us to make a first-order guess on the role of some of these transcription regulators in regulating important virulence factors such as those involved in three type III secretion systems and biosynthesis of pyochelin, exopolysaccharide (EPS) and phospholipase C. This paper has demonstrated an efficient and systematic genome-wide scale prediction of the AraC/XylS family that can be applied to other protein families. PMID:18391231

  2. PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries.

    PubMed

    Mauban, Joseph R H; Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

    2015-06-15

    Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction.

  3. Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes.

    PubMed

    Bonilla, Julia B; Cid, M Belén; Contreras, F-Xabier; Goñi, Félix M; Martín-Lomas, Manuel

    2006-02-01

    The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs. PMID:16315198

  4. A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding.

    PubMed

    Khan, Hanif M; He, Tao; Fuglebakk, Edvin; Grauffel, Cédric; Yang, Boqian; Roberts, Mary F; Gershenson, Anne; Reuter, Nathalie

    2016-03-29

    Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought. PMID:27028646

  5. Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes.

    PubMed

    Bonilla, Julia B; Cid, M Belén; Contreras, F-Xabier; Goñi, Félix M; Martín-Lomas, Manuel

    2006-02-01

    The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.

  6. PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries

    PubMed Central

    Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

    2015-01-01

    Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K+ concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca2+ release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca2+ waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca2+ concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca2+ entry and promote vasoconstriction. PMID:25888510

  7. New chromogenic plating media for detection and enumeration of pathogenic Listeria spp.--an overview.

    PubMed

    Reissbrodt, Rolf

    2004-08-15

    In recent years a number of selective chromogenic plating media for pathogenic Listeria spp. have been developed and marketed. Their advantages are direct detection and enumeration of pathogenic Listeria spp. utilizing cleavage of substrates by the virulence factor phosphatidylinositol-phospholipase C (PI-PLC) and, to a lesser extent, by phosphatidylcholin-phospholipase C (PC-PLC). There are two groups of such media: the first utilizes cleavage by PI-PLC of L-alpha-phosphatidyl-inositol, forming a white precipitation zone around the colony, combined with the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside for detection of beta-d-glucosidase, which occurs in all Listeria spp. All Listeria spp. produce turquoise colonies on these media which include ALOA , CHROMagar Listeria, BBL CHROMagar Listeria, and OCLA. The second group of media utilizes 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate, forming blue-turquoise colonies of pathogenic Listeria spp. and white colonies of non-pathogenic Listeria spp. BCM trade mark Listeria monocytogenes plating medium, Rapid'L.mono and LIMONO-Ident-Agar belong to this group. Selective chromogenic L. monocytogenes plating media offer the attraction of rapid economic detection and enumeration of pathogenic Listeria spp. within 24 or 48 h of incubation at 36+/-1 degrees C. This overview summarises the characteristics of these chromogenic plating media, reviews important evaluations, and focuses on replacement of conventional by these chromogenic plating media, particularly for applications in the food industry.

  8. Family Meals

    MedlinePlus

    ... Story" 5 Things to Know About Zika & Pregnancy Family Meals KidsHealth > For Parents > Family Meals Print A ... even more important as kids get older. Making Family Meals Happen It can be a big challenge ...

  9. Family Arguments

    MedlinePlus

    ... Spread the Word Shop AAP Find a Pediatrician Family Life Medical Home Family Dynamics Adoption & Foster Care ... Life Listen Español Text Size Email Print Share Family Arguments Page Content Article Body We seem to ...

  10. Family History

    MedlinePlus

    Your family history includes health information about you and your close relatives. Families have many factors in common, including their genes, ... as heart disease, stroke, and cancer. Having a family member with a disease raises your risk, but ...

  11. Family Privilege

    ERIC Educational Resources Information Center

    Seita, John R.

    2014-01-01

    Family privilege is defined as "strengths and supports gained through primary caring relationships." A generation ago, the typical family included two parents and a bevy of kids living under one roof. Now, every variation of blended caregiving qualifies as family. But over the long arc of human history, a real family was a…

  12. Family Literacy.

    ERIC Educational Resources Information Center

    Washington, Charles W., Ed.

    1996-01-01

    This newsletter theme issue focuses on the impact of learning disabilities within families, specifically families with low literacy skills. It explores the effectiveness of family literacy programs, examines the connection between the field of family literacy and learning disabilities (LD), and offers suggestions on how to work with students with…

  13. Asteroid families

    NASA Technical Reports Server (NTRS)

    Williams, James G.

    1991-01-01

    More than 100 asteroid families are presented in Williams. Several examples of cratering events are known including family numbers 150, 162, 169, and 189. These are recognizable as many small fragments adjacent to and to one side (in three dimensions) of a much larger cratered body. Family numbers 138 and 140 are adjacent in proper element space. In population they are an intermediate step between the long recognizable families and the more frequent less populated families. Family number 164 is the fifth most populous family in the belt. All members are faint and nothing is known of the physical properties.

  14. Comprehensive genomic analysis and expression profiling of diacylglycerol kinase gene family in Malus prunifolia (Willd.) Borkh.

    PubMed

    Li, Yali; Tan, Yanxiao; Shao, Yun; Li, Mingjun; Ma, Fengwang

    2015-05-01

    Diacylglycerol kinase (DGK) is a pivotal enzyme that phosphorylates diacylglycerol (DAG) to form phosphatidic acid (PA). The production of PA from phospholipase D (PLD) and the coupled phospholipase C (PLC)/DGK route is a critical signaling process in animal and plant cells. Next to PLD, DGK is the second most important generator of PA in biotic and abiotic stress responses. We identified 8 DGK members within the apple genome and all of their putative proteins contain one DGK catalytic domain and one DGK accessory domain. Four coding sequences were confirmed by cloning from Malus prunifolia. Phylogenetic and gene structure analyses showed that the apple DGK genes could be assigned to Clusters I, II, or III. Expression analysis of 6 of them revealed that their transcript levels were highest in stems. Some apple DGK genes were also significantly up-regulated in response to salt and drought stresses. This suggested their possible roles in plant defenses against environmental challenges. As a first step toward genome-wide analyses of the DGK genes in woody plants, our results imply that apple DGK genes are involved in the signaling of stress responses. These findings will contribute to further functional dissection of this gene family.

  15. Muslim Families and Family Therapy.

    ERIC Educational Resources Information Center

    Daneshpour, Manijeh

    1998-01-01

    Examines the applicability of the Anglo-American models of family therapy to Muslim immigrant families. The differences in value systems are the Muslim families' preferences for greater connectedness, a less flexible and more hierarchical family structure, and an implicit communication style. Suggests that directions for change for Muslims need to…

  16. Cancer, Families, and Family Counselors.

    ERIC Educational Resources Information Center

    Duffy, Maureen; Gillig, Scott

    2003-01-01

    Examines the role of the family counselor in working with cancer patients and their families. Suggests ways in which the family counselor can work proactively with families in the area of cancer prevention and helping them cope more effectively with its impact on their lives. Uses a clinical case example to illustrate intervention with cancer…

  17. Family Violence and Family Physicians

    PubMed Central

    Herbert, Carol P.

    1991-01-01

    The acronym IDEALS summarizes family physicians' obligations when violence is suspected: to identify family violence; document injuries; educate families and ensure safety for victims; access resources and coordinate care; co-operate in the legal process; and provide support for families. Failure to respond reflects personal and professional experience and attitudes, fear of legal involvement, and lack of knowledge. Risks of intervention include physician burnout, physician overfunctioning, escalation of violence, and family disruption. PMID:21228987

  18. Familial gigantism.

    PubMed

    Herder, Wouter W de

    2012-01-01

    Familial GH-secreting tumors are seen in association with three separate hereditary clinical syndromes: multiple endocrine neoplasia type 1, Carney complex, and familial isolated pituitary adenomas. PMID:22584702

  19. Family Support.

    ERIC Educational Resources Information Center

    Wieck, Colleen, Ed.; McBride, Marijo, Ed.

    1990-01-01

    This "Feature Issue" of the quarterly journal "Impact" presents 19 brief articles on family support systems in the United States for persons with developmental disabilities and their families. Emphasis is on provisions of Public Law 99-457. Articles include: "Family Support in the United States: Setting a Course for the 1990s" (James Knoll);…

  20. Rural Families.

    ERIC Educational Resources Information Center

    Goetz, Kathy, Ed.

    1992-01-01

    This "special focus" journal issue consists of 13 individual articles on the theme of rural family programs relating to school, health services, church, and other institutions. It includes: (1) "Towards a Rural Family Policy" (Judith K. Chynoweth and Michael D. Campbell); (2) "Montana: Council for Families Collaborates for Prevention (Jean…

  1. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    SciTech Connect

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  2. Bacillus thuringiensis membrane-damaging toxins acting on mammalian cells.

    PubMed

    Celandroni, Francesco; Salvetti, Sara; Senesi, Sonia; Ghelardi, Emilia

    2014-12-01

    Bacillus thuringiensis is widely used as a biopesticide in forestry and agriculture, being able to produce potent species-specific insecticidal toxins and considered nonpathogenic to other animals. More recently, however, repeated observations are documenting the association of this microorganism with various infectious diseases in humans, such as food-poisoning-associated diarrheas, periodontitis, bacteremia, as well as ocular, burn, and wound infections. Similar to B. cereus, B. thuringiensis produces an array of virulence factors acting against mammalian cells, such as phosphatidylcholine- and phosphatidylinositol-specific phospholipase C (PC-PLC and PI-PLC), hemolysins, in particular hemolysin BL (HBL), and various enterotoxins. The contribution of some of these toxins to B. thuringiensis pathogenicity has been studied in animal models of infection, following intravitreous, intranasal, or intratracheal inoculation. These studies lead to the speculation that the activities of PC-PLC, PI-PLC, and HBL are responsible for most of the pathogenic properties of B. thuringiensis in nongastrointestinal infections in mammals. This review summarizes data regarding the biological activity, the genetic basis, and the structural features of these membrane-damaging toxins.

  3. Membrane Topology and Transient Acylation of Toxoplasma gondii Glycosylphosphatidylinositols

    PubMed Central

    Kimmel, Jürgen; Smith, Terry K.; Azzouz, Nahid; Gerold, Peter; Seeber, Frank; Lingelbach, Klaus; Dubremetz, Jean-François; Schwarz, Ralph T.

    2006-01-01

    Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-3H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-3H]mannose or UDP-[6-3H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER. PMID:16896225

  4. Bacillus thuringiensis pore-forming toxins trigger massive shedding of GPI-anchored aminopeptidase N from gypsy moth midgut epithelial cells.

    PubMed

    Valaitis, Algimantas P

    2008-06-01

    The insecticidal Cry proteins produced by Bacillus thuringiensis strains are pore-forming toxins (PFTs) that bind to the midgut brush border membrane and cause extensive damage to the midgut epithelial cells of susceptible insect larvae. Force-feeding B. thuringiensis PFTs to Lymantria dispar larvae elicited rapid and massive shedding of a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase N (APN) from midgut epithelial cells into the luminal fluid, and depletion of the membrane-anchored enzyme on the midgut epithelial cells. The amount of APN released into the luminal fluid of intoxicated larvae was dose- and time-dependent, and directly related to insecticidal potency of the PFTs. The induction of toxin-induced shedding of APN was inhibited by cyclic AMP and MAPK kinase (MEK) inhibitors PD98059 and U0126, indicating that signal transduction in the MEK/ERK pathway is involved in the regulation of the shedding process. APN released from epithelial cells appears to be generated by the action of a phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of the GPI anchor based upon detection of a cross-reacting determinant (CRD) on the protein shed into the luminal fluid. Alkaline phosphatase was also released from the gut epithelial cells, supporting the conclusion that other GPI-anchored proteins are released as a consequence of the activation PI-PLC. These observations are the basis of a novel and highly sensitive tool for evaluating the insecticidal activity of new Cry proteins obtained though discovery or protein engineering.

  5. Family Life.

    ERIC Educational Resources Information Center

    Naturescope, 1986

    1986-01-01

    Focuses on various aspects of mammal family life ranging from ways different species are born to how different mammals are raised. Learning activities include making butter from cream, creating birth announcements for mammals, and playing a password game on family life. (ML)

  6. Family Workshops

    ERIC Educational Resources Information Center

    Bennett, Dave; Rees-Jones, Tanny

    1978-01-01

    A Family Workshop is an informal, multidisciplined educational program for adults and children, organized by a team of teachers. This article discusses the Lavender Hill Family Workshop, one of many, which attempts to provide education in various subject areas for adults and for children while also integrating both objectives in order to educate…

  7. Family Empowerment.

    ERIC Educational Resources Information Center

    Sinclair, Mary F., Ed.; And Others

    1992-01-01

    This feature issue of IMPACT focuses on the empowerment of families with a member who has a developmental disability. It presents strategies and models for a collaborative, respectful approach to service provision, and presents the experiences of families in seeking support and assistance. Feature articles include "Two Generations of Disability: A…

  8. Family, Extended

    ERIC Educational Resources Information Center

    Patton, Jessica Rae

    2006-01-01

    Parents are a child's first and most influential teacher. People hear this truism often, yet nowhere has the author seen it more taken to heart than at Tower Street Elementary School. The school's efforts to form a true partnership with students' families--from involving families in the first day of school, to the principal making home visits, to…

  9. Family Potyviridae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The International Committee on the Taxonomy of Viruses potyvirus study group has revised the description of the family Potyviridae for inclusion in the ICTV 9th report. Characteristic features of each genus within the family is presented. Revised criteria for demarcation and nomenclature of viral sp...

  10. FAMILY EMPIDIDAE.

    PubMed

    Rafael, José Albertino; Câmara, Josenir Teixeira

    2016-01-01

    Empididae is one of the biggest families of Diptera with around 3,000 known species. The family is poorly known in Colombia, with only six species reported and this work provides information on their distribution. No endemic genera or species have been recorded to date from Colombia. PMID:27395282

  11. Family Theory and Family Health Research

    PubMed Central

    Doherty, William J.

    1991-01-01

    Different family theories can be applied to different aspects of how families experience health and illness. The family health and illness cycle describes the phases of a family's experience, beginning with health promotion and risk reduction, then family vulnerability and disease onset or relapse, family illness appraisal, family acute response, and finally family adaptation to illness and recovery. For each phase, specific family theories that are most appropriate for guiding family and health research are discussed. PMID:21229056

  12. Nitric oxide inhibits the shedding of the glycosylphosphatidylinositol-anchored dipeptidase from porcine renal proximal tubules.

    PubMed

    Park, Sung Wook; Yoon, Hyun Joong; Lee, Hwanghee Blaise; Hooper, Nigel M; Park, Haeng Soon

    2002-05-15

    NO is related to the pathological condition acute renal failure, in which we previously observed that the level of soluble dipeptidase in urine was decreased. In this study the role of NO in the shedding of the glycosylphosphatidylinositol (GPI)-anchored form of renal dipeptidase (RDPase) was examined. The NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine rapidly inhibited the release of RDPase from porcine kidney proximal tubules. The substrate of NO synthase, l-Arg, also inhibited the release of RDPase, and this effect was reversed by the NO synthase inhibitor N(omega)-nitro-l-arginine methyl ester. Western-blot analyses using antibodies raised against porcine RDPase and the inositol-1,2-cyclic monophosphate moiety formed on phospholipase C cleavage of the GPI anchor demonstrated that SNP mediated its inhibitory effect on the release of RDPase via a GPI-specific phospholipase C (GPI-PLC). Peroxynitrite scavengers (deferoxamine and superoxide dismutase) or reducing agent (dithiothreitol) did not affect SNP's inhibition of the release of RDPase. Exposure to the G-protein activator AlF(-)(4) mimicked the l-Arg effect in the presence of a low concentration of l-Arg, and the effect was completely reversed by U73122, an intracellular phosphatidylinositol-specific PLC (PI-PLC) inhibitor. These results suggest a signal-transduction pathway involving NO, which is produced by NO synthase(s) following activation of a G-protein-coupled PI-PLC, resulting in inhibition of the GPI-PLC that cleaves and releases RDPase. Therefore, this indicates a role for NO as an inhibitory regulator of the shedding of the GPI-anchored RDPase in acute renal failure.

  13. Development of a Novel Selective and Differential Medium for the Isolation of Listeria monocytogenes

    PubMed Central

    Park, Sang-Hyun; Chang, Pahn-Shick; Ryu, Sangryeol

    2014-01-01

    A new medium (lecithin and levofloxacin [LL] medium) is described for the isolation of Listeria monocytogenes from food samples. LL medium includes lecithin from soybeans for the detection of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) produced by L. monocytogenes. Levofloxacin is incorporated to inhibit the growth of microorganisms other than L. monocytogenes, especially Bacillus cereus, shown to possess PI-PLC and PC-PLC activities. L. monocyogenes produced white colonies with a halo on LL medium, whereas Listeria innocua appeared as white colonies without a halo. Levofloxacin at 0.20 mg/liter completely inhibited the growth of B. cereus, while the growth of L. monocytogenes was unaffected. In the second phase of the study, the sensitivity and the specificity of LL medium were compared to those of modified Oxford agar (MOX) and two chromogenic media (Brilliance Listeria agar and CHROMagar Listeria), using a total of 250 food samples. From 200 unspiked food samples, the specificity of LL medium (96.0%) was superior to that of MOX (72.0%) and similar to the specificities of Brilliance Listeria agar (96.5%) and CHROMagar Listeria (94.5%). From 50 spiked food samples, LL medium and CHROMagar Listeria represented the highest sensitivities (96.0%), followed by Brilliance Listeria agar (92.0%) and MOX (54.0%). Also, LL medium showed the highest confirmation rate (98.8%), followed by Brilliance Listeria agar (98.7%), CHROMagar Listeria (98.3%), and MOX (52.0%). On the basis of its good specificity and cost effectiveness, LL medium is useful for the isolation of L. monocytogenes from food samples. PMID:24271177

  14. Family Health and Family Planning.

    ERIC Educational Resources Information Center

    World Health Organization, Copenhagen (Denmark). Regional Office for Europe.

    This document is made up of a selection of some of the papers distributed to participants in courses on "Family Health and Family Planning" which have been organized each year since 1973 by the International Children's Center and the World Health Organization Regional Office for Europe. Six courses, held between 1973 and 1978, brought together a…

  15. Family Issues

    MedlinePlus

    ... not mean that everyone gets along all the time. Conflicts are a part of family life. Many things can lead to conflict, such as illness, disability, addiction, job loss, school problems, and marital issues. Listening to ...

  16. Unusual families.

    PubMed

    Golombok, Susan

    2005-03-01

    The introduction of assisted reproduction has led to unusual forms of procreation. This article describes the social consequences of lesbian motherhood and of families headed by single heterosexual mothers. PMID:15819999

  17. Familial hypercholesterolemia

    MedlinePlus

    A diet low in cholesterol and saturated fat and rich in unsaturated fat may help to control your LDL level. People with a family history of this condition, particularly if both parents carry the defective ...

