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Sample records for human phosphoinositide-specific phospholipase

  1. Cloning and characterization of the human phosphoinositide-specific phospholipase C-beta 1 (PLC beta 1).

    PubMed

    Caricasole, A; Sala, C; Roncarati, R; Formenti, E; Terstappen, G C

    2000-12-15

    Phospholipase C-beta (PLC beta) catalyses the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (IP(2)), a key step in the intracellular transduction of a large number of extracellular signals, including neurotransmitters and hormones modulating diverse developmental and functional aspects of the mammalian central nervous system. Four mammalian isozymes are known (PLC beta 1-4), which differ in their function and expression patterns in vivo. We have characterized the human PLC beta 1 genomic locus (PLC beta 1), cloned two distinct PLC beta 1 cDNAs (PLC beta 1a and b) and analysed their respective expression patterns in a comprehensive panel of human tissues using quantitative TaqMan technology. The two cDNAs derive from transcripts generated through alternative splicing at their 3' end, and are predicted to encode for PLC beta 1 isoforms differing at their carboxy-terminus. The human PLC beta 1 isoforms are co-expressed in the same tissues with a distinctly CNS-specific profile of expression. Quantitative differences in PLC beta 1 isoform expression levels are observed in some tissues. Transient expression of epitope-tagged versions of the two isoforms followed by immunofluorescence revealed localization of the proteins to the cytoplasm and the inner side of the cell membrane. Finally, we characterized the structure of the PLC beta 1 locus and confirmed its mapping to human chromosome 20.

  2. cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C{beta}4 (PLCB4)

    SciTech Connect

    Alvarez, R.A.; Ghalayini, A.J.; Anderson, R.E.

    1995-09-01

    Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC{beta}4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC{beta}4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC{beta}4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by florescence in situ hybridization. 4 refs., 5 figs.

  3. Phosphoinositide-specific phospholipase C in health and disease.

    PubMed

    Cocco, Lucio; Follo, Matilde Y; Manzoli, Lucia; Suh, Pann-Ghill

    2015-10-01

    Phospholipases are widely occurring and can be found in several different organisms, including bacteria, yeast, plants, animals, and viruses. Phospholipase C (PLC) is a class of phospholipases that cleaves phospholipids on the diacylglycerol (DAG) side of the phosphodiester bond producing DAGs and phosphomonoesters. Among PLCs, phosphoinositide-specific PLC (PI-PLC) constitutes an important step in the inositide signaling pathways. The structures of PI-PLC isozymes show conserved domains as well as regulatory specific domains. This is important, as most PI-PLCs share a common mechanism, but each of them has a peculiar role and can have a specific cell distribution that is linked to a specific function. More importantly, the regulation of PLC isozymes is fundamental in health and disease, as there are several PLC-dependent molecular mechanisms that are associated with the activation or inhibition of important physiopathological processes. Moreover, PI-PLC alternative splicing variants can play important roles in complex signaling networks, not only in cancer but also in other diseases. That is why PI-PLC isozymes are now considered as important molecules that are essential for better understanding the molecular mechanisms underlying both physiology and pathogenesis, and are also potential molecular targets useful for the development of innovative therapeutic strategies.

  4. 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.

  5. Multiple roles of phosphoinositide-specific phospholipase C isozymes.

    PubMed

    Suh, Pann-Ghill; Park, Jae-Il; Manzoli, Lucia; Cocco, Lucio; Peak, Joanna C; Katan, Matilda; Fukami, Kiyoko; Kataoka, Tohru; Yun, Sanguk; Ryu, Sung Ho

    2008-06-30

    Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.

  6. Phosphoinositide-specific Phospholipase C β1 gene deletion in bipolar disorder affected patient.

    PubMed

    Lo Vasco, Vincenza Rita; Longo, Lucia; Polonia, Patrizia

    2013-03-01

    The involvement of phosphoinositides (PI) signal transduction pathway and related molecules, such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, in the pathophysiology of mood disorders is corroborated by a number of recent evidences. Our previous works identified the deletion of PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in 4 out 15 patients affected with schizophrenia, and no deletion both in major depression affected patients and in normal controls. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with bipolar disorder. Deletion of PLCB1 was identified in one female patient.

  7. Phosphoinositide-specific phospholipase C in oat roots: association with the actin cytoskeleton.

    PubMed

    Huang, Chiung-Hua; Crain, Richard C

    2009-10-01

    Phosphoinositide-specific phospholipase C (PI-PLC) activities are involved in mediating plant cell responses to environmental stimuli. Two variants of PI-PLC have been partially purified from the roots of oat seedlings; one cytosolic and one particulate. Although the cytosolic enzyme was significantly purified, the activity still co-migrated with a number of other proteins on heparin HPLC and also on size-exclusion chromatography. The partially purified PI-PLC was tested by Western blotting, and we found that actin and actin-binding proteins, profilin and tropomyosin, co-purified with cytosolic phospholipase C. After a non-ionic detergent (Triton X-100) treatment, PI-PLC activities still remained with the actin cytoskeleton. The effects of phalloidin and F-buffer confirmed this association; these conditions, which favor actin polymerization, decreased the release of PI-PLC from the cytoskeleton. The treatments of latrunculin and G-buffer, the conditions that favor actin depolymerization, increased the release of PI-PLC from the cytoskeleton. These results suggest that oat PI-PLC associates with the actin cytoskeleton.

  8. A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants.

    PubMed

    Nakamura, Kimiyo; Sano, Hiroshi

    2009-01-01

    We previously screened genes that were transcriptionally activated during the early stage of wound response in tobacco plants (Nicotiana tabacum), and isolated a particular clone, which encoded a membrane-located protein, designated as NtC7. Upon overexpression in tobacco plants, NtC7 conferred a marked tolerance to osmotic stress, suggesting it to be involved in maintenance of osmotic adjustments. In this study, we searched for proteins which interact with NtC7 by the yeast two-hybrid screening, and isolated a clone encoding phosphoinositide-specific phospholipase C, designated as NtPI-PLC. Physical interaction between NtC7 and C2 domain of NtPI-PLC was confirmed by the pull-down assay. Expression of fused protein to green-fluorescence protein in onion epidermal cell layers indicated both proteins to predominantly localize to the plasma membrane. Their interaction in planta was shown by the bimolecular fluorescence complementation, which exhibited a clear fluorescence of reconstituted yellow fluorescence protein. Transcripts of NtC7 and NtPI-PLC were markedly increased 30 to 60 min after wounding. PI-PLC is one of key enzymes in metabolism of inositol phospholipids, which function in signal transduction and also in response to stresses including osmotic changes. It was shown to localize to plasma-membrane and, to a lesser extent, to cytosol. However, molecular mechanism of membrane localization has remained to be determined, because of the apparent lack of domains for membrane association. The present results suggest that one of such mechanisms is tethering NtPI-PLC to the plasma membrane through interaction with NtC7, which possesses a transmembrane domain at the C-terminus.

  9. Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide.

    PubMed

    Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Fumagalli, Lorenzo; Cocco, L

    2010-04-01

    Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide-specific phospholipase C (PI-PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI-PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI-PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL-6. PI-PLCs expression in LPS treated neonatal rat astrocytes performed by using RT-PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI-PLC beta1, beta4 and gamma1 in all intervals analysed; PI-PLC delta1 at 6, 18 and 24 h; PI-PLC delta3 at 6 h after treatment. PI-PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub-cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI-PLCs expression and distribution may play a role in ongoing inflammation process of CNS.

  10. Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

    PubMed Central

    Béziau, Delphine M.; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R.; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

    2015-01-01

    Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the

  11. Molecular cytogenetic interphase analysis of Phosphoinositide-specific Phospholipase C β1 gene in paraffin-embedded brain samples of major depression patients.

    PubMed

    Lo Vasco, Vincenza Rita; Polonia, Patrizia

    2012-01-01

    Mood disorders represent a major medical need, as their chronic treatments are not effective in all patients. Literature data suggested that phosphoinositides (PI) signal transduction pathway and related molecules such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, might be involved in the pathophysiology of mood disorders, including major depression. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with major depression and in 15 normal controls. No deletions of PLCB1 were identified with the methodology used, which allows to exclude wide gene deletions. The results, the technical aspects of the FISH methodology, and its limitations are discussed.

  12. Expression pattern and sub-cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells.

    PubMed

    Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Panetta, Barbara; Fumagalli, Lorenzo; Cocco, Lucio

    2010-07-01

    Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.

  13. Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes

    PubMed Central

    2004-01-01

    Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs. PMID:15554872

  14. 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.

  15. The physiological roles of primary phospholipase C.

    PubMed

    Yang, Yong Ryoul; Follo, Matilde Y; Cocco, Lucio; Suh, Pann-Ghill

    2013-09-01

    The roles of phosphoinositide-specific phospholipase C (PLC) have been extensively investigated in diverse cell lines and pathological conditions. Among the PLC isozmes, primary PLCs, PLC-β and PLC-γ, are directly activated by receptor activation, unlike other secondary PLCs (PLC-ɛ, PLC-δ1, and PLC-η1). PLC-β isozymes are activated by G protein couple receptor and PLC-γ isozymes are activated by receptor tyrosine kinase (RTK). Primary PLCs are differentially expressed in different tissues, suggesting their specific roles in diverse tissues and regulate a variety of physiological and pathophysiological functions. Thus, dysregulation of phospholipases contributes to a number of human diseases and primary PLCs have been identified as therapeutic targets for prevention and treatment of diseases. Here we review the roles of primary PLCs in physiology and their impact in pathology.

  16. Role of cytosolic phospholipase A2 in cytokine-stimulated prostaglandin release by human gallbladder cells.

    PubMed

    Grossmann, E M; Longo, W E; Mazuski, J E; Panesar, N; Kaminski, D L

    2000-01-01

    Eicosanoids are involved in gallbladder inflammation, epithelial water transport, and mucous secretion. Phospholipase Asubscript2 enzymes liberate arachidonic acid from membrane phospholipids for the synthesis of eicosanoids. The purpose of this study was to determine the effect of selective cytoplasmic and secretory phospholipase A2 inhibitors on basal and stimulated arachidonic acid and prostaglandin E2 release in gallbladder cells. Western immunoblotting was employed to evaluate both cytosolic and secretory phospholipase A2 enzymes in human gallbladder cells. Cells were incubated for 22 hours with (3)H-labeled arachidonic acid. Arachidonic acid and prostaglandin E2 release was then measured in the supernate after 2 hours of exposure to human interleukin-1beta, alone or after pretreatment for 1 hour with the inhibitors. Unstimulated gallbladder cells express both 85 kDa cytosolic and 14 kDa secretory phospholipase A2++. The 85 kDa phospholipase A2 was induced by interleukin-1beta, whereas there was no apparent change in secretory phospholipase A2 enzyme concentrations. Both the secretory phospholipase A2 inhibitor p-bromophenylacyl bromide and the cytosolic phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone decreased basal and interleukin-1beta-stimulated arachidonic acid release. In contrast, only inhibition of cytosolic phospholipase A2 led to a decrease in interleukin-1beta-stimulated prostaglandin E2 release. Basal and interleukin-1beta-stimulated arachidonic acid release appears to be the result of the activity of both cytosolic and secretory phospholipase A2. Interleukin-1beta-stimulated prostaglandin E2 release appears to be dependent on the activity of cytosolic phospholipase A2.

  17. Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction.

    PubMed

    Martelli, A M; Fiume, R; Faenza, I; Tabellini, G; Evangelista, C; Bortul, R; Follo, M Y; Falà, F; Cocco, L

    2005-10-01

    Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.

  18. Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.

    PubMed

    Sato, H; Aono, S; Semba, R; Kashiwamata, S

    1987-11-15

    Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.

  19. Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier

    PubMed Central

    DiStefano, Peter V.; Smrcka, Alan V.; Glading, Angela J.

    2016-01-01

    The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP2 hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability. PMID:27612188

  20. Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier.

    PubMed

    DiStefano, Peter V; Smrcka, Alan V; Glading, Angela J

    2016-01-01

    The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP2 hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability.

  1. Mastoparan-induced phosphatidylcholine hydrolysis by phospholipase D activation in human astrocytoma cells.

    PubMed Central

    Mizuno, K.; Nakahata, N.; Ohizumi, Y.

    1995-01-01

    1. The effect of mastoparan on phosphatidylcholine hydrolysis was examined in 1321N1 human astrocytoma cells. Mastoparan (3-30 microM) caused an accumulation of diacylglycerol (DG) and phosphatidic acd (PA) accompanied by choline release in a concentration- and time-dependent manner. 2. In the presence of 2% n-butanol, mastoparan (3-100 microM) induced phosphatidylbutanol (PBut) accumulation in a concentration- and time-dependent manner, suggesting that mastoparan activates phospholipase D (PLD). Propranolol (30-300 microM), a phosphatidate phosphohydrolase inhibitor, inhibited DG accumulation induced by mastoparan, supporting this idea. 3. Depletion of extracellular free calcium ion did not alter the effect of mastoparan on PLD activity. 4. A protein kinase C (PKC) inhibitor, calphostin C (1 microM), did not inhibit mastoparan-induce PLD activation but the ability of mastoparan to stimulate phospholipase D activity was decreased in the PKC down regulated cells. 5. PLD activity stimulated by mastoparan was not prevented by pretreatment of the cells with pertussis toxin (PT) or C3 ADP-ribosyltransferase. Furthermore, guanine nucleotides did not affect PLD activity stimulation by mastoparan in membrane preparations. 6. Mastoparan stimulated PLD in several cell lines such as RBL-2H3, RBL-1, HL-60, P388, endothelial cells, as well as 1321N1 human astrocytoma cells. 7. These results suggest that mastoparan induces phosphatidylcholine (PC) hydrolysis by activation of PLD, not by activation of phosphatidylcholine-specific phospholipase C (PC-PLC); mastoparan-induced PLD activation is not mediated by G proteins. PMID:8640350

  2. Human Cytomegalovirus Carries a Cell-Derived Phospholipase A2 Required for Infectivity

    PubMed Central

    Allal, Cuider; Buisson-Brenac, Claire; Marion, Vincent; Claudel-Renard, Clotilde; Faraut, Thomas; Dal Monte, Paola; Streblow, Daniel; Record, Michel

    2004-01-01

    Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2α, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity. PMID:15220446

  3. Long-wave ultraviolet light induces phospholipase activation in cultured human epidermal keratinocytes

    SciTech Connect

    Hanson, D.; DeLeo, V. )

    1990-08-01

    Long wave ultraviolet radiation (UVA) has been shown to play an important role in the overall response of skin to solar radiation, including sunburn, tanning, premature aging, and non-melanoma skin cancer. UVA induction of inflammation in human skin is thought to be mediated by membrane lipid derived products. In order to investigate the mechanism of this response we examined the effect of UVA on phospholipid metabolism of human epidermal keratinocytes in culture. Keratinocytes were grown in serum free low calcium medium. The cells were prelabeled with (3H) arachidonic acid or (3H) choline and irradiated with UVA (Honle 2002-Hg vapor lamp). Identification and quantitation of specific membrane phospholipid-derived components was achieved using high-performance liquid chromatography, paper chromatography, and radioimmunoassay. UVA resulted in a linear dose dependent release of (3H) arachidonic acid into medium between 1 and 20 joule/cm2. This response was inhibited in an oxygen-reduced environment. The radiolabel released was predominantly free arachidonate and cyclooxygenase metabolites. Cyclooxygenase metabolites prostaglandin E2 and prostacyclin derivative, 6-keto-prostaglandin F1a, were stimulated following UVA irradiation, but the lipoxygenase metabolite, leukotriene B was not detected. Maximal release was measured immediately after irradiation and changed little over 24 h post-irradiation. UVA stimulated an increase of (3H) choline metabolites glycerophosphorylcholine and phosphorylcholine in media extracts suggesting UVA activation of phospholipase C and phospholipase A2 or diacylglyceride lipase.