  18. Tomorrow's Family

    ERIC Educational Resources Information Center

    Pickett, Robert S.

    1977-01-01

    Author states that "...the traditional form of family which has been the norm in recent times in the West will persist, but will be forced to "move over" to accommodate other forms of domestic life." (Author)

  19. FAMILY PIOPHILIDAE.

    PubMed

    Wolff, Marta; Pérez, Sandra; Grisales, Diana

    2016-01-01

    Piophilidae is a little family poorly known in Colombia, with only Piophila casei (L.) and Stearibia nigriceps Meigen reported so far. This catalogue expands the distribution of these species to other localities in the country. PMID:27395294

  20. FAMILY BIBIONIDAE.

    PubMed

    Falaschi, Rafaela Lopes; Oliveira, Sarah Siqueira; Amorim, Dalton De Souza

    2016-06-14

    The Bibionidae are a family belonging to the suborder Bibionomorpha with four genera and 17 species known from Colombia. This work expands the distribution of these species to other localities in the country.

  1. FAMILY RHAGIONIDAE.

    PubMed

    Santos, Charles Morphy D; Carmo, Daniel D D

    2016-01-01

    The family Rhagionidae is one of the oldest Brachyeran lineages. Its monophyly is still uncertain. There are four rhagionid genera distributed in Neotropical Region but only three species of Chrysopilus are found in Colombia. PMID:27395270

  2. FAMILY PLATYSTOMATIDAE.

    PubMed

    Wendt, Lisiane Dilli

    2016-01-01

    Platystomatidae (Signal Flies) are one of the largest families of Tephritoidea, with about 1200 species and four subfamilies, worldwide distributed. However, Platystomatidae are not well represented in the New World, and in the Neotropical Region only four genera and 26 species, belonging to Platystomatinae, are recorded. The family is a group understudied in Colombia and only one species is recorded to the country. PMID:27395295

  3. Priority targeting of glycosyl-phosphatidylinositol-anchored proteins to the bile-canalicular (apical) plasma membrane of hepatocytes. Involvement of 'late' endosomes.

    PubMed Central

    Ali, N; Evans, W H

    1990-01-01

    1. Liver plasma membranes originating from the sinusoidal, lateral and canalicular surface domains of hepatocytes were covalently labelled with sulpho-N-hydroxysuccinamide-biotin. After solubilization in Triton X-114, treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), two-phase partitioning and 125I-streptavidin labelling of the proteins resolved by PAGE, six major polypeptides (molecular masses 110, 85, 70, 55, 38 and 35 kDa) were shown to be anchored in bile canalicular membrane vesicles by a glycosyl-phosphatidylinositol (G-PI) 'tail'. 2. Permeabilized 'early' and 'late' endocytic vesicles isolated from liver were also examined. Two polypeptides (110 and 35 kDa) were shown to be anchored by a G-PI tail in 'late' endocytic vesicles. 3. Analysis of marker enzymes in bile-canalicular vesicles treated with PI-PLC showed that 5'-nucleotidase and alkaline phosphatase, but not leucine aminopeptidase and ecto-Ca2(+)-ATPase activities were released from the membrane. A low release and recovery of alkaline phosphodiesterase activity was noted. The cleavage from the membrane of 5'-nucleotidase as a 70 kDa polypeptide was confirmed by Western blotting using an antibody to this enzyme. 4. Antibodies raised to proteins released from bile-canalicular vesicles by PI-PLC treatment, and purified by partitioning in aqueous and Triton X-114 phases, localized to the bile canaliculi in thin liver sections. Antibodies to proteins not hydrolysed by this treatment stained by immunofluorescence the sinusoidal and canalicular surface regions of hepatocytes. 5. Antibodies generated to proteins cleaved by PI-PLC treatment of canalicular vesicles were shown to identify, by Western blotting, a major 110 kDa polypeptide in these vesicles. Two polypeptides (55 and 38 kDa) were detected in MDCK and HepG-2 cultured cells. 6. Since two of the six G-PI-anchored proteins targeted to the bile-canalicular plasma membrane were also detected in 'late' endocytic vesicles, the

  4. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

    SciTech Connect

    Buck, M.A.; Fraser, C.M. )

    1990-12-14

    The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of (3H)-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of (3H)-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. (3H)-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties.

  5. BANK1 and BLK Act through Phospholipase C Gamma 2 in B-Cell Signaling

    PubMed Central

    Bernal-Quirós, Manuel; Wu, Ying-Yu

    2013-01-01

    The B cell adaptor protein with ankyrin repeats (BANK1) and the B lymphoid tyrosine kinase (BLK) have been genetically associated with autoimmunity. The proteins of these genes interact physically and work in concert during B-cell signaling. Little is know about their interactions with other B-cell signaling molecules or their role in the process. Using yeast two hybrid (Y2H) we sought for factors that interact with BANK1. We found that the molecular switch PLCg2 interacts with BANK1 and that the interaction is promoted by B-cell receptor (BCR) stimulation. We found further that the kinase activity of BLK enhanced BANK1- PLCg2 binding and that the interaction was suppressed upon BLK depletion. Immunoprecipitation and mutational analysis demonstrated that the interaction between BANK1 and PLCg2 was dependent on specific tyrosine and proline residues on the adaptor protein. Our results provide new information important to understand the role of these two genes in basic B-cell physiology and immune-related diseases. PMID:23555801

  6. Distribution of phospholipase C isozymes in various rat tissues and cultured cells

    SciTech Connect

    Suh, P.G.; Ryu, S.H.; Choi, W.C.; Lee, K.Y.; Rhee, S.G.

    1987-05-01

    Monoclonal antibodies prepared against PLC-I or PLC-II enzyme did not cross-react with the other. Using a pair of antibodies which recognizes 2 different antigenic sites on the same molecule, radioimmunoassays were developed for the quantitation of PLC-I and PLC-II in homogenates of various tissues and cultured cells, prepared by homogenization in a 2 M KCl buffer. The contents of PLC enzymes were measured in 19 rat tissues, in human platelets and in 17 cultured cells. Results indicate that the concentration of PLC-I and PLC-II is very high in brain, PLC-I is localized mainly in brain and partly in seminal vesicles, PLC-II is found in most tissues and cells. PLC-I is highly localized even in brain: 5 different neuroblastoma did not contain PLC-I while 2 glioma and 1 astrocytoma contained significant amounts.

  7. Design and synthesis of phospholipase C and A2-activatable near-infrared fluorescent smart probes.

    PubMed

    Popov, Anatoliy V; Mawn, Theresa M; Kim, Soungkyoo; Zheng, Gang; Delikatny, E James

    2010-10-20

    The primary focus of this work was to develop activatable probes suitable for in vivo detection of phospholipase activity. Phospholipases (PLs) are ubiquitous enzymes that perform a number of critical regulatory functions. They catalyze phospholipid breakdown and are categorized as A(1), A(2) (PLA(2)), C (PLC), and D (PLD) based on their site of action. Here, we report the design, synthesis, and characterization of self-quenching reporter probes that release fluorescent moieties upon cleavage with PLA(2) or PLC. A series of phospholipids were synthesized bearing the NIR fluorophore pyropheophorbide a (Pyro) at the sn-2 position. Fluorescence quenching was achieved by attachment of either a positively charged black hole quencher-3 (BHQ-3) to the phospholipid headgroup or another neutral Pyro moiety at the sn-1 position. The specificity to different phospholipases was modulated by insertion of spacers (C(6), C(12)) between Pyro and the lipid backbone. The specificity of the quenched fluorescent phospholipids was assayed on a plate reader against a number of phospholipases and compared with two commercial probes bearing the visible fluorophore BODIPY. While PyroC(6)-PyroC(6)-PtdCho revealed significant background fluorescence, and a 10% fluorescence increase under the action of PLA(2), Pyro-PtdEtn-BHQ demonstrated high selective sensitivity to PLC, particularly to the PC-PLC isoform, and its sensitivity to PLA(2) was negligible due to steric hindrance at the sn-2 position. In contrast, the C(12)-spacered PyroC(12)-PtdEtn-BHQ demonstrated a remarkable selectivity for PLA(2) and the best relative PLA(2)/PLC sensitivity, significantly outperforming previously known probes. These results open an avenue for future in vivo experiments and for new probes to detect PL activity. PMID:20882956

  8. FAMILY HYBOTIDAE.

    PubMed

    Ale-Rocha, Rosaly; De Freitas-Silva, Rafael Augusto Pinheiro

    2016-01-01

    Hybotidae is a diverse family of the order Diptera, suborder Brachycera, superfamily Empidoidea. Hybotid flies are generally yellow to black, are morphologically diverse, and are distributed worldwide. The monophyly of the family is based on: palpifer and fore tibial gland present, laterotergite bare and R4+5 unbranched. Hybotids are mainly predators and are usually found on the vegetation, logs and other surfaces, or in flight during displacement and forage. This catalogue, based on the study of specimens and available literature records, summarizes and updates the information on the Colombian fauna of Hybotidae, which includes 19 species distributed in six genera. PMID:27395283

  9. Familial leukemias.

    PubMed

    Wiernik, Peter H

    2015-02-01

    Familial leukemia has been described for more than 50 years but only recently have modern genetic techniques allowed for the investigation of the genome. Genome-wide association studies have identified a number of genetic sites that appear to relate to susceptibility to leukemia in certain families and occasionally to susceptibility to a specific leukemia in general. Many questions remain, including susceptibility to what? An oncogenic virus? An environmental chemical? Mutation of another gene induced by a heritable mutation-promoting gene?.Clinically important facts have been learned. Chronic lymphocytic leukemia (CLL) is by far the most common familial leukemia. Patients with CLL have approximately a 10% chance of a first-degree relative developing CLL, and even a greater chance of one developing monoclonal B-cell lymphocytosis which may be an asymptomatic forme fruste of the neoplasm. Furthermore, there may be an increased incidence of breast cancer in familial CLL pedigrees which raises the question of a common etiology for neoplasms in general, or at least a previously unrecognized relationship among them.

  10. Family Hypnotherapy.

    ERIC Educational Resources Information Center

    Araoz, Daniel L.; Negley-Parker, Esther

    1985-01-01

    A therapeutic model to help families activate experiential and right hemispheric functioning through hypnosis is presented in detail, together with a clinical illustration. Different situations in which this model is effective are mentioned and one such set of circumstances is described. (Author)

  11. FAMILY SCIARIDAE.

    PubMed

    Carvalho-Fernandes, Sheila Patrícia

    2016-01-01

    Sciaridae are a widely distributed family with high number of species. They are known as black fungus gnats due to their dark color and feeding activity. This catalogue presents 17 species from Colombia distributed in eight genera, and for each species the geographical distribution is provided.

  12. FAMILY CECIDOMYIIDAE.

    PubMed

    Maia, Valéria Cid

    2016-01-01

    This large family is poorly known in Colombia, where only 44 species have been recorded in 20 genera. All of them are included in Cecidomyiinae, which is the most diverse subfamily of gall midges in number of species and feeding habits, including phytophagous, predaceous and fungivorous species. Most of them are galler. The other subfamilies have never been recorded in this country.

  13. FAMILY SCATHOPHAGIDAE.

    PubMed

    Wolff, Marta; Grisales, Diana; Nihei, Silvio S

    2016-01-01

    Scathophagidae (Diptera, Calyptratae) is an uncommon group of flies. In Colombia there was no scientific record of this family until now. In this paper we report for the first time the genus Scatogera and the species S. primogenita Albuquerque, collected over 3000m. and previously collected in Ecuador. PMID:27395319

  14. Family Caregivers.

    ERIC Educational Resources Information Center

    Frazier, Billie H.

    This document contains a brief bibliography of peer-reviewed literature, with abstracts, on family caregiving. It is one of 12 bibliographies on aging prepared by the National Agricultural Library for its "Pathfinders" series of publications. Topics covered by the other 11 bibliographies include aging parents, adult children, dementia and…

  15. FAMILY SCIARIDAE.

    PubMed

    Carvalho-Fernandes, Sheila Patrícia

    2016-01-01

    Sciaridae are a widely distributed family with high number of species. They are known as black fungus gnats due to their dark color and feeding activity. This catalogue presents 17 species from Colombia distributed in eight genera, and for each species the geographical distribution is provided. PMID:27395255

  16. FAMILY CECIDOMYIIDAE.

    PubMed

    Maia, Valéria Cid

    2016-01-01

    This large family is poorly known in Colombia, where only 44 species have been recorded in 20 genera. All of them are included in Cecidomyiinae, which is the most diverse subfamily of gall midges in number of species and feeding habits, including phytophagous, predaceous and fungivorous species. Most of them are galler. The other subfamilies have never been recorded in this country. PMID:27395254

  17. FAMILY ROPALOMERIDAE.

    PubMed

    Ale-Rocha, Rosaly

    2016-01-01

    Ropalomeridae is a small family with most species distributed in the Neotropical Region, from Mexico to Argentina, and only one Nearctic species. In Colombia, eight species distributed in four genera have been found. This catalogue, based on the study of specimens and available literature records, summarizes and updates the information on the Colombian fauna. PMID:27395300

  18. FAMILY RICHARDIIDAE.

    PubMed

    Wendt, Lisiane Dilli; Ale-Rocha, Rosaly

    2016-01-01

    Richardiidae are a family of "acalyptrate" Diptera represented by ca. 180 species distributed in the New World, mostly in the Neotropical region. The species that occur in Colombia have received little attention from taxonomists, and the great majority of them are known only from their type localities. Currently, 14 genera and 23 species are known to occur in the country. PMID:27395297

  19. Family Disruptions

    MedlinePlus

    ... and Returns Do you or your spouse frequently travel on business? These can be disruptive times for your child and for the family as ... these out-of-town trips. Spend as much time as it takes to explain where you are ... before and during your travels. You need to acknowledge and accept her feelings: " ...

  20. Family Therapy and Disturbed Families.

    ERIC Educational Resources Information Center

    Zuk, Gerald H., Ed.; Boszormenyi-Nagy, Ivan, Ed.

    Presented at a conference at which authors represented major theoretical positions in the field, most of the papers use family therapy as an important source of observations or ideas, or as a means to pinpoint methodological problems. Papers are grouped in sections as follows: four which introduce the reader to the field of specialization, provide…

  1. FAMILY BOMBYLIIDAE.

    PubMed

    Lamas, Carlos José Einicker; Evenhuis, Neal L

    2016-01-01

    Bombyliidae is one of the largest Diptera families with more than 4,500 recognized species worldwide. Their species vary from robust to thin, and may be small to large (2-20mm) and looks like bees or wasps. They also present great variation in color. Adults can often be seen either resting and sunning themselves on trails, rocks or twigs or feeding on flowering plants as they are nectar feeders. All reared bee flies are predators or parasitoids of arthropods. The Colombian fauna of bombyliids comprises at the moment 22 species, and 12 genera, of which, six are endemic species. Nonetheless, this number may be much higher, as Colombia is a megadiverse country and there are not many specimens of this family deposited in collections all over the world. PMID:27395279

  2. Familial Hypercholesterolemia

    PubMed Central

    Bouhairie, Victoria Enchia; Goldberg, Anne Carol

    2015-01-01

    Familial hypercholesterolemia is a common, inherited disorder of cholesterol metabolism that leads to early cardiovascular morbidity and mortality. It is underdiagnosed and undertreated. Statins, ezetimibe, bile acid sequestrants, niacin, lomitapide, mipomersen and LDL apheresis are treatments that can lower LDL cholesterol levels. Early treatment can lead to substantial reduction of cardiovascular events and death in patients with FH. It is important to increase awareness of this disorder in physicians and patients in order to reduce the burden of this disorder. PMID:25939291

  3. Familial hypercholesterolemia

    PubMed Central

    Turgeon, Ricky D.; Barry, Arden R.; Pearson, Glen J.

    2016-01-01

    Objective To summarize the pathophysiology, epidemiology, screening, diagnosis, and treatment of familial hypercholesterolemia (FH). Quality of evidence A PubMed search was conducted (inception to July 2014) for articles on pathophysiology, screening, diagnosis, and management of FH, supplemented with hand searches of bibliographies of guidelines and reviews. A supporting level of evidence for each recommendation was categorized as level I (randomized controlled trial or systematic review of randomized controlled trials), level II (observational study), or level III (expert opinion). The best available evidence is mostly level II or III. Main message Familial hypercholesterolemia affects 1 in 500 Canadians. Risk of a coronary event is high in these patients and is underestimated by risk calculators (eg, Framingham). Clinicians should screen patients according to guidelines and suspect FH in any patient with a premature cardiovascular event, physical stigmata of hypercholesterolemia, or an elevated plasma lipid level. Physicians should diagnose FH using either the Simon Broome or Dutch Lipid Network criteria. Management of heterozygous FH includes reducing low-density lipoprotein levels by 50% or more from baseline with high-dose statins and other lipid-lowering agents. Clinicians should refer any patient with homozygous FH to a specialized centre. Conclusion Familial hypercholesterolemia represents an important cause of premature cardiovascular disease in Canadians. Early identification and aggressive treatment of individuals with FH reduces cardiovascular morbidity and mortality. PMID:26796832

  4. FAMILY MYCETOPHILIDAE.

    PubMed

    Oliveira, Sarah Siqueira; Amorim, Dalton De Souza

    2016-06-14

    The Mycetophilidae include small fungus-gnats which life cycle is associated with fungi, especially of the larvae. The known diversity of the family in the Neotropical region is 1,145 species, but only some very few papers have been published on the Colombian species of Mycetophilidae, with records for the genera Docosia Winnertz, Paraleia Tonnoir, and Dziedzickia Johannsen. This catalogue gathers the information available on mycetophilids from Colombia, including genera and some species that for the first time are mentioned to occur in the country-as Leiella unicincta Edwards and Leiella zonalis Edwards.

  5. Family and family therapy in the Netherlands.

    PubMed

    Wagenaar, Karin; Baars, Jan

    2012-04-01

    This article describes how families are functioning in the Netherlands, and how family therapy is used in mental healthcare. In the open Dutch society, new ideas are easily incorporated, as exemplified by the rapid introduction and growth of family therapy in the 1980s. In recent decades, however, family therapy has lost ground to other treatment models that are more individually orientated, and adhere to stricter protocols. This decline of family therapy has been exacerbated by recent budget cuts in mental healthcare. In regular healthcare institutes family therapy now has a marginal position at best, although family treatment models are used in specific areas such as forensic treatments. In addition, the higher trained family therapists have found their own niches to work with couples and families. We argue that a stronger position of family therapy would be beneficial for patients and for families, in order to counteract the strong individualization of Dutch society. PMID:22515464

  6. Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures

    PubMed Central

    Rodas-Junco, Beatriz A; Cab-Guillen, Yahaira; Muñoz-Sanchez, J Armando; Vázquez-Flota, Felipe; Monforte-Gonzalez, Miriam; Hérnandez-Sotomayor, S M Teresa

    2013-01-01

    Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

  7. Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures.