  4. Mechanism of inhibition of human secretory phospholipase A2 by flavonoids: rationale for lead design

    NASA Astrophysics Data System (ADS)

    Lättig, Jens; Böhl, Markus; Fischer, Petra; Tischer, Sandra; Tietböhl, Claudia; Menschikowski, Mario; Gutzeit, Herwig O.; Metz, Peter; Pisabarro, M. Teresa

    2007-08-01

    The human secretory phospholipase A2 group IIA (PLA2-IIA) is a lipolytic enzyme. Its inhibition leads to a decrease in eicosanoids levels and, thereby, to reduced inflammation. Therefore, PLA2-IIA is of high pharmacological interest in treatment of chronic diseases such as asthma and rheumatoid arthritis. Quercetin and naringenin, amongst other flavonoids, are known for their anti-inflammatory activity by modulation of enzymes of the arachidonic acid cascade. However, the mechanism by which flavonoids inhibit Phospholipase A2 (PLA2) remained unclear so far. Flavonoids are widely produced in plant tissues and, thereby, suitable targets for pharmaceutical extractions and chemical syntheses. Our work focuses on understanding the binding modes of flavonoids to PLA2, their inhibition mechanism and the rationale to modify them to obtain potent and specific inhibitors. Our computational and experimental studies focused on a set of 24 compounds including natural flavonoids and naringenin-based derivatives. Experimental results on PLA2-inhibition showed good inhibitory activity for quercetin, kaempferol, and galangin, but relatively poor for naringenin. Several naringenin derivatives were synthesized and tested for affinity and inhibitory activity improvement. 6-(1,1-dimethylallyl)naringenin revealed comparable PLA2 inhibition to quercetin-like compounds. We characterized the binding mode of these compounds and the determinants for their affinity, selectivity, and inhibitory potency. Based on our results, we suggest C(6) as the most promising position of the flavonoid scaffold to introduce chemical modifications to improve affinity, selectivity, and inhibition of PLA2-IIA by flavonoids.

  5. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

    PubMed Central

    Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

    2016-01-01

    Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

  6. Expression of group XIIA phospholipase A2 in human digestive organs.

    PubMed

    Peuravuori, Heikki; Kollanus, Sinikka; Nevalainen, Timo J

    2014-12-01

    Cellular distribution of group XIIA phospholipase A2 (GXIIA PLA2) was studied in human digestive organs by immunohistochemistry. GXIIA PLA2 protein was detected in epithelial cells of normal gastrointestinal tract, gallbladder and pancreatic acinar cells. The GXIIA PLA2 protein was evenly distributed in the cytoplasm in contrast to secretory granular distribution of GIB PLA2 and GIIA PLA2 in pancreatic acinar cells and small intestinal Paneth cells respectively. Epithelial cells of intestinal glands in Crohn's disease and ulcerative colitis expressed abundant GXIIA PLA2 , whereas inflammatory cells were devoid of the enzyme protein. Tumour cells in colonic adenomas and carcinomas and pancreatic ductogenic carcinomas expressed GXIIA PLA2 protein at varying intensity levels. The putative functions of GXIIA PLA2 remain to be investigated and its role in healthy and diseased digestive organs can only be speculated on at present.

  7. Preservation of bilayer structure in human erythrocytes and erythrocyte ghosts after phospholipase treatment. A 31P-NMR study.

    PubMed

    van Meer, G; de Kruijff, B; op den Kamp, J A; van Deenen, L L

    1980-02-15

    1. Fresh human erythrocytes were treated with lytic and non-lytic combinations of phospholipases A2, C and sphingomyelinase. The 31P-NMR spectra of ghosts derived from such erythrocytes show that, in all cases, the residual phospholipids and lysophospholipids remain organized in a bilayer configuration. 2. A bilayer configuration of the (lyso)phospholipids was also observed after treatment of erythrocyte ghosts with various phospholipases even in the case that 98% of the phospholipid was converted into lysophospholipid (72%) and ceramides (26%). 3. A slightly decreased order of the phosphate group of phospholipid molecules, seen as reduced effective chemical shift anisotropy in the 31P-NMR spectra, was found following the formation of diacyglycerols and ceramides in the membrane of intact erythrocytes. Treatment of ghosts always resulted in an extensive decrease in the order of the phosphate groups. 4. The results allow the following conclusions to made: a. Hydrolysis of phospholipids in intact red cells and ghosts does not result in the formation of non-bilayer configuration of residual phospholipids and lysophospholipids. b. Haemolysis, which is obtained by subsequent treatment of intact cells with sphingomyelinase and phospholipase A2, or with phospholipase C, cannot be ascribed to the formation of non-bilayer configuration of phosphate-containing lipids. c. Preservation of bilayer structure, even after hydrolysis of all phospholipid, shows that other membrane constitutents, e.g. cholesterol and/or membrane proteins play an important role in stabilizing the structure of the erythrocyte membrane. d. A major prerequisite for the application of phospholipases in lipid localization studies, the preservation of a bilayer configuration during phospholipid hydrolysis, is met for the erythrocyte membrane.

  8. Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases

    PubMed Central

    Boynton, Tye O'Hara; Shimkets, Lawrence Joseph

    2015-01-01

    Myxococcus xanthus development requires CsgA, a member of the short-chain alcohol dehydrogenase (SCAD) family of proteins. We show that CsgA and SocA, a protein that can replace CsgA function in vivo, oxidize the 2′-OH glycerol moiety on cardiolipin and phosphatidylglycerol to produce diacylglycerol (DAG), dihydroxyacetone, and orthophosphate. A lipid extract enriched in DAGs from wild-type cells initiates development and lipid body production in a csgA mutant to bypass the mutational block. This novel phospholipase C-like reaction is widespread. SCADs that prevent neurodegenerative disorders, such as Drosophila Sniffer and human HSD10, oxidize cardiolipin with similar kinetic parameters. HSD10 exhibits a strong preference for cardiolipin with oxidized fatty acids. This activity is inhibited in the presence of the amyloid β peptide. Three HSD10 variants associated with neurodegenerative disorders are inactive with cardiolipin. We suggest that HSD10 protects humans from reactive oxygen species by removing damaged cardiolipin before it induces apoptosis. PMID:26338420

  9. Phospholipase D-mTOR requirement for the Warburg effect in human cancer cells.

    PubMed

    Toschi, Alfredo; Lee, Evan; Thompson, Sebastian; Gadir, Noga; Yellen, Paige; Drain, C Michael; Ohh, Michael; Foster, David A

    2010-12-18

    A characteristic of cancer cells is the generation of lactate from glucose in spite of adequate oxygen for oxidative phosphorylation. This property - known as the "Warburg effect" or aerobic glycolysis - contrasts with anaerobic glycolysis, which is triggered in hypoxic normal cells. The Warburg effect is thought to provide a means for cancer cells to survive under conditions where oxygen is limited and to generate metabolites necessary for cell growth. The shift from oxidative phosphorylation to glycolysis in response to hypoxia is mediated by the production of hypoxia-inducible factor (HIF) - a transcription factor family that stimulates the expression of proteins involved in glucose uptake and glycolysis. We reported previously that elevated phospholipase D (PLD) activity in renal and breast cancer cells is required for the expression of the α subunits of HIF1 and HIF2. We report here that the aerobic glycolysis observed in human breast and renal cancer cells is dependent on the elevated PLD activity. Intriguingly, the effect of PLD on the Warburg phenotype was dependent on the mammalian target of rapamycin complex 1 (mTORC1) in the breast cancer cells and on mTORC2 in the renal cancer cells. These data indicate that elevated PLD-mTOR signaling, which is common in human cancer cells, is critical for the metabolic shift to aerobic glycolysis.

  10. 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.

  11. Crystal structure of human secretory phospholipase A2-IIA complex with the potent indolizine inhibitor 120-1032.

    PubMed

    Kitadokoro, K; Hagishita, S; Sato, T; Ohtani, M; Miki, K

    1998-04-01

    Phospholipase A2 is a key enzyme in a number of physiologically important cellular processes including inflammation and transmembrane signaling. Human secretory phospholipase A2-IIA is present at high concentrations in synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. Inhibitors of this enzyme have been suggested to be therapeutically useful non-steroidal anti-inflammatory drugs. The crystal structure of human secretory phospholipase A2-IIA bound to a novel potent indolizine inhibitor (120-1032) has been determined. The complex crystallizes in the space group P3121, with cell dimensions of a = b = 75.8 A and c = 51.3 A. The model was refined to an R-factor of 0. 183 for the intensity data collected to a resolution of 2.2 A. It was revealed that the inhibitor is located near the active site and bound to the calcium ion. Although the binding mode of the 120-1032 inhibitor to human secretory phospholipase A2-IIA is similar to that previously determined for an indole inhibitor LY311299, the specific interactions between the enzyme and the inhibitor in the present complex include the oxycarboxylate group which was introduced in this inhibitor. The oxycarboxylate group in 120-1032 is coordinated to the calcium ion and included in the water-mediated hydrogen bonding to the catalytic Asp49. In addition, the ethyl group in 120-1032 gains hydrophobic contacts with the cavity wall of the hydrophobic channel of the enzyme.

  12. Unlike thyrotropin, thyroid-stimulating antibodies do not activate phospholipase C in human thyroid slices.

    PubMed Central

    Laurent, E; Van Sande, J; Ludgate, M; Corvilain, B; Rocmans, P; Dumont, J E; Mockel, J

    1991-01-01

    The effects of thyroid-stimulating antibodies (TSAb) and of thyrotropin (TSH) were compared, on the generation of cyclic AMP and inositol phosphates (InsP), in human thyroid slices incubated in vitro, and on the Rapoport cyclic AMP bioassay. The TSAb positive sera were obtained from 19 patients with Graves' disease. In 14 experiments with the slices system, TSH significantly increased cyclic AMP accumulation (TSH, 0.03-10 mU/ml) as well as the cyclic AMP-independent inositol trisphosphate (InsP3) generation (TSH, 1-10 mU/ml). In the same 14 experiments, TSAb (0.10-28 mg/ml) enhanced cyclic AMP intracellular levels as expected while they did not induce any InsP accumulation. Even when TSAb increased cyclic AMP levels to the same or higher values as those obtained with TSH concentrations allowing InsP3 generation. TSAb were still unable to activate the phosphatidylinositol-Ca2+ cascade. The patterns of the response curves of TSAb and TSH on cyclic AMP accumulation were different, suggesting that different mechanisms may be involved. In addition, unlike TSH, TSAb were not able to stimulate H2O2 generation, which in human tissue mainly depends on the activation of the phosphatidylinositol-Ca2+ cascade. Immunoglobulins from six additional Graves' patients lacking measurable cyclic AMP-stimulating activity in both slices and cells systems did not activate phospholipase C either. In conclusion, our results show that TSAb do not share all the metabolic actions of TSH on human thyroid tissue. The data provide support for the concept that the pathogenesis of Graves' disease can be fully accounted for by the ability of TSAb to stimulate adenylate cyclase. This work also confirms that TSH activates the cyclic AMP and the phosphatidylinositol cascade by independent pathways in the human thyroid. PMID:1673689

  13. Regulation of human eosinophil degranulation and activation by endogenous phospholipase A2.

    PubMed Central

    White, S R; Strek, M E; Kulp, G V; Spaethe, S M; Burch, R A; Neeley, S P; Leff, A R

    1993-01-01

    The unique granular proteins of eosinophils may have a pathogenetic role in asthma and in the defense against parasitic infestations. However, the mechanisms regulating eosinophil degranulation are largely unknown. We examined the hypothesis that release of these proteins is regulated by endogenous activation of phospholipase A2. Human eosinophils (HE) were isolated from the peripheral blood of 42 subjects either by Percoll density separation or by negative-selection immunomagnetic fractionation. Eosinophil activation was initiated in vitro with 10(-6) M FMLP and 5 micrograms/ml cytochalasin B and was assessed by measurement of eosinophil peroxidase (EPO), leukotriene C4 (LTC4) and superoxide radical (.O2-) secretion. Treatment of HE with 100 microM mepacrine before activation blocked EPO release (2.0 +/- 0.2 vs 10.2 +/- 2.1% cell content for activated HE, P < 0.004, n = 9), .O2- generation (2.6 +/- 0.9 vs 44.2 +/- 10.8 nmol/ml per 10(6) HE, P < 0.002, n = 5), and LTC4 secretion (68.2 +/- 32.2 vs 1,125.2 +/- 526.8 pg/ml per 10(6) HE, P < 0.04, n = 8). Pretreatment of HE with 100 microM 4-bromophenacyl bromide before activation similarly blocked EPO release, .O2- generation and LTC4 secretion. Addition of AA to HE after treatment with 100 microM mepacrine and before subsequent activation reversed the inhibition of both EPO (10.4 +/- 2.2% with 1 microM AA vs 2.0 +/- 0.2% for mepacrine, n = 5, P < 0.02) and LTC4 secretion (695.1 +/- 412.9 with 10 microM AA vs 68.2 +/- 32.2 pg/ml per 10(6) HE for mepacrine, n = 8, P < 0.04), but did not reverse inhibition of .O2- generation by mepacrine. We demonstrate that secretion of preformed cytotoxic proteins and .O2- by eosinophils is regulated endogenously by phospholipase A2. PMID:8387540

  14. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition.

    PubMed

    Wang, Hui; Klein, Michael G; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching

    2016-07-03

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  15. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

    SciTech Connect

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching

    2016-07-01

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  16. Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2*

    PubMed Central

    Olson, Erin D.; Nelson, Jennifer; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Gelb, Michael H.; Judd, Allan M.; Bell, John D.

    2010-01-01

    Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform. PMID:20139082

  17. High specificity of human secretory class II phospholipase A2 for phosphatidic acid.

    PubMed

    Snitko, Y; Yoon, E T; Cho, W

    1997-02-01

    Lysophosphatidic acid (LPA) is a potent lipid second messenger which stimulates platelet aggregation, cell proliferation and smooth-muscle contraction. The phospholipase A2 (PLA2)-catalysed hydrolysis of phosphatidic acid (PA) is thought to be a primary synthetic route for LPA. Of the multiple forms of PLA2 present in human tissues, human secretory class-II PLA2 (hs-PLA2) has been implicated in the production of LPA from platelets and whole blood cells challenged with inflammatory stimuli. To explore further the possibility that hs-PLA2 is involved in the production of LPA, we rigorously measured the phospholipid head group specificity of hs-PLA2 by a novel PLA2 kinetic system using polymerized mixed liposomes. Kinetic analysis of recombinant hs-PLA2 demonstrates that hs-PLA2 strongly prefers PA as substrate over other phospholipids found in the mammalian plasma membrane including phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The order of preference is PA > PE approximately PS > PC. To identify amino acid residues of hs-PLA2 that are involved in its unique substrate specificity, we mutated two residues, Glu-56 and Lys-69, which were shown to interact with the phospholipid head group in the X-ray-crystallographic structure of the hs-PLA2-transition-state-analogue complex. The K69Y mutant showed selective inactivation toward PA whereas the E56K mutant displayed a most pronounced inactivation to PE. Thus it appears that Lys-69 is at least partially involved in the PA specificity of hs-PLA2 and Glu-56 in the distinction between PE and PC. In conjunction with a recent cell study [Fourcade, Simon, Viode, Rugani, Leballe, Ragab, Fournie, Sarda and Chap (1995) Cell 80, 919-927], these studies suggest that hs-PLA2 can rapidly hydrolyse PA molecules exposed to the outer layer of cell-derived microvesicles and thereby produce LPA.

  18. Phospholipases A in Trypanosomatids

    PubMed Central

    Belaunzarán, María Laura; Lammel, Estela María; de Isola, Elvira Luisa Durante

    2011-01-01

    Phospholipases are a complex and important group of enzymes widespread in nature, that play crucial roles in diverse biochemical processes and are classified as A1, A2, C, and D. Phospholipases A1 and A2 activities have been linked to pathogenesis in various microorganisms, and particularly in pathogenic protozoa they have been implicated in cell invasion. Kinetoplastids are a group of flagellated protozoa, including extra- and intracellular parasites that cause severe disease in humans and animals. In the present paper, we will mainly focus on the three most important kinetoplastid human pathogens, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., giving a perspective of the research done up to now regarding biochemical, biological, and molecular characteristics of Phospholipases A1 and A2 and their contribution to pathogenesis. PMID:21603263

  19. Inhibition of phospholipaseD2 increases hypoxia-induced human colon cancer cell apoptosis through inactivating of the PI3K/AKT signaling pathway.

    PubMed

    Liu, Maoxi; Fu, Zhongxue; Wu, Xingye; Du, Kunli; Zhang, Shouru; Zeng, Li

    2016-05-01

    Hypoxia is a common feature of solid tumor, and is a direct stress that triggers apoptosis in many human cell types. As one of solid cancer, hypoxia exists in the whole course of colon cancer occurrence and progression. Our previous studies shown that hypoxia induce high expression of phospholipase D2 (PLD2) and survivin in colon cancer cells. However, the correlation between PLD2 and survivin in hypoxic colon cancer cells remains unknown. In this study, we observed significantly elevated PLD2 and survivin expression levels in colon cancer tissues and cells. This is a positive correlation between of them, and co-expression of PLD2 and survivin has a positive correlation with the clinicpatholic features including tumor size, TNM stage, and lymph node metastasis. We also found that hypoxia induced the activity of PLD increased significant mainly caused by PLD2 in colon cancer cells. However, inhibition the activity of PLD2 induced by hypoxia promotes the apoptosis of human colon cancer cells, as well as decreased the expression of apoptosis markers including survivin and bcl2. Moreover, the pharmacological inhibition of PI3K/AKT supported the hypothesis that promotes the apoptosis of hypoxic colon cancer cells by PLD2 activity inhibition may through inactivation of the PI3K/AKT signaling pathway. Furthermore, interference the PLD2 gene expression leaded to the apoptosis of hypoxic colon cancer cells increased and also decreased the expression level of survivin and bcl2 may through inactivation of PI3K/AKT signaling pathway. These results indicated that PLD2 play antiapoptotic role in colon cancer under hypoxic conditions, inhibition of the activity, or interference of PLD2 gene expression will benefit for the treatment of colon cancer patients.