    PubMed

    Rodas-Junco, Beatriz A; Cab-Guillén, Yahaira; Muñoz-Sánchez, J Armando; Vázquez-Flota, Felipe; Monforte-González, Miriam; Hernández-Sotomayor, S M Teresa

    2013-10-01

    Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

  8. Roles within the Family

    MedlinePlus

    ... Spread the Word Shop AAP Find a Pediatrician Family Life Medical Home Family Dynamics Adoption & Foster Care ... Text Size Email Print Share Roles Within the Family Page Content Article Body Families are not democracies. ...

  9. The Family Hero in Black Alcoholism Families.

    ERIC Educational Resources Information Center

    Brisbane, Francis L.

    1989-01-01

    Uses data from 20 case studies of Black adult female children of alcoholic parents to discuss Family Hero role often assumed by oldest or only female child in Black alcoholism families. Explains how female-dominated survival role of Family Hero in Black families is significantly more related to racial and cultural factors than numbers alone may…

  10. Integrating Family Resilience and Family Stress Theory.

    ERIC Educational Resources Information Center

    Patterson, Joan M.

    2002-01-01

    The construct, family resilience, is defined differently by practitioners and researchers. This study tries to clarify the concept of family resilience. The foundation is family stress and coping theory, particularly the stress models that emphasize adaptation processes in families exposed to major adversities. (JDM)

  11. Whole Family: Whole Child. Broken Family.

    ERIC Educational Resources Information Center

    DeVaul, Sue; Davis, John U.

    A literature review on the family environments of gifted students found that gifted children are more likely to be living in intact families than in divorced families. Children of single parents were more likely to be low-achieving, tardy, absent, truant, discipline problems, suspended, expelled, and dropouts than students in two-parent families.…

  12. Final Evaluation Report: Family to Family Program.

    ERIC Educational Resources Information Center

    Ramey, Luellen; Meyer, David P.

    This evaluation report of the Family to Family Program assesses parental attitudes towards their Family to Family experience and the functioning of their emotionally impaired children. It reviews issues of goal achievement; the impact on the targeted problem; service population demographics; and sustainability. Related topics include…

  13. Family Psychology and Family Therapy in Japan.

    ERIC Educational Resources Information Center

    Kameguchi, Kenji; Murphy-Shigematsu, Stephen

    2001-01-01

    Reviews the development of family psychology and family therapy in Japan, tracing the origins of these movements, explaining how these fields were activated by the problem of school refusal, and describing an approach to family therapy that has been developed to work with families confronting this problem, as well as preventive programs of family…

  14. Reclaiming Family Privilege

    ERIC Educational Resources Information Center

    Seita, John

    2012-01-01

    The pull for family is strong, almost primeval, most likely it is evolutionary, and for those lacking the benefit of family or Family Privilege, the loss of family is painful and profoundly sad. Young people who struggle to cope without stable family connections are profoundly aware of their lack of "Family Privilege." In this article, the author…

  15. Family and family therapy in Russia.

    PubMed

    Bebtschuk, Marina; Smirnova, Daria; Khayretdinov, Oleg

    2012-04-01

    This article represents the information about family and family therapy in the context of culture, traditions and contemporary changes of social situations in Russia. The legislation of family rights are mentioned within items about marriage and family in the Constitution, Civil Code and Family Code of the Russian Federation which has changed during recent years. The definition of family and description of family structure are given through the prism of the current demographic situation, dynamics of statistics of marriage and divorce rates, mental disorders, disabilities and such phenomena as social abandonment. The actual curriculum, teaching of family therapy and its disadvantages, system of continuous education, supervision and initiatives of the Institute of Integrative Family Therapy in improvement of preparing of specialists who can provide qualified psychosocial assistance for the family according to the actual needs of society are noted. The directions of state and private practice of family counselling and therapy both for psychiatric patients and medical patients, for adults and children in a family systemic approach are highlighted with an indication of the spectrum of techniques and methods used by Russian professionals. The main obstacles and perspectives of development of family therapy in Russia are summarized. PMID:22515460

  16. Family and family therapy in Russia.

    PubMed

    Bebtschuk, Marina; Smirnova, Daria; Khayretdinov, Oleg

    2012-04-01

    This article represents the information about family and family therapy in the context of culture, traditions and contemporary changes of social situations in Russia. The legislation of family rights are mentioned within items about marriage and family in the Constitution, Civil Code and Family Code of the Russian Federation which has changed during recent years. The definition of family and description of family structure are given through the prism of the current demographic situation, dynamics of statistics of marriage and divorce rates, mental disorders, disabilities and such phenomena as social abandonment. The actual curriculum, teaching of family therapy and its disadvantages, system of continuous education, supervision and initiatives of the Institute of Integrative Family Therapy in improvement of preparing of specialists who can provide qualified psychosocial assistance for the family according to the actual needs of society are noted. The directions of state and private practice of family counselling and therapy both for psychiatric patients and medical patients, for adults and children in a family systemic approach are highlighted with an indication of the spectrum of techniques and methods used by Russian professionals. The main obstacles and perspectives of development of family therapy in Russia are summarized.

  17. The Changing Family Structure.

    ERIC Educational Resources Information Center

    Bernard van Leer Foundation Newsletter, 1993

    1993-01-01

    This newsletter issue contains feature articles and short reports on how and why family structures are undergoing substantial change in many parts of the world. These articles include: (1) "The Changing Family Structure," a review of how families are changing and why; (2) "Peru: Families in the Andes"; (3) "Thailand: Families of the Garbage Dump";…

  18. Family Literacy and ESL.

    ERIC Educational Resources Information Center

    Red, David L.

    2003-01-01

    Discusses family literacy and English as a Second Language, focusing on types of family literacy programs, issues in family literacy, and future directions in family literacy. Highlights one program and lists the components of three approaches to family literacy: intervention prevention, multiple literacies, and social change. (Author/VWL)

  19. Family Reading Night

    ERIC Educational Resources Information Center

    Hutchins, Darcy; Greenfeld, Marsha; Epstein, Joyce

    2007-01-01

    This book offers clear and practical guidelines to help engage families in student success. It shows families how to conduct a successful Family Reading Night at their school. Family Night themes include Scary Stories, Books We Love, Reading Olympics, Dr. Seuss, and other themes. Family reading nights invite parents to come to school with their…

  20. Family Capital: Implications for Interventions with Families

    ERIC Educational Resources Information Center

    Belcher, John R.; Peckuonis, Edward V.; Deforge, Bruce R.

    2011-01-01

    Social capital has been extensively discussed in the literature as building blocks that individuals and communities utilize to leverage system resources. Similarly, some families also create capital, which can enable members of the family, such as children, to successfully negotiate the outside world. Families in poverty confront serious…

  1. Improving Family Communications

    MedlinePlus

    ... Spread the Word Shop AAP Find a Pediatrician Family Life Medical Home Family Dynamics Adoption & Foster Care ... Listen Español Text Size Email Print Share Improving Family Communications Page Content Article Body How can I ...

  2. Normal Functioning Family

    MedlinePlus

    ... Spread the Word Shop AAP Find a Pediatrician Family Life Medical Home Family Dynamics Adoption & Foster Care ... Español Text Size Email Print Share Normal Functioning Family Page Content Article Body Is there any way ...

  3. Family Reunion Health Guide

    MedlinePlus

    ... Phone (Continued) 1. Send a Kidney Health Message Hi Family, I came across this information and thought ... mails to family members. Before the Reunion 1. Hi family! Taking care of your kidneys is important. ...

  4. Family Activities for Fitness

    ERIC Educational Resources Information Center

    Grosse, Susan J.

    2009-01-01

    This article discusses how families can increase family togetherness and improve physical fitness. The author provides easy ways to implement family friendly activities for improving and maintaining physical health. These activities include: walking, backyard games, and fitness challenges.

  5. Developing Strengths in Families

    ERIC Educational Resources Information Center

    Bowman, Ted

    1976-01-01

    There are few descriptions of growth experiences for total families. This paper describes one such model. It expresses the conviction that families need opportunities to come together with other families to identify strengths, sharpen communication skills, and establish goals. (Author)

  6. Families and family therapy in Hong Kong.

    PubMed

    Tse, Samson; Ng, Roger M K; Tonsing, Kareen N; Ran, Maosheng

    2012-04-01

    Family therapy views humans not as separate entities, but as embedded in a network of relationships, highlighting the reciprocal influences of one's behaviours on one another. This article gives an overview of family demographics and the implementation of family therapy in Hong Kong. We start with a review of the family demographics in Hong Kong and brief notes on families in mainland China. Demographics show that the landscape has changed markedly in the past decade, with more cross-border marriages, an increased divorce rate, and an ageing overall population - all of which could mean that there is increasing demand for professional family therapy interventions. However, only a limited number of professionals are practising the systems-based approach in Hong Kong. Some possible reasons as to why family therapy is not well disseminated and practised are discussed. These reasons include a lack of mental health policy to support family therapy, a lack of systematic family therapy training, and a shortage of skilled professionals. Furthermore, challenges in applying the western model in Chinese culture are also outlined. We conclude that more future research is warranted to investigate how family therapy can be adapted for Chinese families.

  7. Families and family therapy in Hong Kong.

    PubMed

    Tse, Samson; Ng, Roger M K; Tonsing, Kareen N; Ran, Maosheng

    2012-04-01

    Family therapy views humans not as separate entities, but as embedded in a network of relationships, highlighting the reciprocal influences of one's behaviours on one another. This article gives an overview of family demographics and the implementation of family therapy in Hong Kong. We start with a review of the family demographics in Hong Kong and brief notes on families in mainland China. Demographics show that the landscape has changed markedly in the past decade, with more cross-border marriages, an increased divorce rate, and an ageing overall population - all of which could mean that there is increasing demand for professional family therapy interventions. However, only a limited number of professionals are practising the systems-based approach in Hong Kong. Some possible reasons as to why family therapy is not well disseminated and practised are discussed. These reasons include a lack of mental health policy to support family therapy, a lack of systematic family therapy training, and a shortage of skilled professionals. Furthermore, challenges in applying the western model in Chinese culture are also outlined. We conclude that more future research is warranted to investigate how family therapy can be adapted for Chinese families. PMID:22515459

  8. Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes.

    PubMed

    Zapata-Martínez, J; Medina, M F; Gramajo-Bühler, M C; Sánchez-Toranzo, G

    2016-08-01

    Rhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.

  9. NFAT regulates calcium-sensing receptor-mediated TNF production

    SciTech Connect

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  10. Identification of membrane dipeptidase as a major glycosyl-phosphatidylinositol-anchored protein of the pancreatic zymogen granule membrane, and evidence for its release by phospholipase A.

    PubMed

    Hooper, N M; Cook, S; Lainé, J; Lebel, D

    1997-05-15

    Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin-Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.

  11. Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

    SciTech Connect

    Haas, R.; Marshall, T.L.; Rosenberry, T.L.

    1988-08-23

    The presence of a glycoinositol phospholipid anchor Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine ((/sup 125/I)TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The (/sup 125/)I)TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine.

  12. Cytodifferentiation in Tetrahymena vorax is linked to glycosyl-phosphatidylinositol-anchored protein assembly.

    PubMed

    Yang, X; Ryals, P E

    1994-03-15

    The role of glycosyl-PtdIns (GPI)-anchored proteins in the cytodifferentiation of Tetrahymena vorax was examined. Labelling of cells with [3H]myristate or [3H]palmitate followed by electrophoresis showed an array of proteins carrying covalently bound lipids. Electrophoresis of protein from cells labelled with the GPI-anchor components [3H]Ins and [14C]ethanolamine revealed three polypeptides on fluorograms which have apparent molecular masses of approx. 28, 50 and 82 kDa. Labelled lipid associated with these polypeptides was susceptible to release by in vitro exposure to Bacillus thuringiensis PtdIns-specific phospholipase C (PI-PLC). Using labelled fatty acids, cells induced to differentiate showed altered GPI-anchored protein-labelling patterns in comparison with undifferentiated control cells, with a heavily labelled 32 kDa band appearing upon differentiation. Pre-incubation of cells in 10 mM D-mannosamine, an inhibitor of GPI incorporation into protein, resulted in a reduction of the incorporation of label into the three GPI-anchored proteins, nearly complete inhibition of differentiation and a reduction in the rate of digestive vacuole formation. A 50% inhibition of differentiation was obtained using 500 microM mannosamine. The inhibitory impact of D-mannosamine on differentiation could be competitively and completely reversed by the inclusion of D-mannose, but not D-glucose. Neither glucosamine nor tunicamycin inhibited differentiation. Incubation of cells in PI-PLC (5 units/ml) plus the differentiation inducer resulted in an acceleration of differentiation and generally higher percentages of differentiated cells versus controls.

  13. Molecular cloning of gp42, a cell-surface molecule that is selectively induced on rat natural killer cells by interleukin 2: glycolipid membrane anchoring and capacity for transmembrane signaling

    PubMed Central

    1991-01-01

    We have previously shown that in vitro culture of rat natural killer (NK) cells in high concentrations of recombinant interleukin 2 (rIL-2) leads to the expression of a surface glycoprotein with a molecular mass of approximately 42 kD. This glycoprotein, gp42, is not induced on other lymphocytes and thus provides a lineage-specific marker for rIL-2- activated NK cells. We here present the nucleotide sequence for gp42 cDNA. The open reading frame encodes 233 amino acids with three potential sites for N-linked glycosylation. The deduced amino acid sequence lacks an apparent transmembrane domain and instead contains a hydrophobic COOH terminus that is characteristic of glycosylphosphatidylinositol (GPI)-anchored surface proteins. Consistent with this, gp42 is cleaved from the NK-like cell line, RNK- 16, by phosphatidylinositol-specific phospholipase C (PI-PLC), as is gp42 expressed on CHO cells that have been transformed with gp42 cDNA. On rIL-2-activated NK cells, gp42 is resistant to PI-PLC, though our studies suggest that gp42 on these cells is still expressed as a GPI- anchored molecule. Antibody to gp42 stimulates in RNK-16 cells an increase in inositol phosphates and in intracellular calciu, signals that are associated with the activation of lymphocytes, including NK cells. rIL-2-activated NK cells, however, lack this response to gp42 as well as to other stimuli. Thus, gp42, the only NK-specific activation antigen, is a GPI-anchored surface molecule with the capacity to stimulate transmembrane signaling. PMID:1845873

  14. Making Time for Family.

    ERIC Educational Resources Information Center

    Newman, Rita

    1998-01-01

    Reviews Covey's "7 Habits of Effective Families," noting that time plays an important part in each habit. Discusses several important "time" issues for families: time to plan for time, time for awareness of quality time, time to have two-way communication, time for family moments, and time to reflect. Includes suggestions for family activities in…

  15. Families in Transition .

    ERIC Educational Resources Information Center

    Bundy, Michael L., Ed.; Gumaer, James, Ed.

    1984-01-01

    Focuses on disrupted families and the role of the school counselor in helping children adjust. Describes characteristics of healthy families, and discusses the transition to the blended family, effects of divorce groups on children's classroom behavior, counseling children in stepfamilies, single-parent families, and parenting strengths of single…

  16. Black Families. Third Edition.

    ERIC Educational Resources Information Center

    McAdoo, Harriette Pipes, Ed.

    The chapters of this collection explore the experiences of black families in the United States and Africa, today and in the past. They are: (1) "African American Families: A Historical Note" (John Hope Franklin); (2) "African American Families and Family Values" (Niara Sudarkasa); (3) "Old-Time Religion: Benches Can't Say 'Amen'" (William Harrison…

  17. Gavin Families. A Report.

    ERIC Educational Resources Information Center

    Weaver, Paul Neufeld; And Others

    The Family Literacy Project at the Dr. Charles E. Gavin School in Chicago Heights, Illinois, brings together a community college adult education program, an early childhood program, and a local school district. This report of the Prairie State College Family Literacy Project assesses the needs for family literacy among the families in the Gavin…

  18. Familial Ebstein's anomaly.

    PubMed Central

    Rosenmann, A; Arad, I; Simcha, A; Schaap, T

    1976-01-01

    A family is described in which both a father and son are affected with Ebstein's anomaly, while several other family members manifest different cardiac malformations. Five additional instances of familial Ebstein's anomaly were found in the literature and compared with our family. Inspection of possible modes of inheritance in this group of families suggests that Ebstein's anomaly is probably inherited as a polygenic character with a threshold phenomenon. PMID:1018315

  19. Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53/56Lyn and a 120-kDa phosphoprotein.

    PubMed Central

    Sidorenko, S P; Law, C L; Chandran, K A; Clark, E A

    1995-01-01

    The 72-kDa spleen tyrosine kinase (Syk) and Src-family kinase p53/56Lyn (Lyn) contribute to signaling via the B-cell antigen receptor complex. Here we show that Syk and Lyn from human B lymphocytes can interact directly. Syk and Lyn coimmunoprecipitated from mature and activated B-cell lines, and gel-purified Syk and Lyn reassociated in vitro, demonstrating their direct interaction. This Syk-Lyn interaction may be dependent on the stage of B-cell differentiation, since Syk-Lyn associations were not detected in pre-B and myeloma cell lines and Syk from an immature B-cell line did not reassociate with Lyn in vitro. Serine/threonine kinase activity was also associated with Syk. Crosslinking of cell surface IgM led to rapid activation of both tyrosine and serine/threonine protein kinase activities that resulted in phosphorylation in vitro of proteins coprecipitating with Syk--in particular, a serine/threonine phosphorylated protein 120 kDa in size (pp120). Several phosphoproteins, including one of 72 kDa and one of 120 kDa, coprecipitated with phospholipase C-gamma 1 (PLC gamma 1). Sequential immunoprecipitation identified the 72-kDa protein associated with PLC gamma 1 as Syk. The 120-kDa serine/threonine phosphorylated protein that coprecipitated with PLC gamma 1 resembled the Syk-associated pp120 by several criteria. Thus, pp120 may serve as a link between Syk and PLC gamma 1, coupling the B-cell antigen receptor to the phosphatidylinositol pathway. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7831290

  20. Nontraditional family romance.

    PubMed

    Corbett, K

    2001-07-01

    Family stories lie at the heart of psychoanalytic developmental theory and psychoanalytic clinical technique, but whose family? Increasingly, lesbian and gay families, multiparent families, and single-parent families are relying on modern reproductive technologies to form families. The contemplation of these nontraditional families and the vicissitudes of contemporary reproduction lead to an unknowing of what families are, including the ways in which psychoanalysts configure the family within developmental theory. This article focuses on the stories that families tell in order to account for their formation--stories that include narratives about parental union, parental sexuality, and conception. The author addresses three constructs that inform family stories and that require rethinking in light of the category crises posed by and for the nontraditional family: (1) normative logic, (2) family reverie and the construction of a family romance, and (3) the primal scene. These constructs are examined in tandem with detailed clinical material taken from the psychotherapy of a seven-year-old boy and his two mothers.

  1. Nontraditional family romance.

    PubMed

    Corbett, K

    2001-07-01

    Family stories lie at the heart of psychoanalytic developmental theory and psychoanalytic clinical technique, but whose family? Increasingly, lesbian and gay families, multiparent families, and single-parent families are relying on modern reproductive technologies to form families. The contemplation of these nontraditional families and the vicissitudes of contemporary reproduction lead to an unknowing of what families are, including the ways in which psychoanalysts configure the family within developmental theory. This article focuses on the stories that families tell in order to account for their formation--stories that include narratives about parental union, parental sexuality, and conception. The author addresses three constructs that inform family stories and that require rethinking in light of the category crises posed by and for the nontraditional family: (1) normative logic, (2) family reverie and the construction of a family romance, and (3) the primal scene. These constructs are examined in tandem with detailed clinical material taken from the psychotherapy of a seven-year-old boy and his two mothers. PMID:11491437

  2. Putting the "family" back into family therapy.

    PubMed

    Breunlin, Douglas C; Jacobsen, Elizabeth

    2014-09-01

    In this article, we examine the field of family therapy by drawing a distinction between two forms of practice: Whole Family Therapy (WFT), defined as treating the whole family, and Relational Family Therapy (RFT), defined as working with a subsystem of the family or an individual while retaining a systemic lens. Our thesis is that the practice of WFT has been in decline for some time and steps must be taken to keep it from becoming a defunct practice. We consider the trajectory of WFT and RFT throughout the development of family therapy through reference to the people, the literature, training, and practice patterns associated with family therapy. We remind the reader of the many benefits of WFT and suggest that today WFT is likely to be practiced in conjunction with RFT and individual therapy. Since training of family therapists today is largely located in degree-granting programs, we identify constraints to including WFT in such programs. We conclude by offering suggestions that can enhance a program's ability to train students in WFT.

  3. Family Synergy, A Variant Family Organization

    ERIC Educational Resources Information Center

    LaFollette, Patrick

    1975-01-01

    This article describes "Family Synergy" which is a non-profit organization for people interested in or exploring nontraditional marriage and family forms. It acts as a clearinghouse for the exchange of ideas and also provides a supportive community for people actually exploring these new lifestyles. (Author)

  4. Families and Family Study in International Perspective

    ERIC Educational Resources Information Center

    Adams, Bert N.

    2004-01-01

    Many changes are occurring in the world's families. Some observers feel that the changes are destructive, whereas others see them as leading to new opportunities and understanding. Issues in international family studies include regional limitations and the various aspects of doing research cross-culturally. Knowledge regarding certain categories…

  5. Invest in Family*

    PubMed Central

    Shah, Nilesh; De Sousa, Avinash

    2015-01-01

    The family is an integral part of one's life. It is very essential that every individual employed or unemployed invests time therein. The family is a source of support and growth for an individual, and the lack of family support or loneliness may be a causative factor in the genesis of psychiatric disorders, especially depression. In India, family plays a paramount role when it comes to mental health of the individual. Tips on how one should invest time in one's family along with the role of a family in one's personal and social structure are discussed. PMID:25838732

  6. Familial myeloproliferative disease.

    PubMed

    Gilbert, H S

    1998-12-01

    The occurrence of one or more myeloproliferative disease (MPD) syndromes in 42 families is described. MPD appeared in a single generation in 10 families, two generations in 30 families and three generations in two families. In contrast to sparse case reports of familial polycythaemia vera, familial essential thrombocythaemia, or familial agnogenic myeloid metaplasia, in which all the involved members presented with the same MPD, 21 of the 42 families in the present series had members who presented with different MPD variants. The occurrence of multiple disease phenotypes in 'MPD families' is entirely consistent with the accepted theory of MPD as a disease arising from clonal expansion of a pluripotential haematopoietic precursor cell (PHPC) that retains its pluripotentiality and produces an array of inter-related syndromes, each named for the predominant haematic cell type involved in the proliferation. Changes in disease phenotype during the course of MPD and 'hybrid' phenotypes at the time of diagnosis are common. This report challenges the previously accepted belief that PV and other MPD variants are sporadic and randomly-occurring, and that familial occurrence of MPD is rare. The ability to identify 'MPD families' by surveying a large population of patients with MPD through the Internet, as was done in this study, and heightened awareness of familial occurrence and its phenotypic heterogeneity, should facilitate further characterization of the mode of inheritance in familial MPD and the nature of the gene mutations responsible for the dysregulation of haematopoiesis.

  7. Familial Pulmonary Fibrosis

    MedlinePlus

    ... are here: Health Information > Condition Information Familial Pulmonary Fibrosis: Overview When two or more members within the ... Associate Professor View full profile More Familial Pulmonary Fibrosis Information Forms Causes Genetic Counseling Print Page Email ...

  8. Evaluation of family therapy.

    PubMed

    Nielsen, Nanna

    2006-01-01

    Because children of mentally disordered parents have an increased risk of becoming subjects of mental disorder and psychological problems, adult psychiatry has increasingly paid attention to the patients' parental skills. This study includes 31 families with children younger than 18 years of age where the parents were admitted to family therapy in an open psychiatric ward. The average number of sessions was seven, and the diagnoses of severe stress and of adjustment disorder were the most common. The aim of the study was to measure changes in the family climate during the period of therapy. The instrument used was the Family Environment Scale (FES), a self-rating questionnaire that measures the parents' experience of family environment. The change of family environment is related to its influence on children's well-being and development. The study finds that family therapy has resulted in positive changes of the parents' experience of the family climate.

  9. Normative Family Development.

    ERIC Educational Resources Information Center

    Barcai, Avner

    1981-01-01

    Describes the sequentially developmental life stages of healthy, normal families. Provides an exposition of these developmental stages and forms as a guide or normative framework within which to test for dysfunction and pathology in the family process. (Author/JAC)

  10. MSUD Family Support Group

    MedlinePlus

    ... Group The MSUD Family Support Group is a non-profit 501 (c)(3) organization for those with MSUD ... Family Support Group is a 501(c)(3) non-profit organization with no paid staff. Funds are needed ...

  11. Assessing postpartum family functioning.

    PubMed

    Midmer, D; Talbot, Y

    1988-09-01

    The birth of a child requires adaptation and reorganization within the family system in order to accommodate the new family member and to allow the family to continue in its psychosocial development. Knowledge of the normative and transitional changes required at this stage of family life will enhance family practitioners' understanding of some of the common concerns and complaints related to them by various family members during the postpartum period. The Family FIRO model represents a helpful conceptual framework to increase the family physician's understanding of the issues of inclusion, control, and intimacy that are highlighted during the transition to parenthood. The authors briefly present this model and discuss its application to postpartum adjustment and its implications for health-care professionals.

  12. Assessing Postpartum Family Functioning

    PubMed Central

    Midmer, Deana; Talbot, Yves

    1988-01-01

    The birth of a child requires adaptation and reorganization within the family system in order to accommodate the new family member and to allow the family to continue in its psychosocial development. Knowledge of the normative and transitional changes required at this stage of family life will enhance family practitioners' understanding of some of the common concerns and complaints related to them by various family members during the postpartum period. The Family FIRO model represents a helpful conceptual framework to increase the family physician's understanding of the issues of inclusion, control, and intimacy that are highlighted during the transition to parenthood. The authors briefly present this model and discuss its application to postpartum adjustment and its implications for health-care professionals. PMID:21253238

  13. Familial Periodic Paralyses

    MedlinePlus

    ... NINDS NINDS Familial Periodic Paralyses Information Page Synonym(s): Periodic Paralyses Table of Contents (click to jump to sections) What are Familial Periodic Paralyses? Is there any treatment? What is the ...

  14. Importance of Family Routines

    MedlinePlus

    ... Listen Español Text Size Email Print Share The Importance of Family Routines Page Content ​Every family needs ... child to sleep. These rituals can include storytelling, reading aloud, conversation, and songs. Try to avoid exciting ...

  15. Year of the Family.

    ERIC Educational Resources Information Center

    California Agriculture, 1994

    1994-01-01

    This special issue focuses on problems and challenges confronting the California family and on research and extension efforts to provide at least partial answers. Research briefs by staff include "Challenges Confront the California Family" (state trends in poverty, divorce, single-parent families, child abuse, delinquency, teen births, limited…

  16. The Family Leukemia Association

    ERIC Educational Resources Information Center

    Pollitt, Eleanor

    1976-01-01

    An association of families of children with leukemia, the Family Leukemia Association (FLA), was recently established in Toronto. This paper discusses (a) philosophy of the FLA; (b) formative years of this organization; (c) problems encountered by leukemic children and their families; and (d) the FLA's past and future educational and social…

  17. Focus on the Family.

    ERIC Educational Resources Information Center

    Hardy, James M.

    This research attempts to evaluate the YMCA's program in terms of its effect upon the family members it serves. The study was designed to: (1) classify, by descriptive types, the various kinds of YMCA operations which serve the family, identifying their characteristic differences; (2) examine and describe operating practices of family YMCAs…

  18. Families in Multicultural Perspective.

    ERIC Educational Resources Information Center

    Ingoldsby, Bron B., Ed.; Smith, Suzanna, Ed.

    Covering contemporary Third World as well as Western families, this teaching text addresses topics essential for developing a multicultural perspective on the family. It is an ideal text for comparative family courses and includes exercises (as well as exercise guidelines for instructors) developed to challenge students' existing viewpoints and…

  19. Valuing Families. Activity Guide.

    ERIC Educational Resources Information Center

    Glashagel, Jerry; Glashagel, Char

    Developed as a resource for family life education, this activity guide can be used to lead experiential learning situations for intergenerational groups by a counselor, in a course, in a family organization like the YMCA, or in the home. The goals of this guide are to increase the self-esteem of each person and to strengthen the family as a human…

  20. Family Planning & Literacy.

    ERIC Educational Resources Information Center

    International Planned Parenthood Federation, London (England).

    This publication is an International Planned Parenthood Federation (IPPF) annotated bibliography of books and articles concerned with family planning and literacy. The subject is divided into four major listings: (1) Literacy; (2) Education; (3) Literacy and Family Planning; and (4) Functional Literacy/Family Planning Projects and Programs.…

  1. Fatherhood and Family Support.

    ERIC Educational Resources Information Center

    Goetz, Kathy, Ed.

    1996-01-01

    On the assumption that fathers have been relatively absent from family support programs, this publication of the Family Resource Coalition addresses the role of fathers in family support programs, examines the impact of fathers on their children, and describes programs involving fathers successfully. Articles include: (1) "What's Behind the…

  2. Doing Better for Families

    ERIC Educational Resources Information Center

    OECD Publishing (NJ3), 2011

    2011-01-01

    All OECD governments want to give parents more choice in their work and family decisions. This book looks at the different ways in which governments support families. It seeks to provide answers to questions like: Is spending on family benefits going up, and how does it vary by the age of the child? Has the crisis affected public support for…

  3. Changing Family Forms.

    ERIC Educational Resources Information Center

    Seibert, M. Therese; Willetts, Marion C.

    2000-01-01

    Explores the definition of family. Considers three facets of the contemporary family measured by U.S. Census statistics: (1) marriage and divorce trends; (2) declining fertility; and (3) the rise in single-headed families. Addresses the societal changes (economic, cultural, legal, and technological) that have influenced the changes in family…

  4. The Resiliency of Families.

    ERIC Educational Resources Information Center

    Morrison, T. R.

    According to researchers, the family may be changing but it is still one of the central institutions in society. Studies report a shift in more than 20 attitudes and values, most of which relate to the context of family life. Specifically, these include attitudes toward marriage, divorce, childbearing, childrearing, working women, family violence,…

  5. Rape: A Family Crisis.

    ERIC Educational Resources Information Center

    White, Priscilla N.; Rollins, Judith C.

    1981-01-01

    Rape is a crisis shared by the victim and her family. The family's reaction is influenced by cultural views such as viewing rape as sex rather than violence. Adaptive responses can be supported by open expression, education, and family, as well as individual counseling. (JAC)

  6. Therapy with African Families.

    ERIC Educational Resources Information Center

    Nwadiora, Emeka

    1996-01-01

    Informs helping professionals about the unique history and challenges of African families to guide them toward providing ethnically sensitive psychological services to African immigrant families in need. African families undergo great stress when faced with the alienation of being Black and African in a Euro-American culture. (SLD)

  7. Treatment of violent families.

    PubMed Central

    Bell, C. C.; Chance-Hill, G.

    1991-01-01

    Family violence is responsible for a significant proportion of homicides, a major cause of premature deaths in African-Americans. This article reviews the prevalence of family violence and explores associated risk factors. Principles and tips of treatment, along with a cognitive framework to guide the actual therapy, are outlined. Finally, issues of preventing family violence are discussed. PMID:2038079

  8. Rape: A Family Crisis.

    ERIC Educational Resources Information Center

    Feinauer, Leslie

    1982-01-01

    Suggests that, although a young woman who has been sexually assaulted may experience pain and alienation as an individual, family members also experience trauma, often left unresolved while retaining an impact on the family's ability to function. Introduces family therapy as a desirable approach to treatment of the rape victim. (Author/JAC)

  9. Strengths of Remarried Families.

    ERIC Educational Resources Information Center

    Knaub, Patricia Kain; And Others

    1984-01-01

    Focuses on remarried families' (N=80) perceptions of family strengths, marital satisfaction, and adjustment to the remarried situation. Results indicated that although most would like to make some changes, scores on the measurements used were high. A supportive environment was the most important predictor of family strength and success. (JAC)

  10. Families in Transition.

    ERIC Educational Resources Information Center

    Britton, Patti O., Ed.; McGee, Michael, Ed.

    1987-01-01

    This issue of "Emphasis" deals with families in transition, providing some model programs for the new family and some historical perspectives on how families have developed over time. Articles include: (1) "Nostalgia on the Right" (Nancy Theriot); (2) "Heart to Heart" (Nancy Harrington-MacLennan); (3) "The Media Get the Message" (Janet Alyn); (4)…

  11. Family Violence: An Overview.

    ERIC Educational Resources Information Center

    National Center on Child Abuse and Neglect (DHHS/OHDS), Washington, DC.

    Family violence is a widespread problem; research has shown multiple factors are associated with family violence. Types of family violence include spouse abuse; elder abuse and neglect; child abuse and neglect; parent abuse; and sibling abuse. There are three types of spouse abuse: physical abuse, sexual violence, and psychological/emotional…

  12. Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

    PubMed Central

    Kumari, Sudha; Depoil, David; Martinelli, Roberta; Judokusumo, Edward; Carmona, Guillaume; Gertler, Frank B; Kam, Lance C; Carman, Christopher V; Burkhardt, Janis K; Irvine, Darrell J; Dustin, Michael L

    2015-01-01

    Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin ‘foci’. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response. DOI: http://dx.doi.org/10.7554/eLife.04953.001 PMID:25758716

  13. [Men and family planning].

    PubMed

    Vieira, J G

    1993-01-01

    Family planning programs since their beginnings have focused exclusively on women. The importance of male participation in family planning has not been recognized. Today's society demands greater understanding and empathy between spouses, if they are to meet the new and difficult challenges of modern life. Incorporation of men into family planning programs is needed because of the deteriorating live conditions of a large segment of the population and the accelerating decomposition of social structures. Persuading men to participate in family planning should strengthen the couple and increase the probability that decisions about family size will be responsible. Strategies should be designed to interest men in family planning and increase their awareness of their role in fathering happy children who enter the world in more just and humane conditions. Such strategies must combat sex role socialization that begins in infancy. The assignment of responsibility for family planning to the woman excludes men from what should be a fundamental role.

  14. [Health and disadvantaged families.].

    PubMed

    Thibaudeau, M F

    1985-01-01

    Using data obtained from an evaluative study of experimental , health care given to families in two C.L.S.C, this article describes the functioning of a group on under priviledged families in two C.L.S.C. and the health both of the parents and their children below 10 years old. The sample of 85 families comprises an equal number of biparental and monoparental families. Three quarters of those families are on welfare and most of their living conditions are pitiable, half of the mothers come from broken families. Events of daily life involving stress are met with difficulty by most families. The shows that the mothers are under stress throughout the project. The children below 10 years old have high levels of school absenteism and display physical and psychological problems.

  15. [Familial pituitary tumors].

    PubMed

    Yoshimoto, K; Saito, S

    1995-11-01

    Familial pituitary tumors are relatively rare. Most commonly, they occur as a part of multiple endocrine neoplasia type 1 (MEN 1). However, familial pituitary adenomas unrelated MEN 1 (familial pituitary adenomas) are extremely rare. In review of MEN 1 in Japan, 60% of the patients with MEN 1 had pituitary tumors. Only 45 cases of familial pituitary adenomas have been reported from 20 families. In our review of familial pituitary adenomas, 30 (67%) of 45 reported cases are acromegaly or gigantism. This incidence is much higher than 28% in MEN 1 patients with pituitary tumors. Allelic deletions at 11q13 were identified in MEN 1 associated pituitary adenomas and familial pituitary adenomas in two gigantism brothers. PMID:8538028

  16. 24 CFR 982.515 - Family share: Family responsibility.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 4 2012-04-01 2012-04-01 false Family share: Family responsibility... Assistance Payment § 982.515 Family share: Family responsibility. (a) The family share is calculated by subtracting the amount of the housing assistance payment from the gross rent. (b) The family rent to owner...

  17. 24 CFR 982.515 - Family share: Family responsibility.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 4 2013-04-01 2013-04-01 false Family share: Family responsibility... Assistance Payment § 982.515 Family share: Family responsibility. (a) The family share is calculated by subtracting the amount of the housing assistance payment from the gross rent. (b) The family rent to owner...

  18. 24 CFR 982.515 - Family share: Family responsibility.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 4 2014-04-01 2014-04-01 false Family share: Family responsibility... Assistance Payment § 982.515 Family share: Family responsibility. (a) The family share is calculated by subtracting the amount of the housing assistance payment from the gross rent. (b) The family rent to owner...

  19. 24 CFR 982.515 - Family share: Family responsibility.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false Family share: Family responsibility... Assistance Payment § 982.515 Family share: Family responsibility. (a) The family share is calculated by subtracting the amount of the housing assistance payment from the gross rent. (b) The family rent to owner...

  20. 24 CFR 982.515 - Family share: Family responsibility.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Family share: Family responsibility... Assistance Payment § 982.515 Family share: Family responsibility. (a) The family share is calculated by subtracting the amount of the housing assistance payment from the gross rent. (b) The family rent to owner...