  20. Evidence for two forms of phospholipase A2 in human semen

    SciTech Connect

    Antaki, P.; Langlais, J.; Ross, P.; Guerette, P.; Roberts, K.D.

    1988-03-01

    The molecular weight of the active unit of phospholipase A2 (PA2) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA2 activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 degrees C prior to irradiation, the sperm exhibited PA2 activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA2 was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA2 activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at -20 degrees C for 48 hr followed by a heating period of 10 min at 60 degrees C. Long-term storage of spermatozoa at -20 degrees C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 degrees C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 degrees C or -20 degrees C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA2 when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA2 was found and was sensitive to changes in temperature.

  1. Disposition and metabolism of darapladib, a lipoprotein-associated phospholipase A2 inhibitor, in humans.

    PubMed

    Dave, Mehul; Nash, Mike; Young, Graeme C; Ellens, Harma; Magee, Mindy H; Roberts, Andrew D; Taylor, Maxine A; Greenhill, Robert W; Boyle, Gary W

    2014-03-01

    The absorption, metabolism, and excretion of darapladib, a novel inhibitor of lipoprotein-associated phospholipase A2, was investigated in healthy male subjects using [(14)C]-radiolabeled material in a bespoke study design. Disposition of darapladib was compared following single i.v. and both single and repeated oral administrations. The anticipated presence of low circulating concentrations of drug-related material required the use of accelerator mass spectrometry as a sensitive radiodetector. Blood, urine, and feces were collected up to 21 days post radioactive dose, and analyzed for drug-related material. The principal circulating drug-related component was unchanged darapladib. No notable metabolites were observed in plasma post-i.v. dosing; however, metabolites resulting from hydroxylation (M3) and N-deethylation (M4) were observed (at 4%-6% of plasma radioactivity) following oral dosing, indicative of some first-pass metabolism. In addition, an acid-catalyzed degradant (M10) resulting from presystemic hydrolysis was also detected in plasma at similar levels of ∼5% of radioactivity post oral dosing. Systemic exposure to radioactive material was reduced within the repeat dose regimen, consistent with the notion of time-dependent pharmacokinetics resulting from enhanced clearance or reduced absorption. Elimination of drug-related material occurred predominantly via the feces, with unchanged darapladib representing 43%-53% of the radioactive dose, and metabolites M3 and M4 also notably accounting for ∼9% and 19% of the dose, respectively. The enhanced study design has provided an increased understanding of the absorption, distribution, metabolism and excretion (ADME) properties of darapladib in humans, and substantially influenced future work on the compound.

  2. Phospholipase C Activity in Human Polymorphonuclear Leukocytes: Partial Characterization and Effect of Indomethacin

    DTIC Science & Technology

    1988-12-01

    phospholipase C activity alone, and in the presence of 0.5 mM and I mM indomethacin, is plotted according to Lineweaver and Burke as described previously...The data were plotted according to the method of Lineweaver and Burke (26). The values represent the mean + S.E.M. of values derived from neutrophils of 4 subjects. 18

  3. Lipoprotein-associated phospholipase A2 decreases oxidized lipoprotein cellular association by human macrophages and hepatocytes.

    PubMed

    Yang, Ming; Chu, Eugene M; Caslake, Muriel J; Edelstein, Celina; Scanu, Angelo M; Hill, John S

    2010-02-01

    We investigated whether the presence of endogenous or exogenous lipoprotein-associated phospholipase A2 (Lp-PLA2) can modify the cellular association of oxidized low density lipoprotein (oxLDL) and oxidized lipoprotein(a) (oxLp(a)) by human monocyte-derived macrophages (MDM) and hepatocytes (HepG2). Purified recombinant Lp-PLA2 was used as a source of exogenous enzyme whereas Pefabloc (serine esterase inhibitor) was used to inhibit the endogenous Lp-PLA2 activity associated with isolated lipoproteins. Cellular association studies were performed with DiI-labeled oxLDL or oxLp(a) and human monocyte-derived macrophages and HepG2 cells. Active Lp-PLA2 decreased the cellular association of oxLDL and oxLp(a) in macrophages and HepG2 cells by approximately 30-40%, whereas the inactive enzyme did not significantly change oxidized lipoprotein cellular association by either cell type. OxLDL pretreated by Pefabloc increased oxLDL cellular association by MDM and HepG2 cells compared to untreated oxLDL. Therefore, unlike some lipases, Lp-PLA2 did not appear to have any catalytic independent function in oxLDL cellular association. To assess whether the reduced cellular association mediated by Lp-PLA2 was due to the hydrolysis of oxidized phosphatidylcholine (oxPC), we measured the concentration of lysophosphatidylcholine (lysoPC) in lipoprotein fractions after Lp-PLA2 treatment. LysoPC was increased by 20% (0.4 microM) and 87% (0.7 microM) by active Lp-PLA2 compared to inactive Lp-PLA2 for oxLDL and Lp(a), respectively. LysoPC at higher concentration dose-dependently increased the cellular association of oxLDL and oxLp(a) in MDM and HepG2 cells. We conclude that Lp-PLA2 mediates a decrease in oxidized lipoprotein cellular association in human macrophages and HepG2 cells by reducing the concentration of oxPC within these lipoproteins.

  4. Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: minimising venom toxicity.

    PubMed

    Funayama, Jaqueline C; Pucca, Manuela B; Roncolato, Eduardo C; Bertolini, Thaís B; Campos, Lucas B; Barbosa, José E

    2012-03-01

    The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

  5. Phospholipase C-γ1 involved in brain disorders.

    PubMed

    Jang, Hyun-Jun; Yang, Yong Ryoul; Kim, Jung Kuk; Choi, Jang Hyun; Seo, Young-Kyo; Lee, Yong Hwa; Lee, Jeung Eun; Ryu, Sung Ho; Suh, Pann-Ghill

    2013-01-01

    Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) is an important signaling regulator involved in various cellular processes. In brain, PLC-γ1 is highly expressed and participates in neuronal cell functions mediated by neurotrophins. Consistent with essential roles of PLC-γ1, it is involved in development of brain and synaptic transmission. Significantly, abnormal expression and activation of PLC-γ1 appears in various brain disorders such as epilepsy, depression, Huntington's disease and Alzheimer's disease. Thus, PLC-γ1 has been implicated in brain functions as well as related brain disorders. In this review, we discuss the roles of PLC-γ1 in neuronal functions and its pathological relevance to diverse brain diseases.

  6. sPLA2 IB induces human podocyte apoptosis via the M-type phospholipase A2 receptor.

    PubMed

    Pan, Yangbin; Wan, Jianxin; Liu, Yipeng; Yang, Qian; Liang, Wei; Singhal, Pravin C; Saleem, Moin A; Ding, Guohua

    2014-10-22

    The M-type phospholipase A2 receptor (PLA2R) is expressed in podocytes in human glomeruli. Group IB secretory phospholipase A2 (sPLA2 IB), which is one of the ligands of the PLA2R, is more highly expressed in chronic renal failure patients than in controls. However, the roles of the PLA2R and sPLA2 IB in the pathogenesis of glomerular diseases are unknown. In the present study, we found that more podocyte apoptosis occurs in the kidneys of patients with higher PLA2R and serum sPLA2 IB levels. In vitro, we demonstrated that human podocyte cells expressed the PLA2R in the cell membrane. After binding with the PLA2R, sPLA2 IB induced podocyte apoptosis in a time- and concentration-dependent manner. sPLA2 IB-induced podocyte PLA2R upregulation was not only associated with increased ERK1/2 and cPLA2α phosphorylation but also displayed enhanced apoptosis. In contrast, PLA2R-silenced human podocytes displayed attenuated apoptosis. sPLA2 IB enhanced podocyte arachidonic acid (AA) content in a dose-dependent manner. These data indicate that sPLA2 IB has the potential to induce human podocyte apoptosis via binding to the PLA2R. The sPLA2 IB-PLA2R interaction stimulated podocyte apoptosis through activating ERK1/2 and cPLA2α and through increasing the podocyte AA content.

  7. A novel approach to the design of inhibitors of human secreted phospholipase A2 based on native peptide inhibition.

    PubMed

    Church, W B; Inglis, A S; Tseng, A; Duell, R; Lei, P W; Bryant, K J; Scott, K F

    2001-08-31

    Human Type IIA secreted phospholipase A(2) (sPLA(2)-IIA) is an important modulator of cytokine-dependent inflammatory responses and a member of a growing superfamily of structurally related phospholipases. We have previously shown that sPLA(2)-IIA is inhibited by a pentapeptide sequence comprising residues 70-74 of the native sPLA(2)-IIA protein and that peptides derived from the equivalent region of different sPLA(2)-IIA species specifically inhibit the enzyme from which they are derived. We have now used an analogue screen of the human pentapeptide (70)FLSYK(74) in which side-chain residues were substituted, together with molecular docking approaches that modeled low-energy conformations of (70)FLSYK(74) bound to human sPLA(2)-IIA, to generate inhibitors with improved potency. Importantly, the modeling studies showed a close association between the NH(2) and COOH termini of the peptide, predicting significant enhancement of the potency of inhibition by cyclization. Cyclic compounds were synthesized and indeed showed 5-50-fold increased potency over the linear peptide in an Escherichia coli membrane assay. Furthermore, the potency of inhibition correlated with steady-state binding of the cyclic peptides to sPLA(2)-IIA as determined by surface plasmon resonance studies. Two potential peptide interaction sites were identified on sPLA(2)-IIA from the modeling studies, one in the NH(2)-terminal helix and the other in the beta-wing region, and in vitro association assays support the potential for interaction of the peptides with these sites. The inhibitors were effective at nanomolar concentrations in blocking sPLA(2)-IIA-mediated amplification of cytokine-induced prostaglandin synthesis in human rheumatoid synoviocytes in culture. These studies provide an example where native peptide sequences can be used for the development of potent and selective inhibitors of enzyme function.

  8. Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase.

    PubMed

    Nelson, Jennifer; Francom, Lyndee L; Anderson, Lynn; Damm, Kelly; Baker, Ryan; Chen, Joseph; Franklin, Sarah; Hamaker, Amy; Izidoro, Izadora; Moss, Eric; Orton, Mikayla; Stevens, Evan; Yeung, Celestine; Judd, Allan M; Bell, John D

    2012-05-01

    Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.

  9. Phospholipases and their industrial applications.

    PubMed

    De Maria, L; Vind, J; Oxenbøll, K M; Svendsen, A; Patkar, S

    2007-02-01

    Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A1, A2, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions--white biotechnology is a strong alternative.

  10. 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.

  11. The affinities of human platelet and Acanthamoeba profilin isoforms for polyphosphoinositides account for their relative abilities to inhibit phospholipase C.

    PubMed Central

    Machesky, L M; Goldschmidt-Clermont, P J; Pollard, T D

    1990-01-01

    In light of recent work implicating profilin from human platelets as a possible regulator of both cytoskeletal dynamics and inositol phospholipid-mediated signaling, we have further characterized the interaction of platelet profilin and the two isoforms of Acanthamoeba profilin with inositol phospholipids. Profilin from human platelets binds to phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) with relatively high affinity (Kd approximately 1 microM for PIP2 by equilibrium gel filtration), but interacts only weakly (if at all) with phosphatidylinositol (PI) or inositol trisphosphate IP3) in small-zone gel-filtration assays. The two isoforms of Acanthamoeba profilin both have a lower affinity for PIP2 than does human platelet profilin, but the more basic profilin isoform from Acanthamoeba (profilin-II) has a much higher (approximately 10-microM Kd) affinity than the acidic isoform (profilin-I, 100 to 500-microM Kd). None of the profilins bind to phosphatidylserine (PS) or phosphatidylcholine (PC) in small-zone gel-filtration experiments. The differences in affinity for PIP2 parallel the ability of these three profilins to inhibit PIP2 hydrolysis by soluble phospholipase C (PLC). The results show that the interaction of profilins with PIP2 is specific with respect to both the lipid and the proteins. In Acanthamoeba, the two isoforms of profilin may have specialized functions on the basis of their identical (approximately 10 microM) affinities for actin monomers and different affinities for PIP2. PMID:1966040

  12. Human Phospholipase D Activity Transiently Regulates Pyrimidine Biosynthesis in Malignant Gliomas

    PubMed Central

    Mathews, Thomas P.; Hill, Salisha; Rose, Kristie L.; Ivanova, Pavlina T.; Lindsley, Craig W.; Brown, H. Alex

    2015-01-01

    Cancer cells reorganize their metabolic pathways to fuel demanding rates of proliferation. Oftentimes, these metabolic phenotypes lie downstream of prominent oncogenes. The lipid signaling molecule phosphatidic acid (PtdOH), which is produced by the hydrolytic enzyme phospholipase D (PLD), has been identified as a critical regulatory molecule for oncogenic signaling in many cancers. In an effort to identify novel regulatory mechanisms for PtdOH, we screened various cancer cell lines, assessing whether treatment of cancer models with PLD inhibitors altered production of intracellular metabolites. Preliminary findings lead us to focus on how deoxyribonucleoside triphosphates (dNTPs) are altered upon PLD inhibitor treatment in gliomas. Using a combination of proteomics and small molecule intracellular metabolomics, we show herein that PtdOH acutely regulates the production of these pyrimidine metabolites through activation of CAD via mTOR signaling pathways independently of Akt. These changes are responsible for decreases in dNTP production after PLD inhibitor treatment. Our data identify a novel regulatory role for PLD activity in specific cancer types. PMID:25646564

  13. Interaction of low molecular weight group IIA phospholipase A2 with apoptotic human T cells: role of heparan sulfate proteoglycans.

    PubMed

    Boilard, Eric; Bourgoin, Sylvain G; Bernatchez, Chantale; Poubelle, Patrice E; Surette, Marc E

    2003-06-01

    Human group IIA phospholipase A2 (hIIA PLA2) is a 14 kDa secreted enzyme associated with inflammatory diseases. A newly discovered property of hIIA PLA2 is the binding affinity for the heparan sulfate proteoglycan (HSPG) glypican-1. In this study, the binding of hIIA PLA2 to apoptotic human T cells was investigated. Little or no exogenous hIIA PLA2 bound to CD3-activated T cells but significant binding was measured on activated T cells induced to undergo apoptosis by anti-CD95. Binding to early apoptotic T cells was greater than to late apoptotic cells. The addition of heparin and the hydrolysis of HSPG by heparinase III only partially inhibited hIIA PLA2 binding to apoptotic cells, suggesting an interaction with both HSPG and other binding protein(s). Two low molecular weight HSPG were coimmunoprecipitated with hIIA PLA2 from apoptotic T cells, but not from living cells. Treatment of CD95-stimulated T cells with hIIA PLA2 resulted in the release of arachidonic acid but not oleic acid from cells and this release was blocked by heparin and heparinase III. Altogether, these results suggest a role for hIIA PLA2 in the release of arachidonic acid from apoptotic cells through interactions with HSPG and its potential implication in the progression of inflammatory diseases.

  14. Activation of group IVC phospholipase A2 by polycyclic aromatic hydrocarbons induces apoptosis of human coronary artery endothelial cells

    PubMed Central

    Richards, Sean M.; Elgayyar, Mona A.; Menn, Fu-Minn; Vulava, Vijay M.; McKay, Larry; Sanseverino, John; Sayler, Gary; Tucker, Dawn E.; Leslie, Christina C.; Lu, Kim P.; Ramos, Kenneth S.

    2016-01-01

    Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A2 (PLA2) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA2 in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA2 inhibited both 3H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA2. Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA2. PMID:21132278

  15. Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis.

    PubMed

    Yagami, Tatsurou; Ueda, Keiichi; Asakura, Kenji; Hata, Satoshi; Kuroda, Takayuki; Sakaeda, Toshiyuki; Takasu, Nobuo; Tanaka, Kazushige; Gemba, Takefumi; Hori, Yozo

    2002-01-01

    Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.