  1. Rethinking family power.

    PubMed

    Kranichfeld, M L

    1987-03-01

    The family power literature, in its macrolevel focus on marital decision-making, has emphasized the kind of family power that is generally conferred on men and is based on extrafamilial roles and performances. Kranichfeld argues that for the last 2 decades, reviews of family power literature have been fundamentally shaped by the abiding interest in the relative power of men and women, rather than in power in the family per se. Researchers have continued to assume that family power is generated by acquiring skills, resources, and status outside the family rather than by acquiring skills for relating to others within the family. Much of the family power literature has focused on marriage and marital decision-making, but Kranichfeld argues that it is power within the parent-child relationship that is more complex, enduring, and significant. An understanding of the depth and reach of the kind of power that women hold, taken together without knowledge of how men's relationships to families are changing in our society, allows the possibility that women's positions are more secure than they sometimes seem. Many of the painful life situations that women experience because their powerlessness at a more macro level of society (desertion, physical abuse, teenage pregnancy) occur within the context of the romantic tie, and women's investment and power in vertical bonds are a source of support that reduces women's dependence on and vulnerability to the horizontal tie. Women occupy positions at the very center of the family, in contrast to men, who are becoming increasingly isolated from the family. From this perspective, the normally male power to make the decision about what kind of car to buy or where to spend the family vacation is nearly reduced to insignificance. The message seems to be that when it comes to securing family power, nothing can substitute for investment, attention, connection, and care.

  2. Creating a Family Health History

    MedlinePlus

    ... Health History? Click for more information A Family Tree for Health A family health history is a ... family members grew up. It's like a family tree for health. Click for more information What a ...

  3. The Growth of a Family

    PubMed Central

    Carroll, June C.; Biringer, Anne

    1991-01-01

    Caring for a family during pregnancy and birth is an ideal opportunity for family physicians to assess family functioning and help the family adjust to the birth of a new child. Stress and support systems can influence the course of pregnancy, including obstetric and perinatal outcomes. A family-centered approach can help patients during this critical stage of family development. PMID:21229107

  4. Strengthening family systems.

    PubMed

    Starn, J

    1993-01-01

    The childbearing year is a psychosocial transition that involves changes in roles and status for each member of the family. This change is perhaps most significant in the transition that occurs with the birth of the first child. Thus, childbirth education can be looked upon as an opportunity to strengthen family systems through anticipatory guidance and skill building that family members may use throughout the life cycle.

  5. State of family planning.

    PubMed

    Schreiber, Courtney A; Traxler, Sarah

    2015-06-01

    Family planning and reproductive health services are uniquely impacted by policy and politics in the United States. Recent years have witnessed an unprecedented number of abortion restrictions, and research funding has decreased in related areas. Despite this, both the science and the implementation of improved family planning and abortion methods have progressed in the past decade. This article reviews the current state of family planning, as well as technologies and patient care opportunities for the future. PMID:25860324

  6. Asteroid family ages

    NASA Astrophysics Data System (ADS)

    Spoto, Federica; Milani, Andrea; Knežević, Zoran

    2015-09-01

    A new family classification, based on a catalog of proper elements with ∼384,000 numbered asteroids and on new methods is available. For the 45 dynamical families with >250 members identified in this classification, we present an attempt to obtain statistically significant ages: we succeeded in computing ages for 37 collisional families. We used a rigorous method, including a least squares fit of the two sides of a V-shape plot in the proper semimajor axis, inverse diameter plane to determine the corresponding slopes, an advanced error model for the uncertainties of asteroid diameters, an iterative outlier rejection scheme and quality control. The best available Yarkovsky measurement was used to estimate a calibration of the Yarkovsky effect for each family. The results are presented separately for the families originated in fragmentation or cratering events, for the young, compact families and for the truncated, one-sided families. For all the computed ages the corresponding uncertainties are provided, and the results are discussed and compared with the literature. The ages of several families have been estimated for the first time, in other cases the accuracy has been improved. We have been quite successful in computing ages for old families, we have significant results for both young and ancient, while we have little, if any, evidence for primordial families. We found 2 cases where two separate dynamical families form together a single V-shape with compatible slopes, thus indicating a single collisional event. We have also found 3 examples of dynamical families containing multiple collisional families, plus a dubious case: for these we have obtained discordant slopes for the two sides of the V-shape, resulting in distinct ages. We have found 2 cases of families containing a conspicuous subfamily, such that it is possible to measure the slope of a distinct V-shape, thus the age of the secondary collision. We also provide data on the central gaps appearing in

  7. Strengthening Families: Exploring the Impacts of Family Camp Experiences on Family Functioning and Parenting

    ERIC Educational Resources Information Center

    Garst, Barry A.; Baughman, Sarah; Franz, Nancy K.; Seidel, Richard W.

    2013-01-01

    Research suggests that family camp experiences can enhance family relationships. Families often participate in family camp experiences for a vacation, as part of a therapeutic and/or intervention strategy, or to gain general enrichment or engagement. To better understand the impacts of family camp experiences on family functioning, a mixed-methods…

  8. The Family Constellation Scale.

    ERIC Educational Resources Information Center

    Lemire, David

    The Family Constellation Scale (FC Scale) is an instrument that assesses perceived birth order in families. It can be used in counseling to help initiate conversations about various traits and assumptions that tend to characterize first-born, middle-born children, youngest-born, and only children. It provides both counselors and clients insights…

  9. America's Family Time Famine.

    ERIC Educational Resources Information Center

    Mattox, Jr., William R.

    1990-01-01

    Parents spend increasingly less time with their children because of the pressures of dual careers and single parenthood. Economic pressures and social values have affected sharing of family time. Studies show both parents and children consider spending time together the most important element in improving family life. (BC)

  10. A Family Disease.

    ERIC Educational Resources Information Center

    Harrington, Virginia Clarke

    1983-01-01

    Discusses alcoholism, its effect upon the children in the family, and what teachers can do to help children of alcoholics. Suggests that teachers be caring, nonjudgmental, and empathetic; learn what alcoholism is, how it affects the family, and how to identify affected children in classroom; and provide information on alcoholic help groups. (DMM)

  11. Families Falling Apart.

    ERIC Educational Resources Information Center

    Moynihan, Daniel Patrick

    1990-01-01

    Reviews trends in Black male unemployment, out-of-wedlock births, and the number of Aid to Families with Dependent Children cases over the past 25 years. Argues that family breakdown is creating a state of urban social chaos that could lead to martial law. (FMW)

  12. Uninsured Rural Families

    ERIC Educational Resources Information Center

    Ziller, Erika C.; Coburn, Andrew F.; Anderson, Nathaniel J.; Loux, Stephenie L.

    2008-01-01

    Context: Although research shows higher uninsured rates among rural versus urban individuals, prior studies are limited because they do not examine coverage across entire rural families. Purpose: This study uses the Medical Expenditure Panel Survey (MEPS) to compare rural and urban insurance coverage within families, to inform the design of…

  13. Marriage or Family Therapy.

    ERIC Educational Resources Information Center

    Haley, Jay

    1984-01-01

    Reviews the differences between family therapy and marriage counseling in terms of professional organization, theory, and practice. Suggests that training in marriage therapy does not appear adequate for family therapy. The goal of the therapy field should be more consensus in theory and a single profession of therapists. (JAC)

  14. Balancing Work & Family.

    ERIC Educational Resources Information Center

    Hutchinson Community Junior Coll., KS.

    This curriculum is based on what students need to know, to be able to do, and to be like in order to be competent in the work of the family. Each of the 12 units follows a uniform format that includes the following: perennial problem (one faced over and over by successive generations of families); practical problem (the organizing scheme for how…

  15. THE URBAN NEGRO FAMILY.

    ERIC Educational Resources Information Center

    DOUGLASS, JOSEPH H.

    IN TRACING THE MOVEMENT OF THE NEGRO FAMILY TOWARD A MIDDLE-CLASS ORIENTATION AND TOWARD URBANIZATION, THIS ARTICLE NOTES THAT THE PATTERN IS BECOMING SIMILAR TO THAT OF THE GENERAL AMERICAN FAMILY. NEGROES HAVE LEFT THEIR SOUTHERN RURAL FARMS FOR BOTH SOUTHERN AND NORTHERN URBAN AREAS AND HAVE TENDED TO SETTLE IN THE INNER CORE OF THE LARGEST…

  16. Balancing Family and Work.

    ERIC Educational Resources Information Center

    Yahnke, Sally; And Others

    The purpose of this monograph is to present a series of activities designed to teach strategies needed for effectively managing the multiple responsibilities of family and work. The guide contains 11 lesson plans dealing with balancing family and work that can be used in any home economics class, from middle school through college. The lesson…

  17. Therapy for Family Systems.

    ERIC Educational Resources Information Center

    Rosmann, Michael R.

    A family therapy model, based on a conceptualization of the family as a behavioral system whose members interact adaptively so that an optimal level of functioning is maintained within the system, is described. The divergent roots of this conceptualization are discussed briefly, as are the treatment approaches based on it. The author's model,…

  18. Marinating the Family.

    ERIC Educational Resources Information Center

    Hensel, Karen A.

    1982-01-01

    Describes the New York Aquarium's program specifically designed for family learning and teaching. The program's goal is to create an environment where child-parent roles are dropped and where the philosophy that no one of us is as smart as all of us prevails. Strategies for family involvement are outlined. (MH)

  19. Selecting Family Interventions.

    ERIC Educational Resources Information Center

    Watts, Richard E.

    Just as counseling approaches designed for individuals have their theory-specific techniques, family counseling approaches also have theory-specific interventions and strategies. Whatever presenting problem the family brings to counseling, one or more of four essential components (communication, problem solving, roles and boundaries) is typically…

  20. Closure Issues with Families.

    ERIC Educational Resources Information Center

    Craig, Steven E.; Bischof, Gary H.

    Closure of the counseling relationship constitutes both an ending and a beginning. Although closure signifies the ending of the present counseling relationship, many family counselors conceptualize closure as the start of a working relationship between counselor and family that may be summoned in future times of crisis or during a difficult life…

  1. Family History Resources.

    ERIC Educational Resources Information Center

    Bookmark, 1991

    1991-01-01

    The 12 articles in this issue focus on the theme of family history resources: (1) "Introduction: Family History Resources" (Joseph F. Shubert); (2) "Work, Credentials, and Expectations of a Professional Genealogist" (Coreen P. Hallenbeck and Lewis W. Hallenbeck); (3) "Computers and Genealogy" (Theresa C. Strasser); (4) "Finding Historical Records…

  2. Family/Individual Health.

    ERIC Educational Resources Information Center

    Texas Tech Univ., Lubbock. Home Economics Curriculum Center.

    This document contains teacher's materials for a six-unit secondary education vocational home economics course on personal and family health. The units cover: (1) personal health and wellness (including the decisions and other factors that influence health, principles of personal health, and stress management); (2) family health (including coping…

  3. Patent Family Databases.

    ERIC Educational Resources Information Center

    Simmons, Edlyn S.

    1985-01-01

    Reports on retrieval of patent information online and includes definition of patent family, basic and equivalent patents, "parents and children" applications, designated states, patent family databases--International Patent Documentation Center, World Patents Index, APIPAT (American Petroleum Institute), CLAIMS (IFI/Plenum). A table noting country…

  4. Black Families. Interdisciplinary Perspectives.

    ERIC Educational Resources Information Center

    Cheatham, Harold E., Ed.; Stewart, James B., Ed.

    Since the early 1960s, the black family has been characterized as pathological. This six-part collection of 18 research studies presents alternative approaches to understanding the special characteristics of black families. Part I, "Theoretical and Methodological Perspectives," comprises a comparison of the pioneering work of W. E. B. Du Bois and…

  5. Explaining Family Interactions.

    ERIC Educational Resources Information Center

    Fitzpatrick, Mary Anne, Ed.; Vangelisti, Anita L., Ed.

    A detailed review of current research and state-of-the-art ideas concerning both communication processes and family functioning is presented in this collection of articles. The volume is organized around three sections. Part 1, "The Development of Family Communication Patterns," contains: (1) "Communication in Infancy" (Marguerite Stevenson…

  6. Reaching Rural Families.

    ERIC Educational Resources Information Center

    Bernard van Leer Foundation Newsletter, 1995

    1995-01-01

    This newsletter issue focuses on programming undertaken to address the health and educational needs of rural families in developing and developed nations. After examining the nature of rural families and rural poverty, the newsletter discusses: (1) the Mon Women's Organization in Thailand; (2) The "Contact With Kids" parent education project in…

  7. Changing Families, Changing Workplaces

    ERIC Educational Resources Information Center

    Bianchi, Suzanne M.

    2011-01-01

    American families and workplaces have both changed dramatically over the past half-century. Paid work by women has increased sharply, as has family instability. Education-related inequality in work hours and income has grown. These changes, says Suzanne Bianchi, pose differing work-life issues for parents at different points along the income…

  8. Contextual Family Therapy.

    ERIC Educational Resources Information Center

    Frank, Catherine

    1984-01-01

    Examines the major principles and goals of contextual therapy and methods employed in its clinical application. A second article presents an interview with Dr. Ivan Boszormenyi-Nagy, who developed contextual family therapy. The interview ranges from Dr. Nagy's early training to the theoretical and clinical foundations of contextual family therapy.…

  9. Family-Friendly Art

    ERIC Educational Resources Information Center

    Williams, Patterson; Garcia, Maria

    2004-01-01

    In the late 1980s, the Denver Art Museum initiated efforts to make the museum a destination for families. From 1997 to 2001, with a generous grant from The Pew Charitable Trusts, these efforts came to fruition. From the moment they walk through the doors, families' needs are anticipated. For example, they can pick up a welcoming brochure, Free…

  10. Assessment of Troubled Families.

    ERIC Educational Resources Information Center

    Combs-Orme, Terri; Thomas, Katherine H.

    1997-01-01

    Tests the utility of four standardized instruments used in assessing 105 families that sought services in a juvenile corrections setting for their teenage children. Results demonstrate that parents and adolescents can complete standardized assessment instruments and that the information provided can help in understanding distressed families. (RJM)

  11. Golden Matrix Families

    ERIC Educational Resources Information Center

    Fontaine, Anne; Hurley, Susan

    2011-01-01

    This student research project explores the properties of a family of matrices of zeros and ones that arises from the study of the diagonal lengths in a regular polygon. There is one family for each n greater than 2. A series of exercises guides the student to discover the eigenvalues and eigenvectors of the matrices, which leads in turn to…

  12. Feminism and family therapy.

    PubMed

    Goldner, V

    1985-03-01

    Feminism has had a profound effect on contemporary culture and on thinking in most academic fields, including psychoanalysis. Interestingly, until very recently it had made virtually no impact on the theory and practice of family therapy. This paper proposes an explanation for this peculiar phenomenon and argues that family therapy has been considerably handicapped by its insularity from the feminist critique. Utilizing feminist scholarship in psychoanalysis, history, and sociology, the paper analyzes the structural contradictions in family life that family therapists have essentially ignored and then outlines their clinical implications. Key points in the discussion include the argument that systems theory is an inadequate explanatory matrix from which to build a theory of the family, that the archetypal "family case" of the overinvolved mother and peripheral father is best understood, not as a clinical problem, but as the product of a historical process two hundred years in the making, and that power relations between men and women in families function in terms of paradoxical, incongruous hierarchies that reflect the complex interpenetration between the structure of family relations and the world of work. This conceptual model then provides the basis for an analysis and critique of sexual politics as they emerge in the prototypical clinical situation.

  13. Family Perspectives on Prematurity

    ERIC Educational Resources Information Center

    Zero to Three (J), 2003

    2003-01-01

    In this article, seven families describe their experiences giving birth to and raising a premature baby. Their perspectives vary, one from another, and shift over time, depending on each family's circumstances and the baby's developmental course. Experiences discussed include premature labor, medical interventions and the NICU, bringing the baby…

  14. Family Support and Education

    ERIC Educational Resources Information Center

    Goldstein, Lou Ann

    2013-01-01

    Family involvement is essential to the developmental outcome of infants born into Neonatal Intensive Care Unit (NICU). In this article, evidence has been presented on the parent's perspective of having an infant in the NICU and the context of family. Key points to an educational assessment are also reviewed. Throughout, the parent's concerns and…

  15. Families under Economic Pressure.

    ERIC Educational Resources Information Center

    Elder, Glen H., Jr.; And Others

    The economic decline of rural America has widespread consequences for families, children, and education. Broad changes in farming and in the rural nonfarm sector have pushed the poverty rate for rural areas in the 1980s higher than the central cities rate. Projections indicate that by the mid-1990s, one-half of all farm families in the midwest may…

  16. Family Assessment Process Manual.

    ERIC Educational Resources Information Center

    Patterson, Jerri; And Others

    The five-step family assessment process presented in this manual is designed to facilitate the accurate, organized collection of information necessary for the development of an intervention plan for families with children. The materials contain the five forms that are to be completed as part of the assessment process, including a: (1) family…

  17. [Focus: Family Communication].

    ERIC Educational Resources Information Center

    Barnes, Richard E., Ed.

    1977-01-01

    This issue of the "Journal of the Wisconsin Communication Association" focuses on family communication and contains the following articles: "Marital Typologies: An Alternative Approach to the Study of Communication in Enduring Relations" by Mary Anne Fitzpatrick, "Intimate Communication and the Family" by Marilyn D. LaCourt, and "A Study in…

  18. Families, Risk, and Competence.

    ERIC Educational Resources Information Center

    Lewis, Michael, Ed.; Feiring, Candice, Ed.

    The problems of studying families arise from the difficulty in studying systems in which there are multiple elements interacting with each other and with the child. This book attests to the growing sophistication of the conceptualization and measurement techniques for understanding family processes. Chapters in the first part of the book, "The…

  19. Population and family planning.

    PubMed

    Weinberger, C W

    1974-01-01

    The author agrees with the United Teheran Declaration of Human Rights which declared the right of each couple to choose the size of their own family. Government policies must preserve and increase informed choice for all couples. The Supreme Court recognized the right of all married couples to practice birth control in a 1965 decision; the Court extended this right to all persons in 1972. The 1970 Family Planning Services and Population Research Act involved the United States government in family planning support here and abroad. By 1973 organized family planning programs were serving more than 3.2 million women. They had been extended geographically too. Fertility research is being conducted. Before 1980 family planning programs will be integrated into general health care systems with comprehensive health care insurance covering the bills.