  16. Group XV phospholipase A2, a lysosomal phospholipase A2

    PubMed Central

    Shayman, James A.; Kelly, Robert; Kollmeyer, Jessica; He, Yongqun; Abe, Akira

    2010-01-01

    A phospholipase A2 was identified from MDCK cell homogenates with broad specificity toward glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. The phospholipase has the unique ability to transacylate short chain ceramides. This phospholipase is calcium-independent, localized to lysosomes, and has an acidic pH optimum. The enzyme was purified from bovine brain and found to be a water-soluble glycoprotein consisting of a single peptide chain with a molecular weight of 45 kDa. The primary structure deduced from the DNA sequences is highly conserved between chordates. The enzyme was named lysosomal phospholipase A2 (LPLA2) and subsequently designated group XV phospholipase A2. LPLA2 has 49 percent of amino acid sequence identity to lecithin cholesterol acyltransferase and is a member of the αβ-hydrolase superfamily. LPLA2 is highly expressed in alveolar macrophages. A marked accumulation of glycerophospholipids and extensive lamellar inclusion bodies, a hallmark of cellular phospholipidosis, is observed in alveolar macrophages in LPLA2−/− mice. This defect can also be reproduced in macrophages that are exposed to cationic amphiphilic drugs such as amiodarone. In addition, older LPLA2−/− mice develop a phenotype similar to human autoimmune disease. These observations indicate that LPLA2 may play a primary role in phospholipid homeostasis, drug toxicity, and host defense. PMID:21074554

  17. Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor.

    PubMed

    Granata, Francescopaolo; Petraroli, Angelica; Boilard, Eric; Bezzine, Sofiane; Bollinger, James; Del Vecchio, Luigi; Gelb, Michael H; Lambeau, Gerard; Marone, Gianni; Triggiani, Massimo

    2005-01-01

    Secreted phospholipases A(2) (sPLA(2)) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA(2)s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA(2)s induced TNF-alpha and IL-6 release in a concentration-dependent manner by increasing their mRNA expression. This effect was independent of their enzymatic activity because 1) the capacity of sPLA(2)s to mobilize arachidonic acid from HLM was unrelated to their ability to induce cytokine production; and 2) two catalytically inactive isoforms of group IB sPLA(2) (bromophenacyl bromide-inactivated human sPLA(2) and the H48Q mutant of the porcine sPLA(2)) were as effective as the catalytically active sPLA(2)s in inducing cytokine production. HLM expressed the M-type receptor for sPLA(2)s at both mRNA and protein levels, as determined by RT-PCR, immunoblotting, immunoprecipitation, and flow cytometry. Me-indoxam, which decreases sPLA(2) activity as well as binding to the M-type receptor, suppressed sPLA(2)-induced cytokine production. Incubation of HLM with the sPLA(2)s was associated with phosphorylation of ERK1/2, and a specific inhibitor of this pathway, PD98059, significantly reduced the production of IL-6 elicited by sPLA(2)s. In conclusion, two distinct sPLA(2)s produced in the human lung stimulate cytokine production by HLM via a mechanism that is independent of their enzymatic activity and involves activation of the ERK1/2 pathway. HLM express the M-type receptor, but its involvement in eliciting cytokine production deserves further investigation.

  18. Corticotropin-releasing factor induces phosphorylation of phospholipase C-gamma at tyrosine residues via its receptor 2beta in human epidermoid A-431 cells.

    PubMed

    Kiang, J G; Ding, X Z; Gist, I D; Jones, R R; Tsokos, G C

    1998-12-18

    This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-gamma via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2beta. The levels of the CRF receptor type 2beta were not altered in cells treated with activators of protein kinase C, Ca2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-beta3, -gamma1, or -gamma2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-gamma1 and -gamma2 were phosphorylated. The increase in phospholipase C-gamma phosphorylation was concentration-dependent with an EC50 of 4.2+/-0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2beta that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2iminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated the phospholipase C-gamma phosphorylation. In summary

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

    PubMed Central

    Dutta, Mouparna; Ghosh, Anindya S.; Oda, Masataka; Venkatramani, Ravindra; Rao, Basuthkar J.; Dandekar, Abhaya M.; Goñi, Félix M.

    2015-01-01

    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

  20. 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

  1. Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes

    SciTech Connect

    Samanta, Uttamkumar; Kirby, Stephen D.; Srinivasan, Prabhavathi; Cerasoli, Douglas M.; Bahnson, Brian J.

    2009-09-02

    The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P{sub R} and P{sub S} stereoisomers at the P-chiral center. The tabun complex displayed only the P{sub R} stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.

  2. The lipid requirement of the (Ca2+ + Mg2+)-ATPase in the human erythrocyte membrane, as studied by various highly purified phospholipases.

    PubMed

    Roelofsen, B; Schatzmann, H J

    1977-01-04

    1. When complete hydrolysis of glycerophosphlipids and sphingomyelin in the outer membrane leaflet is brought about by treatment of intact red blood cells with phospholipase A2 and sphingomyelinase C, the (Ca2+ + Mg2+)-ATPase activity is not affected. 2. Complete hydrolysis of sphingomyelin, by treatment of leaky ghosts with spingomyelinase C, does not lead to an inactivation of the (Ca2+ + Mg2+)-ATPase. 3. Treatment of ghosts with phospholipase A2 (from either procine pancreas of Naja naja venom), under conditions causing an essentially complete hydrolysis of the total glycerophospholipid fraction of the membrane, results in inactivation of the (Ca2+ + Mg2+)-ATPase by some 80--85%. The residual activity is lost when the produced lyso-compounds (and fatty acids) are removed by subsequent treatment of the ghosts with bovine serum albumin. 4. The degree of inactivation of the (Ca2+ + Mg2+)-ATPase, caused by treatment of ghosts with phospholipase C, is directly proportional to the percentage by which the glycerophospholipid fraction in the inner membrane layer is degraded. 5. After essentially complete inactivation of the (Ca2+ + Mg2+)-ATPase by treatment of ghosts with phospholipase C from Bacillus cereus, the enzyme is reactivated by the addition of any of the glycerophospholipids, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine or lysophosphatidylcholine, but not by addition of sphingomyeline, free fatty acids or the detergent Triton X-100. 6. It is concluded that only the glycerophospholipids in the human erythrocyte membrane are involved in the maintenance of the (Ca2+ + Mg2+)-ATPase activity, and in particular that fraction of these phospholipids located in the inner half of the membrane.

  3. PHOSPHOLIPASE D (PLD) DRIVES CELL INVASION, TUMOR GROWTH AND METASTASIS IN A HUMAN BREAST CANCER XENOGRAPH MODEL

    PubMed Central

    Henkels, Karen M.; Boivin, Gregory P.; Dudley, Emily S.; Berberich, Steven J.; Gomez-Cambronero, Julian

    2014-01-01

    Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based shRNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, p<0.05) and their onset delayed when compared to control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (FIPI and NOPT). These inhibitors led to significant (>70%, p<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid (PA), WASp, Grb2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows that PLD has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target. PMID:23752189

  4. 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

  5. Group V secreted phospholipase A2 is upregulated by IL-4 in human macrophages and mediates phagocytosis via hydrolysis of ethanolamine phospholipids.

    PubMed

    Rubio, Julio M; Rodríguez, Juan P; Gil-de-Gómez, Luis; Guijas, Carlos; Balboa, María A; Balsinde, Jesús

    2015-04-01

    Studies on the heterogeneity and plasticity of macrophage populations led to the identification of two major polarization states: classically activated macrophages or M1, induced by IFN-γ plus LPS, and alternatively activated macrophages, induced by IL-4. We studied the expression of multiple phospholipase A2 enzymes in human macrophages and the effect that polarization of the cells has on their levels. At least 11 phospholipase A2 genes were found at significant levels in human macrophages, as detected by quantitative PCR. None of these exhibited marked changes after treating the cells with IFN-γ plus LPS. However, macrophage treatment with IL-4 led to strong upregulation of the secreted group V phospholipase A2 (sPLA2-V), both at the mRNA and protein levels. In parallel with increasing sPLA2-V expression levels, IL-4-treated macrophages exhibited increased phagocytosis of yeast-derived zymosan and bacteria, and we show that both events are causally related, because cells deficient in sPLA2-V exhibited decreased phagocytosis, and cells overexpressing the enzyme manifested higher rates of phagocytosis. Mass spectrometry analyses of lipid changes in the IL-4-treated macrophages suggest that ethanolamine lysophospholipid (LPE) is an sPLA2-V-derived product that may be involved in regulating phagocytosis. Cellular levels of LPE are selectively maintained by sPLA2-V. By supplementing sPLA2-V-deficient cells with LPE, phagocytosis of zymosan or bacteria was fully restored in IL-4-treated cells. Collectively, our results show that sPLA2-V is required for efficient phagocytosis by IL-4-treated human macrophages and provide evidence that sPLA2-V-derived LPE is involved in the process.

  6. Exploitation of a Novel Binding Pocket in Human Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Discovered through X-ray Fragment Screening.

    PubMed

    Woolford, Alison J-A; Pero, Joseph E; Aravapalli, Sridhar; Berdini, Valerio; Coyle, Joseph E; Day, Philip J; Dodson, Andrew M; Grondin, Pascal; Holding, Finn P; Lee, Lydia Y W; Li, Peng; Manas, Eric S; Marino, Joseph; Martin, Agnes C L; McCleland, Brent W; McMenamin, Rachel L; Murray, Christopher W; Neipp, Christopher E; Page, Lee W; Patel, Vipulkumar K; Potvain, Florent; Rich, Sharna; Rivero, Ralph A; Smith, Kirsten; Somers, Donald O; Trottet, Lionel; Velagaleti, Ranganadh; Williams, Glyn; Xie, Ren

    2016-06-09

    Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

  7. Involvement of phospholipase D in store-operated calcium influx in vascular smooth muscle cells.

    PubMed

    Walter, M; Tepel, M; Nofer, J R; Neusser, M; Assmann, G; Zidek, W

    2000-08-11

    In non-excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a 'store-operated' mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide-specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store-operated Ca2+ entry in VSMC.

  8. Phospholipase C δ-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells

    PubMed Central

    Leung, David W; Tompkins, Chris; Brewer, Jim; Ball, Alexey; Coon, Mike; Morris, Valerie; Waggoner, David; Singer, Jack W

    2004-01-01

    Background The expression of the rodent phosphoinositide-specific phospholipase C δ-4 (PLCδ4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCδ4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCδ4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCδ4 on cell signaling and proliferation in this study. Results The cDNA for human PLCδ4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCδ4 selectively activates protein kinase C-φ and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells. MCF-7 cells stably expressing PLCδ4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCδ4 are not reversible by siRNA. Conclusion Overexpression or dysregulated expression of PLCδ4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCδ4 are not reversible, PLCδ4 itself is not a suitable drug target, but enzymes in pathways activated by PLCδ4 are potential therapeutic targets for oncogenic intervention. PMID:15140260

  9. Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation.

    PubMed

    Jia, Xiaodong; Chen, Fangyan; Pan, Weihua; Yu, Rentao; Tian, Shuguang; Han, Gaige; Fang, Haiqin; Wang, Shuo; Zhao, Jingya; Li, Xianping; Zheng, Dongyu; Tao, Sha; Liao, Wanqing; Han, Xuelin; Han, Li

    2014-06-01

    The internalization of Aspergillus fumigatus into lung epithelial cells is critical for the infection process in the host. Gliotoxin is the most potent toxin produced by A. fumigatus. However, its role in A. fumigatus internalization into the lung epithelial cells is still largely unknown. In the present study, the deletion of the gliP gene regulating the production of gliotoxin in A. fumigatus suppressed the internalization of conidia into the A549 lung epithelial cells, and this suppression could be rescued by the exogenous addition of gliotoxin. At lower concentrations, gliotoxin enhanced the internalization of the conidia of A. fumigatus into A549 cells; in contrast, it inhibited the phagocytosis of J774 macrophages in a dose-dependent manner. Under a concentration of 100 ng/ml, gliotoxin had no effect on A549 cell viability but attenuated ROS production in a dose-dependent manner. Gliotoxin significantly stimulated the phospholipase D activity in the A549 cells at a concentration of 50 ng/ml. This stimulation was blocked by the pretreatment of host cells with PLD1- but not PLD2-specific inhibitor. Morphological cell changes induced by gliotoxin were observed in the A549 cells accompanying with obvious actin cytoskeleton rearrangement and a moderate alteration of phospholipase D distribution. Our data indicated that gliotoxin might be responsible for modulating the A. fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement.

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

    PubMed Central

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

    1995-01-01

    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

  11. 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.

  12. Expression, purification and characterization of a human serine-dependent phospholipase A2 with high specificity for oxidized phospholipids and platelet activating factor.

    PubMed Central

    Rice, S Q; Southan, C; Boyd, H F; Terrett, J A; MacPhee, C H; Moores, K; Gloger, I S; Tew, D G

    1998-01-01

    Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution. PMID:9494101

  13. Human- and mouse-inducible nitric oxide synthase promoters require activation of phosphatidylcholine-specific phospholipase C and NF-kappa B.

    PubMed Central

    Spitsin, S. V.; Farber, J. L.; Bertovich, M.; Moehren, G.; Koprowski, H.; Michaels, F. H.

    1997-01-01

    BACKGROUND: The production of nitric oxide by type II inducible nitric oxide synthase (type II NOS) gene is controlled at least in part by transcriptional activation. Although the murine and human type II NOS genes share significant sequence homology, they differ in the induction stimuli required for activation. MATERIALS AND METHODS: The A549 human and murine RAW 264.7 cell lines were cultured in the presence of inducers of the type II NOS gene and exposed to specific inhibitors of phosphatidyl choline-specific phospholipase C, NF-kappa B, and endocytosis, as well as to reagents that deplete stores of ATP or prevent the acidification of endosomes. The effect of these reagents on the induction of the type II NOS gene transcription, translation, and NO expression was studied using electromobility shift assays, Western blotting, and the detection of NO as nitrates, as appropriate. Additionally, the ability of the native human type II NOS NF-kappa B recognition sequence to bind NF-kappa B was compared with a concensus sequence and with a mutated oligomer. RESULTS: Type II NOS production by both human and mouse cells could be prevented by the addition of the specific inhibitor of phosphatidylcholine-specific phospholipase C, D609, and of agents that interfere with the activation of NF-kappa B. Both mouse and human cells also required acidic endosome formation and the production of 1,2-diacylglycerol for type II NOS expression. Additionally, the native human type II NOS NF-kappa B recognition sequence bound NF-kappa B with significantly less affinity than did the recognition sequence derived from the human immunoglobulin light-chain gene promoter. CONCLUSIONS: These experiments show that whereas mouse cells can be activated by lipopolysaccharide to produce nitric oxide, and human cells require activation by a mixture of cytokines to produce nitric oxide, the intracellular activation pathway following receptor binding of these heterologous stimuli is shared. Additionally

  14. Phospholipases in arterial tissue

    PubMed Central

    Eisenberg, S.; Stein, Y.; Stein, O.

    1969-01-01

    The role of phospholipases in the regulation of the changing phospholipid composition of normal human aortae with age was studied. Portions of grossly and histologically lesion-free ascending aortae from 16 females and 29 males obtained at autopsy, were analyzed for deoxyribonucleic acid (DNA), phospholipid, and cholesterol content and phospholipid composition. Enzymic activity toward four substrates, lecithin (LE), phosphatidyl ethanolamine, lysolecithin, and sphingomyelin (SP), was determined on portions of the same homogenate. By regression analysis for correlation between all determinations and age the following results were obtained: (a) total phospholipids and choleserol increased linearly with age; (b) the increase in sphingomyelin accounted for about 70% of the phospholipid increment; (c) hydrolysis of lecithin and phosphatidyl ethanolamine increased markedly with age, that of lysolecithin only moderately; (d) hydrolysis of sphingomyelin decreased with age; and (e) an inverse relation between the SP/LE ratio and age and sphingomyelinase/lecithinase activity and age was obtained. These results were interpreted to indicate that a causal relation exists between the fall in sphingomyelinase activity, both absolute and relative to lecithinase activity, and the accumulation of sphingomyelin with age. PMID:5355343

  15. Bacterial Sphingomyelinases and Phospholipases as Virulence Factors.

    PubMed

    Flores-Díaz, Marietta; Monturiol-Gross, Laura; Naylor, Claire; Alape-Girón, Alberto; Flieger, Antje

    2016-09-01

    Bacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases.

  16. Loss of function variants in human PNPLA8 encoding calcium-independent phospholipase A2γ recapitulate the mitochondriopathy of the homologous null mouse

    PubMed Central

    Saunders, Carol J.; Moon, Sung Ho; Liu, Xinping; Thiffault, Isabelle; Coffman, Keith; LePichon, Jean-Baptiste; Taboada, Eugenio; Smith, Laurie D.; Farrow, Emily G.; Miller, Neil; Gibson, Margaret; Patterson, Melanie; Kingsmore, Stephen F.; Gross, Richard W.