  20. Family intervention for schizophrenia

    PubMed Central

    Pharoah, Fiona; Mari, Jair; Rathbone, John; Wong, Winson

    2014-01-01

    Background People with schizophrenia from families that express high levels of criticism, hostility, or over involvement, have more frequent relapses than people with similar problems from families that tend to be less expressive of emotions. Forms of psychosocial intervention, designed to reduce these levels of expressed emotions within families, are now widely used. Objectives To estimate the effects of family psychosocial interventions in community settings for people with schizophrenia or schizophrenia-like conditions compared with standard care. Search strategy We updated previous searches by searching the Cochrane Schizophrenia Group Trials Register (September 2008). Selection criteria We selected randomised or quasi-randomised studies focusing primarily on families of people with schizophrenia or schizoaffective disorder that compared community-orientated family-based psychosocial intervention with standard care. Data collection and analysis We independently extracted data and calculated fixed-effect relative risk (RR), the 95% confidence intervals (CI) for binary data, and, where appropriate, the number needed to treat (NNT) on an intention-to-treat basis. For continuous data, we calculated mean differences (MD). Main results This 2009-10 update adds 21 additional studies, with a total of 53 randomised controlled trials included. Family intervention may decrease the frequency of relapse (n = 2981, 32 RCTs, RR 0.55 CI 0.5 to 0.6, NNT 7 CI 6 to 8), although some small but negative studies might not have been identified by the search. Family intervention may also reduce hospital admission (n = 481, 8 RCTs, RR 0.78 CI 0.6 to 1.0, NNT 8 CI 6 to 13) and encourage compliance with medication (n = 695, 10 RCTs, RR 0.60 CI 0.5 to 0.7, NNT 6 CI 5 to 9) but it does not obviously affect the tendency of individuals/families to leave care (n = 733, 10 RCTs, RR 0.74 CI 0.5 to 1.0). Family intervention also seems to improve general social impairment and the levels of

  1. Family allowance and family planning in Chile.

    PubMed Central

    Plank, S J

    1978-01-01

    Family allowances designed to promote maternal and child health and welfare could be self-defeating if they stimulated otherwise unwanted births, as often assumed. That assumption, with its public health and demographic implications, needs testing. An attempt to test it was made in Chile in 1969--1970 through interviews with 945 wives receiving an allowance and 690 non-recipients. Recipients practiced contraception significantly more than did non-recipients. This was not explained by wives' educational attainment or employment, the couples' earnings, or number of living children, but was associated with a 50 per cent greater utilization of professional prenatal care by recipients during the most recent pregnancy; women with such care (regardless of allowance status) were 75 per cent more likely than others to control their fertility. Prenatal care was probably sought more by recipients in part because an additional stipend was provided as soon as pregnancy was confirmed, usually at clinics with integrated family planning. Greater family income, attributable to the allowance, probably also contributed to the recipients' better prenatal attention and to contraceptive practice. Noteworthy, too, was the finding that with the number of living children controlled, contraceptive practice was significantly greater amoung couples who had never lost a child. PMID:717610

  2. Families Get Involved! Learning Partners.

    ERIC Educational Resources Information Center

    Office of Educational Research and Improvement (ED), Washington, DC. Media and Information Services.

    Noting that families who are involved in their children's education make a difference in their child's performance, this two-page information sheet encourages families to get involved by listing the benefits of family involvement on one side and the ways adult family members can help in the school on the other. As a result of family participation:…

  3. Family Oriented Geographic Field Experience.

    ERIC Educational Resources Information Center

    Williams, Karen Ann Lalk

    This paper describes a program of geographic education through field experience trips for family groups. Developed at Delta College in Michigan, the approach is unique because it emphasizes learning experiences for families rather than for individual students. The family is interpreted to include nuclear families, single-parent families with…

  4. Canadian Families (Les Familles Canadiennes).

    ERIC Educational Resources Information Center

    Vanier Inst. of the Family, Ottawa (Ontario).

    Structural changes that have taken place in Canadian families in recent decades are described in this booklet. Topical sections are as follows: (1) What Counts in Canadian Families (importance of (importance of family); (2) The Family--Variations on a Theme origins, family structure, seniors aged 60 and over, how lives are spent, religion); (3)…

  5. Ethical Issues in Family Work.

    ERIC Educational Resources Information Center

    Kaplan, David M.

    For more than half a decade, the author edited a quarterly ethics column focusing on family work, first in the "International Association of Marriage and Family Counselors Newsletter" and later in "The Family Journal: Counseling and Therapy for Couples and Families." These columns responded to ethical dilemmas in family work submitted by…

  6. Family Centered Maternity Care

    PubMed Central

    Enkin, Murray W.

    1973-01-01

    Current practices of obstetrical care tend to hinder rather than facilitate family development and maturation. A program of family centred maternity care is described. Husbands are invited to prenatal visits, and are involved in intensive preparation for labor and delivery. Their presence and active participation in labor, delivery, and postpartum course are encouraged. This, along with a rooming-in policy for the baby, and the utilization of the postpartum period for an intensive training in parenthood, appears to produce a safe and satisfying obstetrical experience for the family. PMID:20468914

  7. Induction of TRIM22 by IFN-γ Involves JAK and PC-PLC/PKC, but Not MAPKs and pI3K/Akt/mTOR Pathways.

    PubMed

    Gao, Bo; Xu, Wei; Wang, Yaxin; Zhong, Linmao; Xiong, Sidong

    2013-10-01

    Tripartite motif (TRIM) 22 plays an important role in interferons (IFNs)-mediated antiviral activity. We previously demonstrated that interferon regulatory factor-1 (IRF-1) played a central role in IFN-γ-induced TRIM22 expression via binding to a special cis-element named 5' extended IFN-stimulating response element (5'eISRE). In this study, we sought to identify the signaling pathways involved in TRIM22 induction by IFN-γ. By using various pharmacological inhibitors, it was found that the activity of tyrosine kinase and phosphatidylcholine-phospholipase C (PC-PLC), but not phosphatidylinositol-phospholipase C (PI-PLC) and phospholipase D (PLD), was required for IFN-γ-induced TRIM22 expression in HepG2 cells. Tyrosine kinase Janus kinase (JAK), not SRC and PYK2, played an indispensable role in TRIM22 induction. Inhibition of protein kinase C (PKC) activity also significantly attenuated IFN-γ induction of TRIM22. Although treatment with IFN-γ resulted in the stimulation of mitogen-activated protein kinases (MAPKs) (p38, ERK, and JNK) and pI3K/Akt/mTOR pathways in HepG2 cells, the inhibition of their activity did not affect IFN-γ-stimulated TRIM22 expression. Further studies showed that overexpression of JAK1 and PKCα activated TRIM22 promoter activity in a 5'eISRE-dependent manner, and inhibition of not only JAK but also PC-PLC/PKC pathways significantly attenuated IFN-γ-induced IRF-1 expression in HepG2 cells. Taken together, these data indicated that IFN-γ induced TRIM22 expression via activation of JAK and PC-PLC/PKC signaling pathways, which involved the cis-element 5'eISRE and the transactivator IRF-1.

  8. Induction of TRIM22 by IFN-γ Involves JAK and PC-PLC/PKC, but Not MAPKs and pI3K/Akt/mTOR Pathways

    PubMed Central

    Gao, Bo; Xu, Wei; Wang, Yaxin; Zhong, Linmao

    2013-01-01

    Tripartite motif (TRIM) 22 plays an important role in interferons (IFNs)-mediated antiviral activity. We previously demonstrated that interferon regulatory factor-1 (IRF-1) played a central role in IFN-γ-induced TRIM22 expression via binding to a special cis-element named 5′ extended IFN-stimulating response element (5′eISRE). In this study, we sought to identify the signaling pathways involved in TRIM22 induction by IFN-γ. By using various pharmacological inhibitors, it was found that the activity of tyrosine kinase and phosphatidylcholine-phospholipase C (PC-PLC), but not phosphatidylinositol-phospholipase C (PI-PLC) and phospholipase D (PLD), was required for IFN-γ-induced TRIM22 expression in HepG2 cells. Tyrosine kinase Janus kinase (JAK), not SRC and PYK2, played an indispensable role in TRIM22 induction. Inhibition of protein kinase C (PKC) activity also significantly attenuated IFN-γ induction of TRIM22. Although treatment with IFN-γ resulted in the stimulation of mitogen-activated protein kinases (MAPKs) (p38, ERK, and JNK) and pI3K/Akt/mTOR pathways in HepG2 cells, the inhibition of their activity did not affect IFN-γ-stimulated TRIM22 expression. Further studies showed that overexpression of JAK1 and PKCα activated TRIM22 promoter activity in a 5′eISRE-dependent manner, and inhibition of not only JAK but also PC-PLC/PKC pathways significantly attenuated IFN-γ-induced IRF-1 expression in HepG2 cells. Taken together, these data indicated that IFN-γ induced TRIM22 expression via activation of JAK and PC-PLC/PKC signaling pathways, which involved the cis-element 5′eISRE and the transactivator IRF-1. PMID:23659673

  9. Family medical leave as a resilience resource for family caregivers.

    PubMed

    Swanke, Jayme; Zeman, Laura Dreuth

    2009-01-01

    Case managers mobilize family networks to care for patients. Family medical leave can be a resource for case managers who seek to enhance resilience among family caregivers. The Family Medical Leave Act, passed in 1993, was the first U.S. policy to regulate employee leaves from work for family care purposes (29 CFR 825.102). This policy offers family caregivers increased flexibility and equality. Current and emerging policies also can reduce financial strain. The discussion examines how case managers can integrate family medical leave into best-practice models to support patients and family caregivers.

  10. Families in the Military

    MedlinePlus

    ... have led to deployment of large numbers of military personnel (active duty, Reserves, National Guard). As a result ... worries and plans for the future. Let your child know that the family member is making a ...

  11. Familial combined hyperlipidemia

    MedlinePlus

    ... fats. It can cause early heart attacks. Diabetes , alcoholism, and hypothyroidism make the condition worse. Risk factors include a family history of high cholesterol and early coronary artery disease.

  12. Family Caregiver Alliance

    MedlinePlus

    ... A Complex Web: Family Caregiving and Healthcare [Editor's note: This blog was originally posted on the the Atlas of Caregiving website on June ... 2016 Taking Care of SoMEone Else November 14, 2016 Grupo ...

  13. Collaboration in Family Therapy

    PubMed Central

    Tuerk, Elena Hontoria; McCart, Michael R.; Henggeler, Scott W.

    2015-01-01

    This article summarizes and illustrates the collaboration strategies used by several family therapies. The strategies used within multisystemic therapy (MST) are emphasized because it has demonstrated high rates of treatment completion and favorable outcomes in multiple clinical trials. Many of the collaboration strategies in family work are common to other forms of evidence-based psychotherapy (e.g., reflective listening, empathy, reframing, and displays of authenticity and flexibility); however, some strategies are unique to family systems treatments, such as the identification of strengths across multiple systems in the youth’s social ecology and the maintenance of a family (versus a child) focus during treatment. A case example illustrates collaboration and engagement in the context of MST. PMID:23616297

  14. Family Adjustment to Aphasia

    MedlinePlus

    ... this time. Seek additional counseling services as necessary. Communication Skills Family members also can help the person ... aphasia develop new skills to compensate for the communication problems. Some suggestion include: Continue to talk to ...

  15. Family Demands, Social Support and Family Functioning in Taiwanese Families Rearing Children with Down Syndrome

    ERIC Educational Resources Information Center

    Hsiao, C-Y.

    2014-01-01

    Background: Down syndrome (DS) affects not only children but also their families. Much remains to be learned about factors that influence how families of children with DS function, especially families in non-Western populations. The purpose of this cross-sectional, correlational study was to examine how family demographics, family demands and…

  16. The changing American family.

    PubMed

    Thornton, A; Freedman, D

    1983-10-01

    This Bulletin documents recent changes in American family patterns resulting both from longterm trends in urbanization, industrialization, and economic growth and the disruption of the Great Depression and World War 2, as well as changed attitudes toward marriage, parenthood, divorce, and the roles of women. Following a postwar boom in the 1950s and 1960s, marriage rates have now fallen to levels observed in the early 20th century. Since 1970, the number of unmarried couples living together has more than tripled to 1.9 million in 1983. The divorce rate has now stabilized after more than doubling since 1960, but at the current level, 1/2 of all recent marriages will end in divorce. Most divorced persons remarry fairly quickly, often creating complex families of "step-relatives." With 19% of households with minor children now headed by a women with no husband present, up to 1/2 of all children will live for sometime in a fatherless family before age 18. Over 1/2 of all married women, including 49% of married mothers of preschool children, now hold a paid job outside the home. Working wives boost a family's income by an average 40% but still are expected to shoulder most responsiblility for home and childcare. White women now in their 20s say they expect to have an average of 2 children, but are delaying childbearing to such an extent that 29% could end up childless. Most of the elderly live on their own but usually near children whom they see frequently. Despite changes in traditional family patterns, Americans consistently report that a happy marriage and good family are the most important aspects of life. And though most Americans now live with few or no family members, they maintain active contact with a large network of family.

  17. [Counseling of immigrant families].

    PubMed

    Pavkovic, G

    2001-04-01

    The quality and efficiency in the process of psychological counseling with migrant families depends on the counselor's insight in the constellations of migration and culture. Both have a special influence on the way of coping with problems and the relation between client and counselor during the therapy. It is therefore necessary to adapt the own therapeutical approach to the migrant's experiences and abilities of comprehension. The author presents one example of intercultural family counseling by three short case explorations. PMID:11382173

  18. Swedish Family Policy.

    ERIC Educational Resources Information Center

    Herrstrom, Staffan

    1986-01-01

    Family policy remains one of the leading issues of Swedish domestic politics. All parties are agreed that families with children must be given a better deal in the wake of the economic crisis. But how is this to be done and how quickly can it be achieved? Is the expansion of day nursery facilities to be speeded up, or are parents to be given a…

  19. Increase in family allowances.

    PubMed

    1989-01-01

    In July 1989 the family allowance structure in Australia was changed from a 4-rate to a 2-rate structure. The new rates were increased to $A9 a week for the 1st 3 children and $A12 for each additional child. The Family Allowance Supplment rate for children 13-15 years old was raised from $A31 to $A34.10/week. PMID:12344544

  20. Welfare Policies and Black Families.

    ERIC Educational Resources Information Center

    Trader, Harriet Peat

    1979-01-01

    The family is an important resource for minority persons, and many minority families depend on public welfare for their survival. This article offers a compact analysis of how welfare policies often work to the disadvantage of poor Black families. (Author)

  1. [Apothecaries' arms of Voltaire's family].

    PubMed

    Chaigneau, M

    1998-01-01

    Apothecaries of Voltaire's family can be divided into two groups. Marceton Family: Claude Marceton, Hierosme and Pierre Testefolle, from Thouars. Arouet family: Jehan Arouet, Pierre Rochard and Jean Gougeard. Five of those apothecaries beand arms. PMID:11625327

  2. Administration for Children and Families

    MedlinePlus

    ... Visit the Children's Bureau Website The Family Room Blog RSS Feed Ensuring Equal Access for All November ... families View more posts from the Family Room Blog Back to Top Home About What We Do ...

  3. Family Planning in Thailand.

    PubMed

    1980-12-01

    Until 1958, when the World Bank Economic Mission reported that Thailand's high rate of population growth was adversely affecting its development efforts, Thailand had a pronatalist policy. Government concern led to a formal declaration of voluntary family planning support in 1970. The National Family Planning Program (NFPP) under the auspices of the Ministry of Public Health has the following objectives: reduce the population growth rate to 2.5% per annum by the end of 1976; inform and motivate women about family planning, using methods of mass communication; increase availability of family planning services throughout the country; and to integrate family planning activities with maternal and child health services. Activities include training and supervision of NFPP personnel, research and evaluation, and the coordination of public and private family planning organizations. The NFPP has been successful in reducing the growth rate to 2.55% in 1976 and in surpassing its target contraceptive acceptor level of 1,975,000 to a level of 2,490,850. Future concerns of NFPP include its dependence on foreign financial support, and its need to encourage local funding.

  4. Familial pituitary tumors.

    PubMed

    Alband, Neda; Korbonits, Márta

    2014-01-01

    Pituitary adenomas are benign intracranial neoplasms that present a major clinical concern due to hormone overproduction and/or tumor mass effects. The majority of pituitary adenomas occur sporadically; however, familial cases are increasingly being recognized, such as multiple endocrine neoplasia type 1 (MEN1), Carney complex (CNC), and familial isolated pituitary adenoma (FIPA). Familial pituitary tumors appear to differ from their sporadic counterparts both in their genetic basis and in clinical characteristics. Evidence suggests that, especially in MEN1 and FIPA, tumors are more aggressive and affect patients at a younger age, therefore justifying the importance of early diagnosis, while in Carney complex pituitary hyperplasia is common. The genetic alterations responsible for the formation of familial pituitary syndromes include the MEN1 gene, responsible for about 80% of MEN1 cases, the regulatory subunit of the protein kinase A, PRKAR1A, responsible for about 70% of Carney complex cases, and AIP, the gene coding the aryl hydrocarbon receptor interacting protein, responsible for about 20% of FIPA cases. Rarely other genes have also been found responsible for familial pituitary adenoma cases. McCune-Albright syndrome (MAS) also has a genetic origin due to mosaic mutations in the G protein-coupled α subunit coded by the GNAS1 gene. In this chapter, we summarize the genetic and clinical characteristics of these familial pituitary syndromes and MAS. PMID:25248598

  5. Mandolin Family Instruments

    NASA Astrophysics Data System (ADS)

    Cohen, David J.; Rossing, Thomas D.

    The mandolin family of instruments consists of plucked chordophones, each having eight strings in four double courses. With the exception of the mandobass, the courses are tuned in intervals of fifths, as are the strings in violin family instruments. The soprano member of the family is the mandolin, tuned G3-D4-A4-E5. The alto member of the family is the mandola, tuned C3-G3-D4-A4. The mandola is usually referred to simply as the mandola in the USA, but is called the tenor mandola in Europe. The tenor member of the family is the octave mandolin, tuned G2-D3-A3-E4. It is referred to as the octave mandolin in the USA, and as the octave mandola in Europe. The baritone member of the family is the mandocello, or mandoloncello, tuned C2-G2-D3-A3. A variant of the mandocello not common in the USA is the five-course liuto moderno, or simply liuto, designed for solo repertoire. Its courses are tuned C2-G2-D3-A3-E4. A mandobass was also made by more than one manufacturer during the early twentieth century, though none are manufactured today. They were fretted instruments with single string courses tuned E1-A1-D2-G2. There are currently a few luthiers making piccolo mandolins, tuned C4-G4-D5-A5.

  6. Pediatric family-centered rehabilitation.

    PubMed

    Hostler, S L

    1999-08-01

    Family-centered rehabilitation programs are derived from a philosophy of heath care delivery known as family-centered care. The principles of family-centered care are presented with clinical examples. Its origins are reviewed, and the 10-year process of implementation of family-centered care practice and policy at a children's rehabilitation center are described. Profound changes in behavior are required of the health care professionals as meaningful collaboration with families develops. Key elements of a family-centered rehabilitation program include meaningful participation by families in medical decision making and an institutional culture flexible enough to respond to the ongoing collaboration between families and practitioners.