    2015-01-01

    Mitochondriopathies are a group of clinically heterogeneous genetic diseases caused by defects in mitochondrial metabolism, bioenergetic efficiency, and/or signaling functions. The large majority of proteins involved in mitochondrial function are encoded by nuclear genes, with many yet to be associated with human disease. We performed exome sequencing on a young girl with a suspected mitochondrial myopathy that manifested as progressive muscle weakness, hypotonia, seizures, poor weight gain, and lactic acidosis. She was compound heterozygous for two frameshift mutations, p. Asn112HisfsX29 and p. Leu659AlafsX4, in the PNPLA8 gene, which encodes mitochondrial calcium independent phospholipase A2γ (iPLA2γ). Western blot analysis of affected muscle displayed the absence of PNPLA8 protein. iPLA2s are critical mediators of a variety of cellular processes including growth, metabolism, and lipid second messenger generation, exerting their functions through catalyzing the cleavage of the acyl groups in glycerophospholipids. The clinical presentation, muscle histology and the mitochondrial ultrastructural abnormalities of this proband are highly reminiscent of Pnpla8 null mice. Although other iPLA2–related diseases have been identified, namely infantile neuroaxonal dystrophy and neutral lipid storage disease with myopathy, this is the first report of PNPLA8-related disease in a human. We suggest PNPLA8 join the increasing list of human genes involved in lipid metabolism associated with neuromuscular diseases due to mitochondrial dysfunction. PMID:25512002

  17. 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

  18. Coincident regulation of PKCdelta in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling.

    PubMed

    Hall, Kellie J; Jones, Matthew L; Poole, Alastair W

    2007-09-15

    PKC (protein kinase C)d plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCd. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCd with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCd is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCd. Both thrombin and PMA induce recruitment of PKCd to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCd, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCd is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.

  19. Identification of an autoantigen on the surface of apoptotic human T cells as a new protein interacting with inflammatory group IIA phospholipase A2.

    PubMed

    Boilard, Eric; Bourgoin, Sylvain G; Bernatchez, Chantale; Surette, Marc E

    2003-10-15

    One of the most studied secreted phospholipases A2 (sPLA2), the group IIA sPLA2, is found at high levels in inflammatory fluids of patients with autoimmune diseases. A characteristic of group IIA sPLA2 is its preference for negatively charged phospholipids, which become exposed on the extracellular leaflet of apoptotic cell membranes. We recently showed that low molecular weight heparan sulfate proteoglycans (HSPGs) and uncharacterized detergent-insoluble binding site(s) contribute to the enhanced binding of human group IIA PLA2 (hGIIA) to apoptotic human T cells. Using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry we now identify vimentin as the major HSPG-independent binding protein of hGIIA on apoptotic primary T lymphocytes. Vimentin is partially exposed on the surface of apoptotic T cells and binds hGIIA via its rod domain in a calcium-independent manner. Studies with hGIIA mutants showed that specific motifs in the interfacial binding surface are involved in the interaction with vimentin. The sPLA2 inhibitor LY311727, but not heparin, inhibited this interaction. In contrast, heparin but not LY311727 abrogated the binding of hGIIA to cellular HSPGs. Importantly, vimentin does not inhibit the catalytic activity of hGIIA. Altogether, the results show that vimentin, in conjunction with HSPGs, contributes to the enhanced binding of hGIIA to apoptotic T cells.

  20. Effect of Retinoic Acid on Gene Expression in Human Conjunctival Epithelium: Secretory phospholipase A2 mediates retinoic acid induction of MUC16.

    PubMed Central

    Hori, Yuichi; Spurr-Michaud, Sandra J.; Russo, Cindy Leigh; Argüeso, Pablo; Gipson, Ilene K.

    2005-01-01

    Purpose. How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. We sought to identify vitamin A responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial cell line (HCjE) grown with all-trans-retinoic acid (RA). The analysis showed that secretory phospholipase A2 Group IIA (sPLA2-IIA) was the gene most upregulated by RA, followed by the membrane-associated mucin MUC16 at a later time point. Since eicosanoids, the product of arachidonic acid generated by the phospholipase A2 family, have been shown to increase mucin production, we sought to determine if sPLA2 mediates the RA induction of MUC16. Methods. HCjE cells were cultured with or without RA for 3, 6, 24 and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips (HG-U133A; Affymetrix) and analyzed using Rosetta Resolver software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA2 is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad spectrum PLA2 inhibitor, aristolochic acid (ArA) or the specific sPLA2-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis. Results. After RA addition, 28 transcripts were upregulated and 6 downregulated by over 2.0-fold (p < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA2, significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA2 upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA2-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (p < 0.01). Conclusion. The retinoic acid-associated upregulation of

  1. Dissociation Between Fatty Liver and Insulin Resistance in Humans Carrying a Variant of the Patatin-Like Phospholipase 3 Gene

    PubMed Central

    Kantartzis, Konstantinos; Peter, Andreas; Machicao, Fausto; Machann, Jürgen; Wagner, Silvia; Königsrainer, Ingmar; Königsrainer, Alfred; Schick, Fritz; Fritsche, Andreas; Häring, Hans-Ulrich; Stefan, Norbert

    2009-01-01

    OBJECTIVE In a genome-wide association scan, the rs738409 C>G single nucleotide polymorphism (SNP) in the patatin-like phospholipase 3 gene (PNPLA3) was strongly associated with increased liver fat but not with insulin resistance estimated from fasting values. We investigated whether the SNP determines liver fat independently of visceral adiposity and whether it may even play a role in protecting from insulin resistance. RESEARCH DESIGN AND METHODS Liver fat was measured by 1H magnetic resonance spectroscopy and total and visceral fat by magnetic resonance tomography in 330 subjects. Insulin sensitivity was estimated during an oral glucose tolerance test and the euglycemic-hyperinsulinemic clamp (n = 222). PNPLA3 and tumor necrosis factor-α mRNA and triglyceride content were measured in liver biopsies from 16 subjects. RESULTS Liver fat correlated strongly with insulin sensitivity (P < 0.0001) independently of age, sex, total fat, and visceral fat. G allele carriers of the SNP rs738409 had higher liver fat (P < 0.0001) and an odds ratio of 2.38 (95% CI 1.37–4.20) for having fatty liver compared to C allele homozygotes. Interestingly, insulin sensitivity (oral glucose tolerance test: P = 0.99; clamp: P = 0.32), serum C-reactive protein levels, lipids, or liver enzymes (all P > 0.14) were not different among the genotypes. Additional adjustment for liver fat actually revealed increased insulin sensitivity in more obese carriers of the G allele (P = 0.01). In liver biopsies triglyceride content correlated positively with expression of the proinflammatory gene tumor necrosis factor-α in C allele homozygotes (n = 6, P = 0.027) but not in G allele carriers (n = 10, P = 0.149). CONCLUSIONS PNPLA3 may be an important key to understand the mechanisms discriminating fatty liver with and without metabolic consequences. PMID:19651814

  2. Induction of progesterone receptor A form attenuates the induction of cytosolic phospholipase A2alpha expression by cortisol in human amnion fibroblasts.

    PubMed

    Guo, Chunming; Ni, Xiaotian; Zhu, Ping; Li, Wenjiao; Zhu, Xiaoou; Sun, Kang

    2010-05-01

    Cytosolic phospholipase A2alpha (cPLA(2alpha), now known as PLA2G4A) is the enzyme catalyzing the formation of the rate-limiting substrate, arachidonic acid, for prostaglandin (PG) synthesis. The increasing expression of PLA2G4A toward term gestation in human amnion fibroblasts is believed to be the crucial event in parturition. Human amnion fibroblasts produce cortisol, progesterone and express glucocorticoid receptor (GR), progesterone receptor A (PGRA) form at term. The roles of progesterone and PGRA in the induction of PLA2G4A by cortisol via GR in the amnion fibroblasts remain largely unknown. Using cultured human term amnion fibroblasts, we found that cortisol induced the expression of PGRA, which was attenuated by inhibiting PG synthesis with indomethacin. Knockdown of PGRA expression or inhibition of endogenous progesterone production with trilostane significantly enhanced the induction of PLA2G4A by cortisol, whereas overexpression of PGRA attenuated the induction of PLA2G4A by cortisol. Although exogenous progesterone did not alter PLA2G4A expression under basal conditions, it attenuated cortisol-induced PLA2G4A expression at concentrations about tenfold higher, which might be achieved by competition with cortisol for GR. In conclusion, PGRA in the presence of endogenous progesterone is a transdominant repressor of the induction of PLA2G4A by cortisol. High level of progesterone may compete with cortisol for GR, thus further inhibiting the induction of PLA2G4A by cortisol. Moreover, increased PG synthesis by cortisol may feed back on the expression of PGRA leading to attenuation of cortisol-induced PLA2G4A expression. The above findings may be pertinent to the inconsistent effects of glucocorticoids on parturition in humans.

  3. Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)

    SciTech Connect

    Baldassare, J.J.; Henderson, P.A.; Fisher, G.J. )

    1989-01-10

    The effects of thrombin and GTP{gamma}S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous ({sup 3}H)inositol-labeled membranes or with lipid vesicles containing either ({sup 3}H)phosphatidylinositol or ({sup 3}H)phosphatidylinositol 4,5-bisphosphate. GTP{gamma}S (1 {mu}M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP{sub 3}), inositol bisphosphate (IP{sub 2}), or inositol phosphate (IP) from ({sup 3}H)inositol-labeled membranes. IP{sub 2} and IP{sub 3}, but not IP, from ({sup 3}H)inositol-labeled membranes were, however, stimulated 3-fold by GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). A higher concentration of GTP{gamma}S (100 {mu}M) alone also stimulated IP{sub 2} and IP{sub 3}, but not IP, release. In the presence of 1 mM calcium, release of IP{sub 2} and IP{sub 3} was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) by platelet membrane associated PLC was also markedly enhanced by GTP{gamma}S (100 {mu}M) or GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP{sub 2} was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP{gamma}S (100 {mu}M) or calcium (1 mM) dependent PIP{sub 2} breakdown, while TPA inhibited GTP{gamma}S-dependent but not calcium-dependent phospholipase C activity.

  4. Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes

    PubMed Central

    Guijas, Carlos; Pérez-Chacón, Gema; Astudillo, Alma M.; Rubio, Julio M.; Gil-de-Gómez, Luis; Balboa, María A.; Balsinde, Jesús

    2012-01-01

    Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60–70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2α (cPLA2α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA2α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA2α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis. PMID:22949356

  5. Production of Vascular Endothelial Growth Factors from Human Lung Macrophages Induced by Group IIA and Group X Secreted Phospholipases A2

    PubMed Central

    Granata, Francescopaolo; Frattini, Annunziata; Loffredo, Stefania; Staiano, Rosaria I.; Petraroli, Angelica; Ribatti, Domenico; Oslund, Rob; Gelb, Michael H.; Lambeau, Gerard; Marone, Gianni; Triggiani, Massimo

    2010-01-01

    Angiogenesis and lymphangiogenesis mediated by vascular endothelial growth factors (VEGFs) are main features of chronic inflammation and tumors. Secreted phospholipases A2 (sPLA2s) are overexpressed in inflammatory lung diseases and cancer and they activate inflammatory cells by enzymatic and receptor-mediated mechanisms. We investigated the effect of sPLA2s on the production of VEGFs from human macrophages purified from the lung tissue of patients undergoing thoracic surgery. Primary macrophages express VEGF-A, VEGF-B, VEGF-C, and VEGF-D at both mRNA and protein level. Two human sPLA2s (group IIA and group X) induced the expression and release of VEGF-A and VEGF-C from macrophages. Enzymatically-inactive sPLA2s were as effective as the active enzymes in inducing VEGF production. Me-Indoxam and RO092906A, two compounds that block receptor-mediated effects of sPLA2s, inhibited group X-induced release of VEGF-A. Inhibition of the MAPK p38 by SB203580 also reduced sPLA2-induced release of VEGF-A. Supernatants of group X-activated macrophages induced an angiogenic response in chorioallantoic membranes that was inhibited by Me-Indoxam. Stimulation of macrophages with group X sPLA2 in the presence of adenosine analogs induced a synergistic increase of VEGF-A release and inhibited TNF-α production through a cooperation between A2A and A3 receptors. These results demonstrate that sPLA2s induce production of VEGF-A and VEGF-C in human macrophages by a receptor-mediated mechanism independent from sPLA2 catalytic activity. Thus, sPLA2s may play an important role in inflammatory and/or neoplastic angiogenesis and lymphangiogenesis. PMID:20357262

  6. Crystal Structures of Human Group-VIIA Phospholipase A2 Inhibited by Organophosphorus Nerve Agents Exhibit Non-aged Complexes ☆,☆☆

    PubMed Central

    Samanta, Uttamkumar; Kirby, Stephen D.; Srinivasan, Prabhavathi; Cerasoli, Douglas M.; Bahnson, Brian J.

    2009-01-01

    The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of PR and PS stereoisomers at the P-chiral center. The tabun complex displayed only the PR stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents. PMID:19394314

  7. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

    PubMed Central

    Faure, Grazyna; Gowda, Veerabasappa T; Maroun, Rachid C

    2007-01-01

    Background The snake venom group IIA secreted phospholipases A2 (SVPLA2), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA2-inhibitors from snake serum; and (iii) coagulation factors present in human blood. Results Using surface plasmon resonance (SPR) protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors. PMID:18062812

  8. Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation

    PubMed Central

    Escoffier, Jessica; Yassine, Sandra; Lee, Hoi Chang; Martinez, Guillaume; Delaroche, Julie; Coutton, Charles; Karaouzène, Thomas; Zouari, Raoudha; Metzler-Guillemain, Catherine; Pernet-Gallay, Karin; Hennebicq, Sylviane; Ray, Pierre F.; Fissore, Rafael; Arnoult, Christophe

    2015-01-01

    We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca2+ oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCζ in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCζ and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCζ is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca2+ oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCζ in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCζ in globozoospermic sperm is thus consistent and reinforces the role of PLCζ as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2

  9. Taiwan cobra phospholipase A2 suppresses ERK-mediated ADAM17 maturation, thus reducing secreted TNF-α production in human leukemia U937 cells.

    PubMed

    Chen, Ying-Jung; Lin, Hui-Chen; Chen, Ku-Chung; Lin, Shinne-Ren; Cheng, Tian-Lu; Chang, Long-Sen

    2014-08-01

    The goal of this study was to explore the signaling pathway regulating the processing of proADAM17 into ADAM17 in Taiwan cobra phospholipase A2 (PLA2)-treated human leukemia U937 cells. PLA2 induced reactive oxygen species (ROS)-elicited p38 MAPK activation and ERK inactivation in U937 cells. Catalytically inactive bromophenacylated PLA2 (BPB-PLA2) and PLA2 mutants evoked Ca(2+)-mediated p38 MAPK activation, and the level of phosphorylated ERK remained unchanged. PLA2 treatment reduced mature ADAM17 expression and secreted TNF-α (sTNF-α) production. Co-treatment of SB202190 (p38 MAPK inhibitor) and catalytically inactive PLA2 increased ERK phosphorylation, ADAM17 maturation and sTNF-α production. Nevertheless, mRNA levels of ADAM17 and TNF-α were insignificantly altered after PLA2 and SB202190/BPB-PLA2 treatment. ADAM17 activity assay and knock-down of ADAM17 revealed that ADAM17 was involved in sTNF-α production. Restoration of ERK activation increased the processing of proADAM17 into ADAM17 in PLA2-treated cells, while inactivation of ERK reduced ADAM17 maturation in untreated and SB202190/BPB-PLA2-treated cells. Removal of cell surface heparan sulfate abrogated PLA2 and SB202190/BPB-PLA2 effect on ADAM17 maturation. Taken together, the present data reveal that PLA2 suppresses ERK-mediated ADAM17 maturation, thus reducing sTNF-α production in U937 cells. Moreover, the binding with heparan sulfate is crucial for the PLA2 effect.

  10. Formation of 8-iso-PGF(2alpha) and thromboxane A(2) by stimulation with several activators of phospholipase A(2) in the isolated human umbilical vein.