  7. Changing families, changing workplaces.

    PubMed

    Bianchi, Suzanne M

    2011-01-01

    American families and workplaces have both changed dramatically over the past half-century. Paid work by women has increased sharply, as has family instability. Education-related inequality in work hours and income has grown. These changes, says Suzanne Bianchi, pose differing work-life issues for parents at different points along the income distribution. Between 1975 and 2009, the labor force rate of mothers with children under age eighteen increased from 47.4 percent to 71.6 percent. Mothers today also return to work much sooner after the birth of a child than did mothers half a century ago. High divorce rates and a sharp rise in the share of births to unmarried mothers mean that more children are being raised by a single parent, usually their mother. Workplaces too have changed, observes Bianchi. Today's employees increasingly work nonstandard hours. The well-being of highly skilled workers and less-skilled workers has been diverging. For the former, work hours may be long, but income has soared. For lower-skill workers, the lack of "good jobs" disconnects fathers from family obligations. Men who cannot find work or have low earnings potential are much less likely to marry. For low-income women, many of whom are single parents, the work-family dilemma is how to care adequately for children and work enough hours to support them financially. Jobs for working-class and lower middle-class workers are relatively stable, except in economic downturns, but pay is low, and both parents must work full time to make ends meet. Family income is too high to qualify for government subsidized child care, but too low to afford high-quality care in the private market. These families struggle to have a reasonable family life and provide for their family's economic well-being. Bianchi concludes that the "work and family" problem has no one solution because it is not one problem. Some workers need more work and more money. Some need to take time off around the birth of a child

  8. Changing families, changing workplaces.

    PubMed

    Bianchi, Suzanne M

    2011-01-01

    American families and workplaces have both changed dramatically over the past half-century. Paid work by women has increased sharply, as has family instability. Education-related inequality in work hours and income has grown. These changes, says Suzanne Bianchi, pose differing work-life issues for parents at different points along the income distribution. Between 1975 and 2009, the labor force rate of mothers with children under age eighteen increased from 47.4 percent to 71.6 percent. Mothers today also return to work much sooner after the birth of a child than did mothers half a century ago. High divorce rates and a sharp rise in the share of births to unmarried mothers mean that more children are being raised by a single parent, usually their mother. Workplaces too have changed, observes Bianchi. Today's employees increasingly work nonstandard hours. The well-being of highly skilled workers and less-skilled workers has been diverging. For the former, work hours may be long, but income has soared. For lower-skill workers, the lack of "good jobs" disconnects fathers from family obligations. Men who cannot find work or have low earnings potential are much less likely to marry. For low-income women, many of whom are single parents, the work-family dilemma is how to care adequately for children and work enough hours to support them financially. Jobs for working-class and lower middle-class workers are relatively stable, except in economic downturns, but pay is low, and both parents must work full time to make ends meet. Family income is too high to qualify for government subsidized child care, but too low to afford high-quality care in the private market. These families struggle to have a reasonable family life and provide for their family's economic well-being. Bianchi concludes that the "work and family" problem has no one solution because it is not one problem. Some workers need more work and more money. Some need to take time off around the birth of a child

  9. Fatal familial insomnia: a new Austrian family.

    PubMed

    Almer, G; Hainfellner, J A; Brücke, T; Jellinger, K; Kleinert, R; Bayer, G; Windl, O; Kretzschmar, H A; Hill, A; Sidle, K; Collinge, J; Budka, H

    1999-01-01

    We present clinical, pathological and molecular features of the first Austrian family with fatal familial insomnia. Detailed clinical data are available in five patients and autopsy in four patients. Age at onset of disease ranged between 20 and 60 years, and disease duration between 8 and 20 months. Severe loss of weight was an early symptom in all five patients. Four patients developed insomnia and/or autonomic dysfunction, and all five patients developed motor abnormalities. Analysis of the prion protein (PrP) gene revealed the codon 178 point mutation and methionine homozygosity at position 129. In all brains, neuropathology showed widespread cortical astrogliosis, widespread brainstem nuclei and tract degeneration, and olivary 'pseudohypertrophy' with vacuolated neurons, in addition to neuropathological features described previously, such as thalamic and olivary degeneration. Western blotting of one brain and immunocytochemistry in four brains revealed quantitative and regional dissociation between PrP(res)(the protease resistant form of PrP) deposition and histopathology. In the cerebellar cortex of one patient, PrP(res) deposits were prominent in the molecular layer and displayed a peculiar patchy and strip-like pattern with perpendicular orientation to the surface. In another patient, a single vacuolated neuron in the inferior olivary nuclei contained prominent intravacuolar granular PrP(res) deposits, resembling changes of brainstem neurons in bovine spongiform encephalopathy.

  10. Familial pancreatic cancer.

    PubMed

    Klein, A P; Hruban, R H; Brune, K A; Petersen, G M; Goggins, M

    2001-01-01

    Pancreatic cancer is the fourth leading cause of cancer death in both men and women in the United States and will be responsible for an estimated 28,900 deaths in 2001. Relatively little is known of its etiology, and the only well-established risk factor is cigarette smoking. Studies over the past 3 decades have shown that 4%-16% of patients with pancreatic cancer have a family history of the disease. A small fraction of this aggregation can be accounted for in inherited cancer syndromes, including familial atypical multiple-mole melanoma, Peutz-Jeghers syndrome, hereditary breast-ovarian cancer, hereditary pancreatitis, and hereditary nonpolyposis colorectal cancer. These syndromes arise as a result of germline mutations in the BRCA2, pl6 (familial atypical multiple-mole melanoma), mismatch repair (hereditary nonpolyposis colorectal cancer), and STK11 (Peutz-Jeghers syndrome) genes. In addition, hereditary plays a role in predisposing certain patients with apparently sporadic pancreatic cancer. Many patients with pancreatic cancers caused by a germline mutation in a cancer-causing gene do not have a pedigree that is suggestive of a familial cancer syndrome. A recent prospective analysis of the pedigrees in the National Familial Pancreatic Tumor Registry found that individuals with a family history of pancreatic cancer in multiple first-degree relatives have a high risk of pancreatic cancer themselves. The identification of such high-risk individuals will help clinicians target screening programs and develop preventive interventions with the hope of reducing the mortality of pancreatic cancer in these families.

  11. Family planning Indonesia.

    PubMed

    Singarimbun, M

    1968-06-01

    The growth of family planning activities in Indonesia in the Postwar period is traced; and future prospects for family planning are assessed. Transmigration projects initiated by the Dutch and supported by President Sukarno after Indonesian independence as a means of decreasing population pressure on the island of Java, are identified as the only official response to the population problem until 1965. In the face of the government's opposition to the idea of birth control as a population control measure, the activities of the Indonesian Planned Parenthood Association (IPPA) after its founding in 1957 were limited to advising mothers on spacing of their children for health reasons. Statements made in support of a national family planning program by government officials at a 1967 IPPA Congress and on other occasions are noted. The major components of an approved national family planning program to start in 1969 are described. However, the government's policy as of late 1967 and early 1968 is characterized as one of mainly benevolent encouragement and help to voluntary organizations. The chief impediment to family planning in Indonesia is said to be a lack of motivation and the force of traditional values that favor large families. On the positive side are: 1) Studies showing considerable interest in birth control by the rural population; 2) A long history of traditional birth control practices; 3) The absence of outright opposition by religious groups to the principle of family planning. However, financial costs, the need for the training of personnel, and a general unawareness of the magnitude of the task lying ahead constitute other formidable obstacles.

  12. Characterization of inositolphospholipids in Trypanosoma cruzi trypomastigote forms.

    PubMed

    Uhrig, M L; Couto, A S; Colli, W; de Lederkremer, R M

    1996-05-20

    In vivo labeling experiments with [3H]palmitic acid, [3H]inositol, and [3H]glucose allowed the identification of two main classes of inositolphospholipids (IPLs) from the trypomastigote stage of Trypanosoma cruzi. Purification of these compounds was achieved by ion-exchange chromatography, high performance liquid chromatography and thin layer chromatography. Specific phosphatidyl-inositol phospholipase C digestion, dephosphorylation and acid methanolysis showed a ceramide structure for the lower migrating IPL1. Palmitoyldihydrosphingosine and palmitoylsphingosine were detected by reverse-phase thin-layer chromatography. On the other hand, IPL2 showed to be a mixture of diacylglycero- and alkylacylglycero-phospholipids in a 1:1 ratio. After PI-PLC digestion, the lipids were separated by preparative TLC and individually analysed. The diacylglycerol contained mainly C18:0 fatty acid together with a low amount of C16:0. Hexadecylglycerol esterified with the C18:0 fatty acid was the only alkylacylglycerol detected. The C18:2 and C18:1 fatty acids, preponderant in the PI molecules of epimastigote forms, were not detected in trypomastigote forms. This is the first report on inositol phospholipids, putative precursors of lipid anchors in the infective stage of T. cruzi. PMID:8679689

  13. Pseudomonas aeruginosa pyocyanin modulates mucin glycosylation with sialyl-Lewisx to increase binding to airway epithelial cells

    PubMed Central

    Choi, Woosuk; Choe, Shawn; Miao, Jinfeng; Xu, Ying; Powell, Rebecca; Lin, Jingjun; Kuang, Zhizhou; Gaskins, H Rex; Lau, Gee W.

    2015-01-01

    Cystic fibrosis (CF) patients battle life-long pulmonary infections with the respiratory pathogen Pseudomonas aeruginosa (PA). An overabundance of mucus in CF airways provides a favorable niche for PA growth. When compared to that of non-CF individuals, mucus of CF airways is enriched in sialyl-Lewisx, a preferred binding receptor for PA. Notably, the levels of sialyl-Lewisx directly correlate with infection severity in CF patients. However, the mechanism by which PA causes increased sialylation remains uncharacterized. In this study, we examined the ability of PA virulence factors to modulate sialyl-Lewisx modification in airway mucins. We found pyocyanin (PCN) to be a potent inducer of sialyl-Lewisx in both mouse airways and in primary and immortalized CF and non-CF human airway epithelial cells. PCN increased the expression of C2/4GnT and ST3Gal-IV, two of the glycosyltransferases responsible for the stepwise biosynthesis of sialyl-Lewisx, through a TNF-α-mediated phosphoinositol-specific phospholipase C (PI-PLC) dependent pathway. Furthermore, PA bound more efficiently to airway epithelial cells pre-exposed to PCN through a flagellar cap-dependent manner. Importantly, antibodies against sialyl-Lewisx and anti-TNF-α attenuated PA binding. These results indicate that PCN secretes PCN to induce a favorable environment for chronic colonization of CF lungs by increasing the glycosylation of airway mucins with sialyl-Lewisx. PMID:26555707

  14. Nogo-A at CNS paranodes is a ligand of Caspr: possible regulation of K(+) channel localization.

    PubMed

    Nie, Du-Yu; Zhou, Zhi-Hong; Ang, Beng-Ti; Teng, Felicia Y H; Xu, Gang; Xiang, Tao; Wang, Chao-Yang; Zeng, Li; Takeda, Yasuo; Xu, Tian-Le; Ng, Yee-Kong; Faivre-Sarrailh, Catherine; Popko, Brian; Ling, Eng-Ang; Schachner, Melitta; Watanabe, Kazutada; Pallen, Catherine J; Tang, Bor Luen; Xiao, Zhi-Cheng

    2003-11-01

    We report Nogo-A as an oligodendroglial component congregating and interacting with the Caspr-F3 complex at paranodes. However, its receptor Nogo-66 receptor (NgR) does not segregate to specific axonal domains. CHO cells cotransfected with Caspr and F3, but not with F3 alone, bound specifically to substrates coated with Nogo-66 peptide and GST-Nogo-66. Binding persisted even after phosphatidylinositol- specific phospholipase C (PI-PLC) removal of GPI-linked F3 from the cell surface, suggesting a direct interaction between Nogo-66 and Caspr. Both Nogo-A and Caspr co-immunoprecipitated with Kv1.1 and Kv1.2, and the developmental expression pattern of both paralleled compared with Kv1.1, implicating a transient interaction between Nogo-A-Caspr and K(+) channels at early stages of myelination. In pathological models that display paranodal junctional defects (EAE rats, and Shiverer and CGT(-/-) mice), distances between the paired labeling of K(+) channels were shortened significantly and their localization shifted toward paranodes, while paranodal Nogo-A congregation was markedly reduced. Our results demonstrate that Nogo-A interacts in trans with axonal Caspr at CNS paranodes, an interaction that may have a role in modulating axon-glial junction architecture and possibly K(+)-channel localization during development. PMID:14592966

  15. Opportunity NYC--Family Rewards: Qualitative Study of Family Communication

    ERIC Educational Resources Information Center

    Fraker, Carolyn A.; Greenberg, David

    2011-01-01

    Aimed at low-income families in six of New York City's highest-poverty communities, the Family Rewards program ties cash rewards to a pre-specified set of activities. This paper presents the qualitative findings from interviews with 77 families. It examines how families incorporated the program into their households, and specifically the…

  16. Family Support & Health Care: Working Together for Healthy Families.

    ERIC Educational Resources Information Center

    Lalley, Jacqueline, Ed.; Ahsan, Nilofer, Ed.

    1998-01-01

    This report of the Family Resource Coalition of America examines partnerships between family support programs and health care providers, forged to ensure that the comprehensive needs of families are met. The report begins with two articles, "Family Support and the Emerging Health System" and "Social and Economic Issues Affecting Health--A…

  17. We Are Family: Using Diverse Family Structure Literature with Children

    ERIC Educational Resources Information Center

    Gilmore, Deanna Peterschick; Bell, Kari

    2006-01-01

    The structure of the American family has changed over the years. Although the traditional father, mother, child structure still dominates, other family patterns are emerging. In this article the authors present: (1) current statistics relating to diverse family structures; (2) reasons for using diverse family structure literature with children;…

  18. Family Functioning in Families with Older Institutionalized Retarded Offspring.

    ERIC Educational Resources Information Center

    Kazak, Anne E.

    1989-01-01

    Psychological distress, marital satisfaction, family adaptability, and cohesion are explored in 41 families with mentally retarded (MR) institutionalized youth and 38 comparison families. Multivariate analyses found no differences between groups, but univariate analyses revealed greater cohesion in families with MR offspring and stressed the…

  19. Engaging Families in In-Home Family Intervention

    ERIC Educational Resources Information Center

    Thompson, Ronald W.; Koley, Sarah

    2014-01-01

    Boys Town has created a program called In-Home Family Services to deliver help to families in stress. In-home family intervention programs have become widely used to help more families who are at risk and experiencing difficulties with a wide range of problems including domestic violence, child behavior problems, parent-child and family…

  20. Family planning in Singapore.

    PubMed

    Kanagaratnam, K

    1968-01-01

    Since the initial voluntary efforts of the Singapore Family Planning Association in 1949, family planning in Singapore has made important progress. This effort extended over the years until the end of 1965 when the government accepted full responsibility for family planning on a national scale. In September 1965, the government announced a 5-year National Family Planning Program with the goal of reducing the birthrate from 32/1000 in 1964 to below 20/1000 by 1970. This would result in a growth rate of not more than 1.5%. The government program aims at reaching 60% of married women in the reproductive age range of 15-45. It is estimated that out of 450,000 in this age range, some 300,000 are married. The target is 180,000 in 5 years. The Singapore Family Planning & Population Board was established by an Act of Parliament and charged with responsibility for the implementation of the 5-year plan. The national program offers a menu card of all family planning methods except abortion. Initial focus was on the IUD as the method of choice for 80%. Oral contraception (OC) was the preferred alternative for the remaining 20%. Other conventonal methods also were available. A few months after the plan began in 1966, the IUD became unacceptable to Singapore women. Its side effects of bleeding, cramps, perforation, and pregnancy were exaggerated by rumors. By the middle of 1966, attendance and acceptors in the national program had declined. Emphasis in the national program was changed to OCs, which now are the mainstay of family planning. Currently, nearly 65% of the acceptors use OCs. The program also demonstrates the importance, especially in urban areas, of the tremendous impact of a postpartum family planning service. Over 70% of the births in Singapore take place at the Kandang Kerbau Maternity Hospitals. Government midwives deliver another 5%. All these women are contacted by a team of family planning workers in the postpartum period and are offered family planning. Nearly

  1. Family dynamics in biblical times: Joseph as a family psychotherapist.

    PubMed

    Ben-Noun, Liubov Louba

    2003-06-01

    Families have existed for thousands of years and are the most enduring form of small social group. This article discusses the family of Jacob as described in the Bible, and analyses the dynamics of the family system, the relationships between family members and the ways in which they interact and respond to stressful events. Such events disrupted the equilibrium of the family and led to a severe crisis, but this was eventually resolved peacefully and family members were reconciled. The story of Joseph, Jacob's son, unfolds as possibly the earliest description of a psychotherapeutic intervention to resolve a long-standing family crisis.

  2. Effective family planning programs.

    PubMed

    Rosenfield, A G

    1973-01-01

    Organizational and content features of various national family planning programs are reviewed. The Thai program is cited as an example of a family planning program organized on a massive unipurpose compaign basis. The Korean and Taiwan programs have utilized special field workers while upgrading the general health care network. 3 major problems with family planning programs are: 1) the lack of experience with such programs; 2) lack of commitment at the highest political levels; and 3) medical conservatism. Utilization of all available contraceptive methods instead of reliance on 1 method would improve most programs. Nursing and auxiliary personnel could be trained to take over the work of physicians in family planning programs. This is already being done with IUD insertion and pill prescription in several programs. The postpartum tubal ligation approach has proven effective and should be extended. There is a place in all national programs for both the private and the commercial sectors. Incentives for clinics, personnel, and acceptors might spread family planning more rapidly.

  3. Effective family planning programs.

    PubMed

    Rosenfield, A G

    1973-01-01

    Organizational and content features of various national family planning programs are reviewed. The Thai program is cited as an example of a family planning program organized on a massive unipurpose compaign basis. The Korean and Taiwan programs have utilized special field workers while upgrading the general health care network. 3 major problems with family planning programs are: 1) the lack of experience with such programs; 2) lack of commitment at the highest political levels; and 3) medical conservatism. Utilization of all available contraceptive methods instead of reliance on 1 method would improve most programs. Nursing and auxiliary personnel could be trained to take over the work of physicians in family planning programs. This is already being done with IUD insertion and pill prescription in several programs. The postpartum tubal ligation approach has proven effective and should be extended. There is a place in all national programs for both the private and the commercial sectors. Incentives for clinics, personnel, and acceptors might spread family planning more rapidly. PMID:12309877

  4. Familial pituitary adenomas.

    PubMed

    Vandeva, S; Vasilev, V; Vroonen, L; Naves, L; Jaffrain-Rea, M-L; Daly, A F; Zacharieva, S; Beckers, A

    2010-12-01

    Pituitary adenomas are benign intracranial neoplasms that present a major clinical concern because of hormonal overproduction or compression symptoms of adjacent structures. Most arise in a sporadic setting with a small percentage developing as a part of familial syndromes such as multiple endocrine neoplasia type 1 (MEN1), Carney complex (CNC), and the recently described familial isolated pituitary adenomas (FIPA) and MEN-4. While the genetic alterations responsible for the formation of sporadic adenomas remain largely unknown, considerable advances have been made in defining culprit genes in these familial syndromes. Mutations in MEN1 and PRKAR1A genes are found in the majority of MEN1 and CNC patients, respectively. About 15% of FIPA kindreds present with mutations of the aryl hydrocarbon receptor-interacting protein (AIP) gene. Mutations in the CDKN1B gene, encoding p27(Kip)¹ were identified in MEN4 cases. Familial tumours appear to differ from their sporadic counterparts not only in genetic basis but also in clinical characteristics. Evidence suggests that, especially in MEN1 and FIPA, they are more aggressive and affect patients at younger age, therefore justifying the importance of early diagnosis. In this review, we summarize the genetic and clinical characteristics of these familial pituitary adenomas. PMID:20961530

  5. [Familial multiple cavernomatosis].