    PubMed

    Sametz, W; Hummer, K; Butter, M; Wintersteiger, R; Juan, H

    2000-09-01

    We investigated the effects of the phospholipase A(2) (PLA(2)) activators calcium ionophore A 23187, hydrogen peroxide (H(2)O(2)), bradykinin (BK), histamine and noradrenaline (NA) on the 8-iso-prostaglandin (PG)F(2alpha) formation in the isolated human umbilical vein and the isolated rabbit ear. For comparison, the influence of these substances on the thromboxane A(2) (TXA(2)) release was also investigated. The release of total (esterified as well as free) 8-iso-PGF(2alpha), free 8-iso-PGF(2alpha) and TXB(2), the stable metabolite of TXA(2), was determined by specific enzyme immunoassays. The results show that bolus injections of 5.4 mmol H(2)O(2), 30 nmol A 23187, 10 nmol BK, 50 nmol histamine and 20 nmol NA caused an increased release of total 8-iso-PGF(2alpha) in the umbilical vein and the rabbit ear. A perfusion with H(2)O(2) at a final concentration of 0.3 mM also increased the release of this isoprostane. Increased formation of free 8-iso-PGF(2alpha) was induced by A 23187 injection and by both modes of H(2)O(2) administration, but not by the other treatments. Bolus injections of A 23187, BK and histamine induced an increased release of TXB(2) in both organs. Both modes of H(2)O(2) administration and NA showed no releasing effects. In conclusion, our results show that the substances used are able to stimulate the formation of 8-iso-PGF(2alpha) concurrently with the release of PGs. This effect might be of pathophysiological relevance in inflammatory and cardiovascular diseases in which an enhanced release of free radicals, BK, histamine or NA play an important role.

  11. Enhanced phospholipase C-gamma1 activity produced by association of independently expressed X and Y domain polypeptides.

    PubMed Central

    Horstman, D A; DeStefano, K; Carpenter, G

    1996-01-01

    The X and Y domains of phospholipase C (PLC)-gamma1, which are conserved in all mammalian phosphoinositide-specific PLC isoforms and are proposed to interact to form the catalytic site, have been expressed as individual hexahistidine-tagged fusion proteins in the baculovirus system. Following coinfection of insect cells with recombinant viruses, association of X and Y polypeptides was demonstrated in coprecipitation assays. When enzyme activity was examined, neither domain possessed catalytic activity when expressed alone; however, coexpression of the X and Y polypeptides produced a functional enzyme. This reconstituted phospholipase activity remained completely dependent on the presence of free Ca2+. The specific activity of the X:Y complex was significantly greater (20- to 100-fold) than that of holoPLC-gamma1 and was only moderately influenced by varying the concentration of substrate. The enzyme activities of holoPLC-gamma1 and the X:Y complex exhibited distinct pH optima. For holoPLC-gamma1 maximal activity was detected at pH 5.0, while activity of the X:Y complex was maximal at pH 7.2. Images Fig. 1 PMID:8755506

  12. The effect of PLC-γ2 inhibitors on the growth of human tumour cells.

    PubMed

    Feng, Linda; Reynisdóttir, Inga; Reynisson, Jóhannes

    2012-08-01

    The phosphoinositide specific-phospholipase C-γ (PLC-γ1 and 2) enzymes are plausible anticancer targets implicated in cell motility important to invasion and dissemination of tumour cells. A host of known PLC-γ2 inhibitors were tested against the NCI60 panel of human tumour cell lines as well as their commercially available structural derivatives. A class of thieno[2,3-b]pyridines showed excellent growth arrest with derivative 3 giving GI(50) = 58 nM for the melanoma MDA-MB-435 cell line. The PLC-γ2 is uniquely expressed in haematopoietic cells and the leukaemia tumour cell lines were growth restricted on average GI(50) = 275 nM by derivative 3 indicating a specific interaction with this isoform. Furthermore, a moderate growth inhibition was found for compound classes of indoles and 1H-pyrazoles. It is likely that the active compounds do not only inhibit the PLC-γ2 isoform but other PLCs as well due to their conserved binding site. The compounds tested were identified by applying the tools of chemoinformatics, which supports the use of in silico methods in drug design.

  13. TNF-α induces cytosolic phospholipase A2 expression via Jak2/PDGFR-dependent Elk-1/p300 activation in human lung epithelial cells.

    PubMed

    Yang, Chuen-Mao; Lee, I-Ta; Chi, Pei-Ling; Cheng, Shin-Ei; Hsiao, Li-Der; Hsu, Chih-Kai

    2014-03-15

    Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.

  14. Anti-CD3 and concanavalin A-induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. Lack of involvement of group IV and group VI phospholipase A2 in remodeling and increased susceptibility of proliferating T cells to CoA-independent transacyclase inhibitor-induced apoptosis.

    PubMed

    Boilard, E; Surette, M E

    2001-05-18

    In this study arachidonate-phospholipid remodeling was investigated in resting and proliferating human T lymphocytes. Lymphocytes induced to proliferate with either the mitogen concanavalin A or with anti-CD3 (OKT3) in combination with interleukin 2 (OKT3/IL-2) showed a greatly accelerated rate of [3H]arachidonate-phospholipid remodeling compared with resting lymphocytes or with lymphocytes stimulated with OKT3 or IL-2 alone. The concanavalin A-stimulated cells showed a 2-fold increase in the specific activity of CoA-independent transacylase compared with unstimulated cells, indicating that this enzyme is inducible. Stimulation with OKT3 resulted in greatly increased quantities of the group VI calcium-independent phospholipase A2 but not of the quantity of group IV cytosolic phospholipase A2. However, group IV phospholipase A2 became phosphorylated in OKT3-stimulated cells, as determined by decreased electrophoretic mobility. Incubation of cells with the group VI phospholipase A2 inhibitor, bromoenol lactone, or the dual group IV/group VI phospholipase A2 inhibitor, methyl arachidonyl fluorophosphonate, did not block arachidonate-phospholipid remodeling resting or proliferating T cells, suggesting that these phospholipases A2 were not involved in arachidonate-phospholipid remodeling. The incubation of nonproliferating human lymphocytes with inhibitors of CoA-independent transacylase had little impact on cell survival. In contrast, OKT3/IL-2-stimulated T lymphocytes were very sensitive to apoptosis induced by CoA-independent transacylase inhibitors. Altogether these results indicate that increased arachidonate-phospholipid remodeling is associated with T cell proliferation and that CoA-independent transacylase may be a novel therapeutic target for proliferative disorders.

  15. Detection and characterization of extracellular phospholipase A sub 2 in pleural effusion of patients with tuberculosis

    SciTech Connect

    Baek, Suk Hwan; Chang, Hyeun Wook ); Takayama, Kiyoshi; Kudo, Ichiro; Inoue, Keizo ); Lee, Hyun Woo; Do Jun Young )

    1991-01-01

    Extracellular phospholipase A{sub 2} activity has been identified in pleural fluid of patients with tuberculosis. This enzyme is a calcium requiring protein and has a pH optimum of 10.0. The enzyme was inhibited by the active site-directed histidine reagent, {rho}-bromophenacyl bromide. Ionic and non-ionic detergents, or the sulfhydryl reagent dithiothreitol, caused loss of enzyme activity. When substrate specificity was tested using 2-(1-{sup 14}C)linoleoyl phospholipids as substrates, phosphatidylethanolamine was the best substrate, followed by phosphatidylserine and phosphatidylcholine. This phospholipase A{sub 2} showed high affinity for heparin, and was recognized by a monoclonal antibody raised against phospholipase A{sub 2} from human synovial fluid. These findings suggest that an extracellular phospholipase A{sub 2}, which may belong to the 14K group II phospholipase A{sub 2} family, exists in the pleural fluid of patients with tuberculosis.

  16. High ω-3:ω-6 fatty acids ratio increases fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in human ectopic endometrial cells

    PubMed Central

    Khanaki, Korosh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi; Darabi, Masoud; Mehdizadeh, Amir; Shabani, Mahdi; Rahimipour, Ali; Nouri, Mohammad

    2014-01-01

    Background: Endometriosis, a common chronic inflammatory disorder, is defined by the atypical growth of endometrium- like tissue outside of the uterus. Secretory phospholipase A2 group IIa (sPLA2-IIa) and fatty acid binding protein4 (FABP4) play several important roles in the inflammatory diseases. Objective: Due to reported potential anti-inflammatory effects of ω-3 and ω-6 fatty acids, the purpose of the present study was to investigate the effects of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) on fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in cultured endometrial cells. Materials and Methods: Ectopic and eutopic endometrial tissues obtained from 15 women were snap frozen. After thawing and tissue digestion, primary mixed stromal and endometrial epithelial cell culture was performed for 8 days in culture mediums supplemented with normal and high ratios of ω-3 and ω-6 PUFA. sPLA2-IIa in the culture medium and FABP4 level was determined using enzyme immuno assay (EIA) technique. Results: Within ectopic endometrial cells group, the level of cellular FABP4 and extracellular sPLA2-IIa were remarkably increased under high ω-3 PUFA exposure compared with control condition (p=0.014 and p=0.04 respectively). Conclusion: ω-3 PUFAs may increase the level of cellular FABP4 and extracellular sPLA2-IIa in ectopic endometrial cells, since sPLAIIa and FABP4 may affect endometriosis via several mechanisms, more relevant studies are encouraged to know the potential effect of increased cellular FABP4 and extracellular sPLA2-IIa on endometriosis. PMID:25709631

  17. Cytosolic phospholipase A2 is coupled to muscarinic receptors in the human astrocytoma cell line 1321N1: characterization of the transducing mechanism.

    PubMed Central

    Bayon, Y; Hernandez, M; Alonso, A; Nuñez, L; Garcia-Sancho, J; Leslie, C; Sanchez Crespo, M; Nieto, M L

    1997-01-01

    The cholinergic agonist carbachol induced the release of arachidonic acid in the 1321N1 astrocytoma cell line, and this was blocked by atropine, suggesting the involvement of muscarinic receptors. To assess the mechanisms of signalling involved in the response to carbachol, a set of compounds characterized by eliciting responses through different mechanisms was tested. A combination of 4beta-phorbol 12beta-myristate 13alpha-acetate and thapsigargin, an inhibitor of endomembrane Ca2+-ATPase that induces a prolonged elevation of cytosolic Ca2+ concentration, induced an optimal response, suggesting at first glance that both protein kinase C (PKC) and Ca2+ mobilization were involved in the response. This was consistent with the observation that carbachol elicited Ca2+ mobilization and PKC-dependent phosphorylation of cytosolic phospholipase A2 (cPLA2; phosphatide sn-2-acylhydrolase, EC 3.1.1.4) as measured by a decrease in electrophoretic mobility. Nevertheless, the release of arachidonate induced by carbachol was unaltered in media containing decreased concentrations of Ca2+ or in the presence of neomycin, a potent inhibitor of phospholipase C which blocks phosphoinositide turnover and Ca2+ mobilization. Guanosine 5'-[gamma-thio]triphosphate added to the cell-free homogenate induced both [3H]arachidonate release and cPLA2 translocation to the cell membrane fraction in the absence of Ca2+, thus suggesting the existence of an alternative mechanism of cPLA2 translocation dependent on G-proteins and independent of Ca2+ mobilization. From the combination of experiments utilizing biochemical and immunological tools the involvement of cPLA2 was ascertained. In summary, these data indicate the existence in the astrocytoma cell line 1321N1 of a pathway involving the cPLA2 which couples the release of arachidonate to the occupancy of receptors for a neurotransmitter, requires PKC activity and G-proteins and might operate in the absence of Ca2+ mobilization. PMID:9173894

  18. A rapid phospholipase A2 bioassay using 14C-oleate-labelled E. coli bacterias.

    PubMed

    Meyer, T; von Wichert, P; Weins, D

    1989-02-01

    Two methods of phospholipase A2 determination using 14C-labelled E. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved 14C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A2 regarding pH and Ca2+ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A2 levels, but that the filter method is superior in terms of sensitivity and workload.

  19. Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment, Molecular Docking, and Enzyme Activity/Binding Assays.

    PubMed

    Djakpa, Helene; Kulkarni, Aditya; Barrows-Murphy, Scheneque; Miller, Greg; Zhou, Weihong; Cho, Hyejin; Török, Béla; Stieglitz, Kimberly

    2016-05-01

    Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non-HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo-phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC50 ) in the nanomolar range with monomeric substrate diC4 PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co-ordinates were available) and results analyzed to develop next-generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high-throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets.

  20. Phospholipase D from Loxosceles laeta Spider Venom Induces IL-6, IL-8, CXCL1/GRO-α, and CCL2/MCP-1 Production in Human Skin Fibroblasts and Stimulates Monocytes Migration.

    PubMed

    Rojas, José M; Arán-Sekul, Tomás; Cortés, Emmanuel; Jaldín, Romina; Ordenes, Kely; Orrego, Patricio R; González, Jorge; Araya, Jorge E; Catalán, Alejandro

    2017-04-05

    Cutaneous loxoscelism envenomation by Loxosceles spiders is characterized by the development of a dermonecrotic lesion, strong inflammatory response, the production of pro-inflammatory mediators, and leukocyte migration to the bite site. The role of phospholipase D (PLD) from Loxosceles in the recruitment and migration of monocytes to the envenomation site has not yet been described. This study reports on the expression and production profiles of cytokines and chemokines in human skin fibroblasts treated with catalytically active and inactive recombinant PLDs from Loxosceles laeta (rLlPLD) and lipid inflammatory mediators ceramide 1-phosphate (C1P) and lysophosphatidic acid (LPA), and the evaluation of their roles in monocyte migration. Recombinant rLlPLD1 (active) and rLlPLD2 (inactive) isoforms induce interleukin (IL)-6, IL-8, CXCL1/GRO-α, and CCL2/monocyte chemoattractant protein-1 (MCP-1) expression and secretion in fibroblasts. Meanwhile, C1P and LPA only exhibited a minor effect on the expression and secretion of these cytokines and chemokines. Moreover, neutralization of both enzymes with anti-rLlPLD1 antibodies completely inhibited the secretion of these cytokines and chemokines. Importantly, conditioned media from fibroblasts, treated with rLlPLDs, stimulated the transmigration of THP-1 monocytes. Our data demonstrate the direct role of PLDs in chemotactic mediator synthesis for monocytes in human skin fibroblasts and indicate that inflammatory processes play an important role during loxoscelism.

  1. Selective Inhibition of Human Group IIA-secreted Phospholipase A2 (hGIIA) Signaling Reveals Arachidonic Acid Metabolism Is Associated with Colocalization of hGIIA to Vimentin in Rheumatoid Synoviocytes*

    PubMed Central

    Lee, Lawrence K.; Bryant, Katherine J.; Bouveret, Romaric; Lei, Pei-Wen; Duff, Anthony P.; Harrop, Stephen J.; Huang, Edwin P.; Harvey, Richard P.; Gelb, Michael H.; Gray, Peter P.; Curmi, Paul M.; Cunningham, Anne M.; Church, W. Bret; Scott, Kieran F.

    2013-01-01

    Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design. PMID:23482564

  2. The chemokine receptor CXCR7 is highly expressed in human glioma cells and mediates antiapoptotic effects.

    PubMed

    Hattermann, Kirsten; Held-Feindt, Janka; Lucius, Ralph; Müerköster, Susanne Sebens; Penfold, Mark E T; Schall, Thomas J; Mentlein, Rolf

    2010-04-15

    The chemokine CXCL12/stromal cell-derived factor-1 and its receptor CXCR4 play a major role in tumor invasion, proliferation, and metastasis. Recently, CXCR7 was identified as a novel, alternate receptor for CXCL12 and CXCL11/I-TAC. Because both chemokines are expressed abundantly in human astrocytomas and glioblastomas, we investigated the occurrence and function of both receptors in astroglial tumors. In situ, CXCR7 is highly expressed on tumor endothelial, microglial, and glioma cells whereas CXCR4 has a much more restricted localization; CXCL12 is often colocalized with CXCR7. CXCR7 transcription in tumor homogenates increased with malignancy. In vitro, CXCR7 was highly expressed in all glioma cell lines investigated whereas CXCR4 was only scarcely transcribed on one of eight lines. In contrast, a tumor stem-like cell line preferentially expressed CXCR4 which diminished upon differentiation, whereas CXCR7 increased drastically. Stimulation of CXCR7-positive glioma cells (CXCR4- and CXCR3-negative) by CXCL12 induced transient phosphorylation of extracellular signal-regulated kinases Erk1/2, indicating that the receptor is functionally active. The phosphoinositide-specific phospholipase C inhibitor U73122 effectively inhibited Erk activation and suggests that the mitogen-activated protein kinase pathway is activated indirectly. Whereas proliferation and migration were little influenced, chemokine stimulation prevented camptothecin- and temozolomide-induced apoptosis. The selective CXCR7 antagonist CCX733 reduced the antiapoptotic effects of CXCL12 as shown by nuclear (Nicoletti) staining, caspase-3/7 activity assays, and cleavage of poly(ADP-ribose) polymerase-1. Thus, CXCR7 is a functional receptor for CXCL12 in astrocytomas/glioblastomas and mediates resistance to drug-induced apoptosis. Whereas CXCR7 is found on "differentiated" glioma cells, the alternate receptor CXCR4 is also localized on glioma stem-like cells.