    PubMed

    Terriza, F; Amrani, Y; Asencio, J J; Goberna, E; Casado, A; Peralta, J I

    1997-04-01

    We present a family study of multiple cavernomatosis which affected a boy of six, his mother and two brothers. It was seen clinically as epileptic crises, focal neurological defects and frequent headaches. In our case, the condition started as a syndrome of intracranial hypertension with progressive headache and vomiting. During the illness, localizing neurological signs due to bleeding were seen. Amongst these were acute left hemiparesia and paralysis of vertical gaze. Other members of the family remain symptom-free. In a search for angiomas at other sites none were found in the patient or his family. Recently the gene giving rise to the familial cerebral cavernosa malformation has been found to be a locus on chromosome 7. We discuss the findings on neuro-imaging, emphasizing the importance of magnetic resonance (MR) both in diagnosis and finding affected asymptomatic family members, because of its great sensitivity and specificity. Angiography is not a suitable technique for this since they behave as hidden malformations. We also point out its importance as a way of following-up the illness and for evaluation of possible complications due to progressive growth or sudden haemorrhage, which may indicate the need for treatment. Finally we emphasize the different characteristics of MR signals in this type of lesion since cavernomatasa malformations are dynamic lesions. PMID:9172920

  6. Understanding Family Interaction Patterns in Families With Alzheimer's Disease.

    PubMed

    Schaber, Patricia; Blair, Kate; Jost, Ellen; Schaffer, Molly; Thurner, Emily

    2016-01-01

    This qualitative study explores the dynamic changes that occur in family interaction patterns when Alzheimer's disease is present. Semi-structured interviews were conducted with 15 participants who have a family member with the disease. Using modified analytic induction, guided by the dimensions of the Family Fundamental Interpersonal Relations Orientation (FIRO) Model, participants shared how Alzheimer's disease affected family structure, control dynamics, and intimacy among family members. Findings demonstrate that (a) families reorganize and restructure based on geographic proximity and shifting roles, act out of filial responsibility, and strive to preserve shared meanings and rituals; (b) decision making increases around care of the person with Alzheimer's disease and shifts to the primary caregiver or other family members based on their abilities; and (c) expressions of intimacy intensify while personality is preserved in the person with the disease. The Family FIRO model can inform practitioners using family-centered care with families with Alzheimer's disease.

  7. Care for bereaved families.

    PubMed

    Fukui, S

    1994-06-01

    Because of support groups and studies on death, a new type of care for bereaved parents has been crafted over the past several decades. Support groups and medical professionals are key to this type of care. For medical professionals this job is difficult at times since individual needs may differ. But by following some basic guidelines, medical professionals can help the parents start the recovery process for they are the ones in contact with the parents at the time of death. The guidelines include giving the families complete medical information, reassurance and a chance to talk. Also, importantly, this care includes treating the baby and family with dignity and acknowledging the magnitude of the loss. In Japan most bereaved parents still go completely unassisted at the time of their baby's death. Formation of support groups and a greater awareness by medical professionals on how to care for these families will solve this problem.

  8. AIDS and family planning.

    PubMed

    1992-01-01

    In 1991, an HIV prevention program advisor and a research/evaluation specialist for family planning programs discussed problems that affected HIV prevention and family planning services in Haiti before and after the coup of the Aristide government. Population activities began aimlessly in 1974 and HIV prevention efforts only began in 1988. After the coup, Haitians lost their newly found hope for meaningful development. All foreign assistance ended and they did not trust the army. In fact, other than essential child survival activities, no health and family planning services operated for several weeks. The situation grew worse after the economic embargo. 3 months after the coup, the US considered adding family planning assistance. Still little movement of condom, family planning, and health supplies left Port-au-Prince for the provinces which adversely affected all health related efforts. Condoms could no longer be distributed easily either in the socially marketed or US supplied condom distribution programs. Before the coup, HIV prevention and family planning programs depended on peer educators to educate the public (this approach made these programs quite successful), but the 2 experts feared that they would not return to those roles and that these programs would need to completely rebuild. Another concern was the large scale urban-rural migration making it difficult for them to continue care. Early in the AIDS epidemic, the Haitian government was on the defensive because the US considered Haitians as a high risk group so it did little to prevent HIV transmission. After 1988, HIV prevention activities in Haiti centered on raising awareness and personalizing the epidemic. The AIDS specialist noted, however, that a major obstacle to increasing knowledge is that AIDS is just 1 of many fatal diseases in Haiti. Moreover few health professionals in Haiti have ever had public health training. PMID:12159262

  9. Family Research Project Progress Report.

    ERIC Educational Resources Information Center

    Bell, David C.; Bell, Linda G.

    This document presents an overview and progress report on the Family Research Project, started in 1974 to (1) study the relationship between family process and individual development of family members, especially children, (2) conceptualize and measure system level variables describing family structure and process, (3) develop microanalytic…

  10. Changes in American Family Life

    ERIC Educational Resources Information Center

    Norton, Arthur J.; Glick, Paul C.

    1976-01-01

    This article attempts to provide a factual, historical perspective on the current family situation of American children. Demographic statistics from recent decades are given which show trends toward small family size, nuclear families, one-parent families, and a higher level of education among parents. (MS)

  11. Stable Black Families. Final Report.

    ERIC Educational Resources Information Center

    Gary, Lawrence E.; And Others

    This document is the final report of a study conducted to determine what factors contribute to strong Black family life and how these strong families solve problems, in order to add to the knowledge base on stable families so as to enhance practical intervention with families in need, and to identify models of self-help strategies used by stable…

  12. Trends in Family Child Care

    ERIC Educational Resources Information Center

    Neugebauer, Roger

    2011-01-01

    The author presents insights from various readers of "ExchangeEveryDay" regarding trends in the world of family child care. Kathleen Reticker of Acre Family Child Care in Lowell, Massachusetts thinks an increasing trend in Family Child Care is the pressure to emulate a Center, instead of seeing family child care as a different model. Over the…

  13. Characteristics of a Healthy Family.

    ERIC Educational Resources Information Center

    Lin, Phylis Lan

    The reason for studying the characteristics of a healthy family is to encourage and strengthen the family and to move toward an enriched family life by using the characteristics as bench marks. Six characteristics are discussed as the essence of a healthy family: (1) commitment; (2) togetherness; (3) appreciation; (4) good communication; (5)…

  14. Gendered Discourse about Family Business

    ERIC Educational Resources Information Center

    Danes, Sharon M.; Haberman, Heather R.; McTavish, Donald

    2005-01-01

    Language patterns of family business owners were explored by identifying discourse styles and emphasized ideas in four presenting contexts: business, family, intersection of family and business, and business success. The content analysis supports the existence of a general discourse style within family businesses and of similarities and…

  15. Work and Family. Special Focus.

    ERIC Educational Resources Information Center

    Goetz, Kathy, Ed.

    1992-01-01

    This newsletter issue focuses on issues concerning families with both parents employed outside the home and describes several employer programs designed to help employees balance their work and family life. The newsletter includes the following articles: (1) "Work and Family: 1992"; (2) "Levi Strauss and Co.--A Work/Family Program in Action"; (3)…

  16. Family Day Care Training Curriculum.

    ERIC Educational Resources Information Center

    Nakatsu, Gail

    California's Family Day Care Training Program was designed to recruit and train in 7 weeks, Lao, Vietnamese, and Chinese refugees to establish their own state-licensed, family day care homes. Topics in the program's curriculum include an introduction to family day care, state licenses for family day care, state licensing requirements for family…

  17. The Power of Family Literacy.

    ERIC Educational Resources Information Center

    National Center for Family Literacy, Louisville, KY.

    This report presents the early findings from the analysis of a family literacy demonstration project under the direction of the National Center for Family Literacy. The data in this report are based upon the experiences of over 300 families who participated in the Toyota Families for Learning Program during the 1992-1993 school year. The first…

  18. Familial hemicrania continua.

    PubMed

    Weatherall, Mark W; Bahra, Anish

    2011-01-01

    There are now three known causative genes for familial hemiplegic migraine and increasing evidence to support a genetic predisposition to the more common types of migraine with and without aura, and for cluster headache. We present the first reported case of familial hemicrania continua. A mother and daughter developed hemicrania continua at the same time of life. Both showed an absolute response to indometacin and at similar doses. Both also suffered from migraine with aura. We discuss the increasing support for a genetic predisposition to dysfunction of the pain system within the brain manifesting as primary headache.

  19. Family-witnessed resuscitation.

    PubMed

    Boucher, Melanie

    2010-09-01

    Family-witnessed resuscitation is a controversial subject for healthcare professionals and support for the practice is not universal (Albarran and Stafford 1999, Kissoon 2006). Research suggests, however, that the advantages of this form of resuscitation for relatives far outweigh the disadvantages, and that hospital staff can support the practice without hindering the clinical care of patients. This article explores the ethical issues raised, as well as the views of patients, families and staff on the subject, and suggests that there should be guidelines on the practice in all emergency departments where it is likely to take place.

  20. Getting a High-Speed Family Connection: Associations between Family Media Use and Family Connection

    ERIC Educational Resources Information Center

    Padilla-Walker, Laura M.; Coyne, Sarah M.; Fraser, Ashley M.

    2012-01-01

    The way families have used the media has substantially changed over the past decade. Within the framework of family systems theory, this paper examines the relations between family media use and family connection in a sample of 453 adolescents (mean age of child = 14.32 years, SD = 0.98, 52% female) and their parents. Results revealed that cell…

  1. Perceived Family Functioning and Family Resources of Hong Kong Families: Implications for Social Work Practice

    ERIC Educational Resources Information Center

    Ma, Joyce L. C.; Wong, Timothy K. Y.; Lau, Luk King; Pun, Shuk Han

    2009-01-01

    This article reports the results of a telephone survey (n = 1,015 respondents) that aims to identify the perceived general family functioning and family resources of Hong Kong Chinese families and their linkage to each other in a rapidly transforming society. The perceived general family functioning of the respondents was average, and the five…

  2. Family/Individual Health.

    ERIC Educational Resources Information Center

    Texas Tech Univ., Lubbock. Home Economics Curriculum Center.

    This curriculum guide consists of materials for use in planning and delivering junior high school homemaking courses focusing on individual and family health. Discussed first are program and curriculum planning. The next chapter focuses on the special needs of handicapped and disadvantaged learners and details strategies for addressing these…

  3. Family First Considerations

    ERIC Educational Resources Information Center

    LaFee, Scott

    2012-01-01

    The typical superintendent these days is male (though the percentage of female superintendents is steadily rising, now accounting for one in four, according to AASA's 2010 decennial study of the superintendency), in his 40s and almost always married with children. When educators become superintendents, the issues of family dynamics and related…

  4. Perspectives on Family Literacy.

    ERIC Educational Resources Information Center

    Literacy Assistance Center, New York, NY.

    This joint publication of the journals of the Literacy Assistance Center (LAC) and the National Even Start Association (NESA) focuses on innovative practices and theory in family literacy education, offers an array of perspectives to members of the literacy community, and critically examines some assumptions about literacy in general, as well as…

  5. Benign familial hyperphosphatasemia

    SciTech Connect

    Siraganian, P.A.; Mulvihill, J.J.; Mulivor, R.A.; Miller, R.W. )

    1989-03-03

    Elevated alkaline phosphatase activity in serum suggests bone or liver disease or a neoplasm but can also indicate pregnancy or another benign condition. A family with benign hyperphosphatasemia was studied to elucidate the genetics and enzyme defect. Serum total alkaline phosphatase activity was greater than the population mean in all six family members, and more than 7 SDs above the mean in two of four offspring. Monoclonal antibodies to three alkaline phosphatase isoenzymes, intestinal, placental, and tissue nonspecific demonstrated markedly increased intestinal alkaline phosphatase levels in all family members and significantly elevated liver/bone/kidney activity in the two offspring. Guanidine hydrochloride denaturation of the liver/bone/kidney component showed high alkaline phosphatase activity from liver in both siblings and from bone in one. The mode of inheritance in this family is obscure, but a complex regulation of the products of two different alkaline phosphatase genes seems likely. Steps toward diagnosis are suggested. Early recognition of this benign biochemical abnormality should help to avoid unnecessary diagnostic tests.

  6. Linking Families, Building Community.

    ERIC Educational Resources Information Center

    Fowler, R. Clarke; Corley, Kathy Klebs

    1996-01-01

    A year-round magnet elementary school in Salem, Massachusetts, has increased its family involvement by borrowing elements from many models. The school has mixed-age groupings, integrated social services, and community outreach strategies such as a parent center, business-sponsored Friday clubs, and celebrations of learning. (MLH)

  7. The Welfare of Families.

    ERIC Educational Resources Information Center

    Stern, Mark J.

    1987-01-01

    Outlines the most current statistics and demographics on the increase in poverty since 1980 in the United States, the increase of single parent families headed by women, and the increasing number of children who live in poverty (currently 21 percent). The reasons for the increased poverty are cited as due to government neglect. (MD)

  8. A Migrant Family.

    ERIC Educational Resources Information Center

    Brimner, Larry Dane

    This book incorporates many photographs portraying the life of a migrant family in a camp near San Diego, California. Houses in the camp are built of salvaged plywood with plastic sheets as roofs. Twelve-year-old Juan and his two younger brothers sleep on an old mattress atop a plywood platform. Juan's mother, stepfather, and younger sister sleep…

  9. Family Feathers. [Videotape Series].

    ERIC Educational Resources Information Center

    1999

    Family Feathers is a set of 18 videotapes for parents of preschool children, created by the Alaska Native Home Base Video Project of the Tlingit and Haida Head Start Program. This series offers culturally relevant solutions to the challenges of parenting, drawing on practical advice from Tlingit and Haida parents, wisdom from elders, and some of…

  10. Healthy Single Parent Families.

    ERIC Educational Resources Information Center

    Hanson, Shirley M. H.

    1986-01-01

    Investigated characteristics of healthy single-parent families. Single parents and their children reported fairly high levels of both physical and mental health. Communication, social support, socioeconomic status, religiousness, and problem solving were also correlated with the mental and physical health of parents and children. (Author/BL)

  11. One-Parent Families.

    ERIC Educational Resources Information Center

    Children's Bureau (DHEW), Washington, DC.

    There are thousands of men and women raising their children without the presence of the other parent. This brief booklet was written to help persons who are faced, or will be faced, with this situation. It begins with some shortened versions of discussions held with single parents and with children of one-parent families. Despite financial and…

  12. Family Support Networks.

    ERIC Educational Resources Information Center

    Frazier, Billie H.

    This document contains a brief bibliography of peer-reviewed literature, with abstracts, on family support networks. It is one of 12 bibliographies on aging prepared by the National Agricultural Library for its "Pathfinders" series of publications. Topics covered by the other 11 bibliographies include aging parents, adult children, dementia and…

  13. Children of Disrupted Families

    PubMed Central

    Robson, Bonnie E.

    1991-01-01

    While some children cope well with divorce, children of divorced parents are at increased risk for suicide, low self-esteem, affective disorders, and general distress. Useful intervention from the family physician usually begins with educating parents about what to expect and about the importance of open communication, co-operation between parents, and consistent discipline. PMID:21228993

  14. All in the Family

    ERIC Educational Resources Information Center

    Lum, Lydia

    2010-01-01

    Even as a little girl, Dr. Nitasha Sharma aspired to become a college professor like her parents, whose careers let the family spend entire summers or longer in either her mother's native Brooklyn, New York, or her father's native India. She dreamed of long vacations as a grown-up and going home for lunch on weekdays. But during a stay in India…

  15. Family reaction to homicide.

    PubMed

    Burgess, A N

    1975-04-01

    This pilot study identifies a two-phased syndrome experienced by families of homicide victims. The crisis phase consists of an acute grief process, including immediate reactions to the homicide, the funeral details, and police investigations. The long-term reorganization phase includes the psychological issues of bereavement and the socio-legal issues of the criminal justice process. PMID:1146971

  16. Not Your Family Farm

    ERIC Educational Resources Information Center

    Tenopir, Carol; Baker, Gayle; Grogg, Jill E.

    2007-01-01

    The information industry continues to consolidate, just as agribusiness has consolidated and now dominates farming. Both the family farm and the small information company still exist but are becoming rarer in an age of mergers, acquisitions, and increased economies of scale. Small companies distinguish themselves by high quality, special themes,…

  17. The Family of Adoption.

    ERIC Educational Resources Information Center

    Pavao, Joyce Maguire

    This book aims to provide a broad framework within which to think about adoption as a whole system, so that everyone involved will learn to feel some empathy for the other members of the adoption process. The book, written by a family and adoption therapist who was adopted as an infant, describes predictable developmental stages and challenges for…

  18. Creating Families of Readers.

    ERIC Educational Resources Information Center

    Graham, Wendy J.

    This document consists of materials developed and used by a project to research and design a prototype plan using the television program "Reading Rainbow" and the resources of the Western New York Public Broadcasting Association to cultivate family literacy. An executive summary presents findings from the six focus group discussions of the…

  19. Incarceration in Fragile Families

    ERIC Educational Resources Information Center

    Wildeman, Christopher; Western, Bruce

    2010-01-01

    Since the mid-1970s the U.S. imprisonment rate has increased roughly fivefold. As Christopher Wildeman and Bruce Western explain, the effects of this sea change in the imprisonment rate--commonly called mass imprisonment or the prison boom--have been concentrated among those most likely to form fragile families: poor and minority men with little…

  20. Changing Families Changing Schools.

    ERIC Educational Resources Information Center

    Evans, Robert

    The decline in civility, social responsibility, and institutional affiliation challenges the nature of schooling. Child development in the 1990s family is under pressure from changes that deny children the three basic essentials (nurture, structure, and latitude) for psychological health, effective learning, and civility, and that require children…