  3. Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

    PubMed

    Drayer, A L; Meima, M E; Derks, M W; Tuik, R; van Haastert, P J

    1995-10-15

    Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.

  4. Lipase and phospholipase biosensors: a review.

    PubMed

    Herrera-López, Enrique J

    2012-01-01

    Recent advances in the field of biology, electronics, and nanotechnology have improved the development of biosensors. A biosensor is a device composed of a biological recognition element and a sensor element. Biosensor applications are becoming increasingly important in areas such as biotechnology, pharmaceutics, food, and environment. Lipases and phospholipases are enzymes which have been used widely in food industry, oleochemical industry, biodegradable polymers, detergents, and other applications. In the medical industry, lipases and phospholipases are used as diagnostic tools to detect triglycerides, cholesterol, and phospholipids levels in blood samples. Therefore, the development of lipase and phospholipase biosensors is of paramount importance in the clinical area. This chapter introduces the reader into the preliminaries of biosensor and reviews recent developments of lipase and phospholipase biosensors.

  5. DAG/PKCδ and IP3/Ca²⁺/CaMK IIβ Operate in Parallel to Each Other in PLCγ1-Driven Cell Proliferation and Migration of Human Gastric Adenocarcinoma Cells, through Akt/mTOR/S6 Pathway.

    PubMed

    Dai, Lianzhi; Zhuang, Luhua; Zhang, Bingchang; Wang, Fen; Chen, Xiaolei; Xia, Chun; Zhang, Bing

    2015-12-01

    Phosphoinositide specific phospholipase Cγ (PLCγ) activates diacylglycerol (DAG)/protein kinase C (PKC) and inositol 1,4,5-trisphosphate (IP3)/Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) axes to regulate import events in some cancer cells, including gastric adenocarcinoma cells. However, whether DAG/PKCδ and IP3/Ca(2+)/CaMK IIβ axes are simultaneously involved in PLCγ1-driven cell proliferation and migration of human gastric adenocarcinoma cells and the underlying mechanism are not elucidated. Here, we investigated the role of DAG/PKCδ or CaMK IIβ in PLCγ1-driven cell proliferation and migration of human gastric adenocarcinoma cells, using the BGC-823 cell line. The results indicated that the inhibition of PKCδ and CaMK IIβ could block cell proliferation and migration of BGC-823 cells as well as the effect of inhibiting PLCγ1, including the decrease of cell viability, the increase of apoptotic index, the down-regulation of matrix metalloproteinase (MMP) 9 expression level, and the decrease of cell migration rate. Both DAG/PKCδ and CaMK IIβ triggered protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/S6 pathway to regulate protein synthesis. The data indicate that DAG/PKCδ and IP3/Ca(2+)/CaMK IIβ operate in parallel to each other in PLCγ1-driven cell proliferation and migration of human gastric adenocarcinoma cells through Akt/mTOR/S6 pathway, with important implication for validating PLCγ1 as a molecular biomarker in early gastric cancer diagnosis and disease surveillance.

  6. Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells

    SciTech Connect

    Fowler, C.J. Karolinska Institutet ); Ahlgren, P.C. ); O'Neill, C. )

    1991-01-01

    IMR-32 and SK-N-MC cells were found to contain ({sup 3}H)quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable ({sup 3}H)8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC{sub 50} value of about 50 {mu}M in both cases. Pirenzepine inhibited the carbachol stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC{sub 50} values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT{sub 1A} receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA{sub 2} values of 5.78 (IMR-32) and 5.61 (SK-N-MC). The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.

  7. Phospholipases in food industry: a review.

    PubMed

    Casado, Víctor; Martín, Diana; Torres, Carlos; Reglero, Guillermo

    2012-01-01

    Mammal, plant, and mainly microbial phospholipases are continuously being studied, experimented, and some of them are even commercially available at industrial scale for food industry. This is because the use of phospholipases in the production of specific foods leads to attractive advantages, such as yield improvement, energy saving, higher efficiency, improved properties, or better quality of the final product. Furthermore, biocatalysis approaches in the food industry are of current interest as non-pollutant and cleaner technologies. The present chapter reviews the most representative examples of the use of phospholipases in food industry, namely edible oils, dairy, and baking products, emulsifying agents, as well as the current trend to the development of novel molecular species of phospholipids with added-value characteristics.

  8. [Several properties of cotton seed phospholipase D].

    PubMed

    Rakhimov, M M; Mad'iarov, Sh R; Abdumalikov, A Kh

    1976-03-01

    Properties of phospholipase D were studied using purified enzyme preparation from cotton seeds. The results obtained differ from those described in literature. It has been shown that the promoting action is exerted not only by diethyl ether and sodium dodecyl sulfate commonly used as initiators, but by some organic solvents in the presence of calcium ions as well. The activation of phospholipase D is also possible in the presence of other bivalent cations, e.g. Sr2+, Ba2+, Mn2+ and Mg2+. It is assumed that the enzyme activation occurs only in the presence of the stable heterogenous system: water-soluble enzyme--phospholipid--non-aqueous phase. Another important factor is the type of modification of the surface of the phospholipid phase, responsible for the enzyme adsorption and its subsequent activation. Comparison is made of the properties of phospholipases D isolated from cotton seeds and some other sources.

  9. Secretion of phospholipase C by Pseudomonas aeruginosa.

    PubMed Central

    Stinson, M W; Hayden, C

    1979-01-01

    The conditions necessary for the secretion of phospholipase C (phosphatidylcholine cholinephosphohydrolase) by Pseudomonas aeruginosa were studied. Enzyme secretion by washed cell suspensions required a carbon source and ammonium, potassium, and calcium ions. The calcium requirement could be substituted by magnesium and strontium but not by copper, manganese, cobalt, or zinc. During growth in liquid medium, cells secreted phospholipase C during late logarithmic and early stationary phases. Secretion was repressed by the addition of inorganic phosphate but not by organic phosphates, glucose, or sodium succinate. Studies with tetracycline indicated that de novo protein synthesis was necessary for the secretion of phospholipase C and that the exoenzyme was not released from a preformed periplasmic pool. Similarly, extraction of actively secreting cells with 0.2 M MgCl2 at pH 8.4 solubilized large quantities of the periplasmic enzyme alkaline phosphatase but insignificant amounts of phospholipase C. Bacteria continued to secrete enzyme for nearly 45 min after the addition of inorganic phosphate or rifampin. Images PMID:114487

  10. Relationship between erythrocyte membrane phase properties and susceptibility to secretory phospholipase A2.

    PubMed

    Best, Katrina B; Ohran, Allison J; Hawes, Andrea C; Hazlett, Theodore L; Gratton, Enrico; Judd, Allan M; Bell, John D

    2002-11-26

    Normally, cell membranes resist hydrolysis by secretory phospholipase A(2). However, upon elevation of intracellular calcium, the cells become susceptible. Previous investigations demonstrated a possible relationship between changes in lipid order caused by increased calcium and susceptibility to phospholipase A(2). To further explore this relationship, we used temperature as an experimental means of manipulating membrane physical properties. We then compared the response of human erythrocytes to calcium ionophore at various temperatures in the range of 20-50 degrees C using fluorescence spectroscopy and two-photon fluorescence microscopy. The steady state fluorescence emission of the environment-sensitive probe, laurdan, revealed that erythrocyte membrane order decreases systematically with temperature throughout this range, especially between 28 and 45 degrees C. Furthermore, the ability of calcium ionophore to induce increased membrane order and susceptibility to phospholipase A(2) depended similarly on temperature. Both responses to calcium influx were enhanced as membrane fluidity increased. Analysis of the spatial distribution of laurdan fluorescence at several temperatures indicated that the ordering effect of intracellular calcium on fluid membranes generates an increase in the number of fluid-solid boundaries. Hydrolysis of the membrane appeared to progress outward from these boundaries. We conclude that phospholipase A(2) prefers to hydrolyze lipids in fluid regions of human erythrocyte membranes, but primarily when those regions coexist with domains of ordered lipids.

  11. Differential phospholipase C-dependent modulation of TASK and TREK two-pore domain K+ channels in rat thalamocortical relay neurons

    PubMed Central

    Bista, Pawan; Pawlowski, Matthias; Cerina, Manuela; Ehling, Petra; Leist, Michael; Meuth, Patrick; Aissaoui, Ania; Borsotto, Marc; Heurteaux, Catherine; Decher, Niels; Pape, Hans-Christian; Oliver, Dominik; Meuth, Sven G; Budde, Thomas

    2015-01-01

    The activity of two-pore domain potassium channels (K2P) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K+ channel (TWIK)-related acid-sensitive K+ (TASK) channels and TWIK-related K+ (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCβ), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) acting downstream of PLCβ was probed. The standing outward current (ISO) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP2 level in TC neurons, we here show that the scavenging of PIP2 (by neomycin) results in an increased muscarinic effect on ISO whereas increased availability of PIP2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP2 but no other phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCβ as well as the application of OAG resulted in membrane depolarization, while PIP2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP2 and DAG in K2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP2 depletion) and TASK (via DAG) channels following MACh

  12. Multiple extracellular phospholipase activities from Prevotella intermedia.

    PubMed

    Bulkacz, Jaime; Faull, Kym F

    2009-06-01

    Enzyme preparations obtained from Prevotella intermedia culture supernatants were partially purified by ammonium sulfate precipitation and ion-exchange column chromatography. Hydrolytic activities were revealed by an assay that uses silicic acid thin layer chromatography to separate the products derived from (14)C-labeled phosphatidyl-choline (PC) hydrolysis. These products were then measured by liquid scintillation spectrometry after iodine visualization. The assays revealed linearity of substrate depletion and product formation with respect to time and protein concentration up to 30 min of incubation. The products had retention times consistent with lyso-phospholipids and phosphoryl-choline. These data strongly suggests the presence of both phospholipase A (PL-A) and phospholipase C (PL-C) activities.

  13. Kinetic Analysis of a Mammalian Phospholipase D

    PubMed Central

    Henage, Lee G.; Exton, John H.; Brown, H. Alex

    2013-01-01

    In mammalian cells, phospholipase D activity is tightly regulated by diverse cellular signals, including hormones, neurotransmitters, and growth factors. Multiple signaling pathways converge upon phospholipase D to modulate cellular actions, such as cell growth, shape, and secretion. We examined the kinetics of protein kinase C and G-protein regulation of mammalian phospholipase D1 (PLD1) in order to better understand interactions between PLD1 and its regulators. Activation by Arf-1, RhoA, Rac1, Cdc42, protein kinase Cα, and phosphatidylinositol 4,5-bisphosphate displayed surface dilution kinetics, but these effectors modulated different kinetic parameters. PKCα activation of PLD1 involves N- and C-terminal PLD domains. Rho GTPases were binding activators, enhancing the catalytic efficiency of a purified PLD1 catalytic domain via effects on Km. Arf-1, a catalytic activator, stimulated PLD1 by enhancing the catalytic constant, kcat. A kinetic description of PLD1 activation by multiple modulators reveals a mechanism for apparent synergy between activators. Synergy was observed only when PLD1 was simultaneously stimulated by a binding activator and a catalytic activator. Surprisingly, synergistic activation was steeply dependent on phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Together, these findings suggest a role for PLD1 as a signaling node, in which integration of convergent signals occurs within discrete locales of the cellular membrane. PMID:16339153

  14. 1-(5-Carboxyindol-1-yl)propan-2-ones as inhibitors of human cytosolic phospholipase A2alpha: synthesis and properties of bioisosteric benzimidazole, benzotriazole and indazole analogues.

    PubMed

    Bovens, Stefanie; Kaptur, Martina; Elfringhoff, Alwine Schulze; Lehr, Matthias

    2009-04-15

    The indole ring systems of the cytosolic phospholipase A(2)alpha (cPLA(2)alpha) inhibitor 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (2) and the isomeric 6-carboxylic acid (3) were replaced by benzimidazole, benzotriazole and indazole scaffolds, respectively. The effect of the structural variations on cPLA(2)alpha inhibitory potency, metabolic stability and solubility was studied. The lead 2 and the indazole-5-carboxylic acid 28 were the metabolically most stable compounds in an assay with rat liver microsomes, while the benzimidazole-5-carboxylic acid derivative 13 possessed the best water solubility (22 microg/mL at pH 7.4). The indazole-5-carboxylic acid 28 revealed the highest cPLA(2)alpha inhibitory potency of the compounds in this series. With an IC(50)-value of 0.005 microM it was about sevenfold more active than the lead 2.

  15. Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.

    PubMed

    Wu, D Q; Lee, C H; Rhee, S G; Simon, M I

    1992-01-25

    High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 11 cDNA showed marked increases in formation of [3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 11 resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha OA did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.

  16. Inactivation of Phospholipase D Diminishes Acinetobacter baumannii Pathogenesis▿ †

    PubMed Central

    Jacobs, Anna C.; Hood, Indriati; Boyd, Kelli L.; Olson, Patrick D.; Morrison, John M.; Carson, Steven; Sayood, Khalid; Iwen, Peter C.; Skaar, Eric P.; Dunman, Paul M.

    2010-01-01

    Acinetobacter baumannii is an emerging bacterial pathogen of considerable health care concern. Nonetheless, relatively little is known about the organism's virulence factors or their regulatory networks. Septicemia and ventilator-associated pneumonia are two of the more severe forms of A. baumannii disease. To identify virulence factors that may contribute to these disease processes, genetically diverse A. baumannii clinical isolates were evaluated for the ability to proliferate in human serum. A transposon mutant library was created in a strain background that propagated well in serum and screened for members with decreased serum growth. The results revealed that disruption of A. baumannii phospholipase D (PLD) caused a reduction in the organism's ability to thrive in serum, a deficiency in epithelial cell invasion, and diminished pathogenesis in a murine model of pneumonia. Collectively, these results suggest that PLD is an A. baumannii virulence factor. PMID:20194595

  17. In vivo Detection of Phospholipase C by Enzyme-Activated Near-infrared Probes

    PubMed Central

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

    2011-01-01

    In this paper 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. Due to 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 IC50 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 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. PMID:22034913

  18. Thyroid hormone status regulates the expression of secretory phospholipases.

    PubMed

    Sharma, Pragya; Levesque, Tania; Boilard, Eric; Park, Edwards A

    2014-01-31

    Thyroid hormone (T3) stimulates various metabolic pathways and the hepatic actions of T3 are mediated primarily through the thyroid hormone receptor beta (TRβ). Hypothyroidism has been linked with low grade inflammation, elevated risk of hepatic steatosis and atherosclerosis. Secretory phospholipases (sPLA2) are associated with inflammation, hyperlipidemia and atherosclerosis. Due to potential linkage between thyroid hormone and sPLA2, we investigated the effect of thyroid hormone status on the regulation of secretory phospholipases in mice, rats and human liver. T3 suppressed the expression of the sPLA2 group IIa (PLA2g2a) gene in the liver of BALB/c mice and C57BL/6 transgenic mice expressing the human PLA2g2a. PLA2g2a was elevated with hypothyroidism and high fat diets which may contribute to the low grade inflammation associated with hypothyroidism and diet induced obesity. We also examined the effects of the TRβ agonist eprotirome on hepatic gene regulation. We observed that eprotirome inhibited the expression of selected sPLA2 genes and furthermore the cytokine mediated induction PLA2g2a was suppressed. In addition, eprotirome induced genes involved in fatty acid oxidation and cholesterol clearance while inhibiting lipogenic genes. Our results indicate that in vivo thyroid hormone status regulates the abundance of sPLA2 and the inhibition of PLA2g2a by T3 is conserved across species. By regulating sPLA2 genes, T3 may impact processes associated with atherosclerosis and inflammation and TRβ agonists may ameliorate inflammation and hyperlipidemia.

  19. Thyroid hormone status regulates the expression of secretory phospholipases

    PubMed Central

    Sharma, Pragya; Levesque, Tania; Boilard, Eric; Park, Edwards A.

    2014-01-01

    Thyroid hormone (T3) stimulates various metabolic pathways and the hepatic actions of T3 are mediated primarily through the thyroid hormone receptor beta (TRβ). Hypothyroidism has been linked with low grade inflammation, elevated risk of hepatic steatosis and atherosclerosis. Secretory phospholipases (sPLA2) are associated with inflammation, hyperlipidemia and atherosclerosis. Due to potential linkage between thyroid hormone and sPLA2, we investigated the effect of thyroid hormone status on the regulation of secretory phospholipases in mice, rats and human liver. T3 suppressed the expression of the sPLA2 group IIa (PLA2g2a) gene in the liver of BALB/c mice and C57BL/6 transgenic mice expressing the human PLA2g2a. PLA2g2a was elevated with hypothyroidism and high fat diets which may contribute to the low grade inflammation associated with hypothyroidism and diet induced obesity. We also examined the effects of the TRβ agonist eprotirome on hepatic gene regulation. We observed that eprotirome inhibited the expression of selected sPLA2 genes and furthermore the cytokine mediated induction PLA2g2a was suppressed. In addition, eprotirome induced genes involved in fatty acid oxidation and cholesterol clearance while inhibiting lipogenic genes. Our results indicate that in vivo thyroid hormone status regulates the abundance of sPLA2 and the inhibition of PLA2g2a by T3 is conserved across species. By regulating sPLA2 genes, T3 may impact processes associated with atherosclerosis and inflammation and TRβ agonists may ameliorate inflammation and hyperlipidemia. PMID:24440706

  20. Effects of dexamethasone on palate mesenchymal cell phospholipase activity

    SciTech Connect

    Bulleit, R.F.; Zimmerman, E.F.

    1984-09-15

    Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with (3H)arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity.

  1. Assay strategies and methods for phospholipases

    SciTech Connect

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is {approximately} as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous.

  2. 1-(3-biaryloxy-2-oxopropyl)indole-5-carboxylic acids and related compounds as dual inhibitors of human cytosolic phospholipase A2α and fatty acid amide hydrolase.

    PubMed

    Zahov, Stefan; Drews, Andreas; Hess, Mark; Schulze Elfringhoff, Alwine; Lehr, Matthias

    2011-03-07

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes that have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (5) is a dual inhibitor of cPLA2α and FAAH. Structure-activity relationship studies revealed that substituents at the indole 3- and 5-positions and replacement of the indole scaffold of this compound by other heterocycles strongly influences the inhibitory potency against cPLA2α and FAAH, respectively. Herein we report the effect of variation of the 4-octyl residue of 5 and an exchange of its carboxylic acid moiety by some bioisosteric functional groups. Several of the compounds assayed were favorably active against both enzymes, and could therefore represent agents with improved analgesic and anti-inflammatory qualities in comparison with selective cPLA2 α and FAAH inhibitors.

  3. Inhibitory Effect of Orientin on Secretory Group IIA Phospholipase A2.

    PubMed

    Bae, Jong-Sup

    2015-08-01

    It is well known that the expression level of secretory group IIA phospholipase A2 (sPLA2-IIA) is elevated in inflammatory diseases and lipopolysaccharide (LPS) upregulates the expression of sPLA2-IIA in human umbilical vein endothelial cells (HUVECs). Orientin, a C-glycosyl flavonoid, is known to have anxiolytic, anti-oxidative, and anti-inflammatory activity. Here, orientin was examined for its effects on the expression and activity of sPLA2-IIA in HUVECs and mouse. Prior treatment of cells or mouse with orientin inhibited LPS-induced expression and activity of sPLA2-IIA. And orientin suppressed the activation of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK) 1/2 by LPS. Therefore, these results suggest that orientin may inhibit LPS-mediated expression of sPLA2-IIA by suppression of cPLA2 and ERK 1/2.

  4. Reminiscence of phospholipase B in Penicillium notatum.

    PubMed

    Saito, Kunihiko

    2014-01-01

    Since the phospholipase B (PLB) was reported as a deacylase of both lecithin and lysolecithin yielding fatty acids and glycerophosphocholine (GPC), there was a question as to whether it is a single enzyme or a mixture of a phospholipase A2 (PLA2) and a lysophospholipase (LPL). We purified the PLB in Penicillium notatum and showed that it catalyzed deacylation of sn-1 and sn-2 fatty acids of 1,2-diacylphospholipids and also sn-1 or sn-2 fatty acids of 1- or 2-monoacylphospholipids (lysophospholipids). Further, it also has a monoacyllipase activity. The purified PLB is a glycoprotein with m.w. of 91,300. The sugar moiety is M9 only and the protein moiety consists of 603 amino acids. PLB, different from PLA2, shows other enzymatic activities, such as transacylase, lipase and acylesterase. PLB activity is influenced by various substances, e.g. detergents, deoxycholate, diethylether, Fe(3+), and endogenous protease. Therefore, PLB might have broader roles than PLA2 in vivo. The database shows an extensive sequence similarity between P. notatum PLB and fungal PLB, cPLA2 and patatin, suggesting a homologous relationship. The catalytic triad of cPLA2, Ser, Asp and Arg, is also present in P. notatum PLB. Other related PLBs, PLB/Lipases are discussed.

  5. Food extracts consumed in Mediterranean countries and East Asia reduce protein concentrations of androgen receptor, phospho-protein kinase B, and phospho-cytosolic phospholipase A(2)alpha in human prostate cancer cells.

    PubMed

    Singh, Jaskirat; Xie, Chanlu; Yao, Mu; Hua, Sheng; Vignarajan, Soma; Jardine, Greg; Hambly, Brett D; Sved, Paul; Dong, Qihan

    2010-04-01

    Active surveillance is an emerging management option for the rising number of men with low-grade, clinically localized prostate cancer. However, 30-40% of men on active surveillance will progress to high-grade disease over 5 y. With the ultimate aim of developing a food-based chemoprevention strategy to retard cancer progression in these otherwise healthy men, we have developed a blend of food extracts commonly consumed in Mediterranean countries and East Asia. The effect of the food extracts known as Blueberry Punch (BBP) on prostate cancer cell growth and key signaling pathways were examined in vitro and in vivo. BBP reduced prostate cancer cell growth in a dose-dependent manner (0.08-2.5%) at 72 h in vitro due to the reduction in cell proliferation and viability. Prostate cancer cell xenograft-bearing mice, administered 10% BBP in drinking water for 2 wk, had a 25% reduction in tumor volume compared with the control (water only). In vitro, BBP reduced protein concentrations in 3 signaling pathways necessary for the proliferation and survival of prostate cancer cells, namely androgen receptor, phospho-protein kinase B/protein kinase B, and phospho-cytosolic phospholipase A(2)alpha. The downstream effectors of these pathways, including prostate-specific antigen and glycogen synthase kinase 3beta, were also reduced. Thus, this palatable food supplement is a potential candidate for testing in clinical trials and may ultimately prove effective in retarding the progression of low-grade, early-stage prostate cancer in men managed by active surveillance.

  6. Ceramides increase the activity of the secretory phospholipase A2 and alter its fatty acid specificity.

    PubMed Central

    Koumanov, Kamen S; Momchilova, Albena B; Quinn, Peter J; Wolf, Claude

    2002-01-01

    Modulation of human recombinant secretory type II phospholipase A(2) activity by ceramide and cholesterol was investigated using model glycerophospholipid substrates composed of phosphatidylethanolamine and phosphatidylserine dispersed in aqueous medium. Enzyme activity was monitored by measurement of released fatty acids using capillary GC-MS. Fatty acids from the sn-2 position of the phospholipids were hydrolysed by the enzyme in proportion to the relative abundance of the phospholipid in the substrate. Addition of increasing amounts of ceramide to the substrate progressively enhanced phospholipase activity. The increased activity was accomplished largely by preferential hydrolysis of polyunsaturated fatty acids, particularly arachidonic acid, derived from phosphatidylethanolamine. The addition of sphingomyelin to the substrate glycerophospholipids inhibited phospholipase activity but its progressive substitution by ceramide, so as to mimic sphingomyelinase activity, counteracted the inhibition. The presence of cholesterol in dispersions of glycerophospholipid-substrate-containing ceramides suppressed activation of the enzyme resulting from the presence of ceramide. The molecular basis of enzyme modulation was investigated by analysis of the phase structure of the dispersed lipid substrate during temperature scans from 46 to 20 degrees C using small-angle synchrotron X-ray diffraction. These studies indicated that intermediate structures created after ceramide-dependent phase separation of hexagonal and lamellar phases represent the most susceptible form of the substrate for enzyme hydrolysis. PMID:11903045

  7. Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis

    PubMed Central

    Lee, Yang Won; Lee, Shin Yung; Lee, Younghoon

    2013-01-01

    Background Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. Objective The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. Methods Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. Results The results of the current study suggest that majority of the patients display expression of lipase RES_0242. Conclusion These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus. PMID:24003273

  8. Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

    PubMed

    Falik Zaccai, Tzipora C; Savitzki, David; Zivony-Elboum, Yifat; Vilboux, Thierry; Fitts, Eric C; Shoval, Yishay; Kalfon, Limor; Samra, Nadra; Keren, Zohar; Gross, Bella; Chasnyk, Natalia; Straussberg, Rachel; Mullikin, James C; Teer, Jamie K; Geiger, Dan; Kornitzer, Daniel; Bitterman-Deutsch, Ora; Samson, Abraham O; Wakamiya, Maki; Peterson, Johnny W; Kirtley, Michelle L; Pinchuk, Iryna V; Baze, Wallace B; Gahl, William A; Kleta, Robert; Anikster, Yair; Chopra, Ashok K

    2017-02-01

    Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

  9. Phospholipase C-β in immune cells.

    PubMed

    Kawakami, Toshiaki; Xiao, Wenbin

    2013-09-01

    Great progress has recently been made in structural and functional research of phospholipase C (PLC)-β. We now understand how PLC-β isoforms (β1-β4) are activated by GTP-bound Gαq downstream of G protein-coupled receptors. Numerous studies indicate that PLC-βs participate in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. The PLC-β3 isoform also interplays with tyrosine kinase-based signaling pathways, to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1, with which PLC-β3 and Stat5 form a multi-molecular signaling platform, named SPS complex. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation.

  10. Streptomyces phospholipase D cloning and production.

    PubMed

    Nakazawa, Yozo

    2012-01-01

    The transphosphatidylation catalytic ability of phospholipase D (PLD, EC 3.1.4.4) is a powerful biochemical tool for the acquisition of rare phospholipids (PLs), e.g., phosphatidylglycerol (PG) and phosphatidylserine (PS), and artificial phospholipids, which do not occur in nature. Specifically, actinomycete PLD recognizes not only the alcohols (i.e., glycerol or serine) corresponding to the polar head groups of natural PLs, but also secondary alcohols, aromatic alcohols, saccharides, N-heterocyclic alcohols, and vitamins as acceptors. Therefore, actinomycete PLD is a valuable enzyme in food, cosmetics, fine chemical and pharmaceutical industries. Here, we describe a protocol for the screening for PLD-producing microorganisms, several PLD assays and methods of PLD production-purification and the strategy of cloning of the Streptomyces PLD gene.

  11. 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.

  12. Cytosolic phospholipase A2: physiological function and role in disease

    PubMed Central

    Leslie, Christina C.

    2015-01-01

    The group IV phospholipase A2 (PLA2) family is comprised of six intracellular enzymes (GIVA, -B, -C, -D, -E, and -F) commonly referred to as cytosolic PLA2 (cPLA2)α, -β, -γ, -δ, -ε, and -ζ. They contain a Ser-Asp catalytic dyad and all except cPLA2γ have a C2 domain, but differences in their catalytic activities and subcellular localization suggest unique regulation and function. With the exception of cPLA2α, the focus of this review, little is known about the in vivo function of group IV enzymes. cPLA2α catalyzes the hydrolysis of phospholipids to arachidonic acid and lysophospholipids that are precursors of numerous bioactive lipids. The regulation of cPLA2α is complex, involving transcriptional and posttranslational processes, particularly increases in calcium and phosphorylation. cPLA2α is a highly conserved widely expressed enzyme that promotes lipid mediator production in human and rodent cells from a variety of tissues. The diverse bioactive lipids produced as a result of cPLA2α activation regulate normal physiological processes and disease pathogenesis in many organ systems, as shown using cPLA2α KO mice. However, humans recently identified with cPLA2α deficiency exhibit more pronounced effects on health than observed in mice lacking cPLA2α, indicating that much remains to be learned about this interesting enzyme. PMID:25838312

  13. Targeting phospholipase D in cancer, infection and neurodegenerative disorders.

    PubMed

    Brown, H Alex; Thomas, Paul G; Lindsley, Craig W

    2017-02-17

    Lipid second messengers have essential roles in cellular function and contribute to the molecular mechanisms that underlie inflammation, malignant transformation, invasiveness, neurodegenerative disorders, and infectious and other pathophysiological processes. The phospholipase D (PLD) isoenzymes PLD1 and PLD2 are one of the major sources of signal-activated phosphatidic acid (PtdOH) generation downstream of a variety of cell-surface receptors, including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and integrins. Recent advances in the development of isoenzyme-selective PLD inhibitors and in molecular genetics have suggested that PLD isoenzymes in mammalian cells and pathogenic organisms may be valuable targets for the treatment of several human diseases. Isoenzyme-selective inhibitors have revealed complex inter-relationships between PtdOH biosynthetic pathways and the role of PtdOH in pathophysiology. PLD enzymes were once thought to be undruggable owing to the ubiquitous nature of PtdOH in cell signalling and concerns that inhibitors would be too toxic for use in humans. However, recent promising discoveries suggest that small-molecule isoenzyme-selective inhibitors may provide novel compounds for a unique approach to the treatment of cancers, neurodegenerative disorders and other afflictions of the central nervous system, and potentially serve as broad-spectrum antiviral and antimicrobial therapeutics.

  14. Cytosolic phospholipase A₂: physiological function and role in disease.

    PubMed

    Leslie, Christina C

    2015-08-01

    The group IV phospholipase A2 (PLA2) family is comprised of six intracellular enzymes (GIVA, -B, -C, -D, -E, and -F) commonly referred to as cytosolic PLA2 (cPLA2)α, -β, -γ, -δ, -ε, and -ζ. They contain a Ser-Asp catalytic dyad and all except cPLA2γ have a C2 domain, but differences in their catalytic activities and subcellular localization suggest unique regulation and function. With the exception of cPLA2α, the focus of this review, little is known about the in vivo function of group IV enzymes. cPLA2α catalyzes the hydrolysis of phospholipids to arachidonic acid and lysophospholipids that are precursors of numerous bioactive lipids. The regulation of cPLA2α is complex, involving transcriptional and posttranslational processes, particularly increases in calcium and phosphorylation. cPLA2α is a highly conserved widely expressed enzyme that promotes lipid mediator production in human and rodent cells from a variety of tissues. The diverse bioactive lipids produced as a result of cPLA2α activation regulate normal physiological processes and disease pathogenesis in many organ systems, as shown using cPLA2α KO mice. However, humans recently identified with cPLA2α deficiency exhibit more pronounced effects on health than observed in mice lacking cPLA2α, indicating that much remains to be learned about this interesting enzyme.

  15. Quercetin-induced downregulation of phospholipase D1 inhibits proliferation and invasion in U87 glioma cells

    SciTech Connect

    Park, Mi Hee; Min, Do Sik

    2011-09-09

    Highlights: {yields} Quercetin, a bioactive flavonoid, suppresses expression and enzymatic activity of phospholipase D1. {yields} Quercetin abolishes NFkB-induced phospholipase D1 expression via inhibition of NFkB transactivation. {yields} Quercetin-induced suppression of phospholipase D1 inhibits invasion and proliferation of human glioma cells. -- Abstract: Phospholipase D (PLD) has been recognized as a regulator of cell proliferation and tumorigenesis, but little is known about the molecules regulating PLD expression. Thus, the identification of small molecules inhibiting PLD expression would be an important advance in PLD-mediated physiology. Quercetin, a ubiquitous bioactive flavonoid, is known to inhibit proliferation and induce apoptosis in a variety of cancer cells. In the present study, we examined the effect of quercetin on the expression of PLD in U87 glioma cells. Quercetin significantly suppressed the expression of PLD1 at the transcriptional level. Moreover, quercetin abolished the protein expression of PLD1 in a time and dose-dependent manner, as well as inhibited PLD activity. Quercetin suppressed NF{kappa}B-induced PLD1 expression via inhibition of NFkB transactivation. Furthermore, quercetin inhibited activation and invasion of metalloproteinase-2 (MMP-2), a key modulator of glioma cell invasion, induced by phosphatidic acid (PA), a product of PLD activity. Taken together these data demonstrate that quercetin abolishes PLD1 expression and subsequently inhibits invasion and proliferation of glioma cells.

  16. Phospholipase and proteinase activities of Candida isolates from denture wearers.

    PubMed

    Marcos-Arias, Cristina; Eraso, Elena; Madariaga, Lucila; Aguirre, Jose Manuel; Quindós, Guillermo

    2011-07-01

    The aim of the present study was to characterise phospholipase and proteinase activities of oral Candida isolates from 100 denture wearers and to study the relationship of these activities with denture stomatitis. Of 100 patients studied, 44 suffered from denture stomatitis. Specimens were collected by swabbing the denture and underlying mucosa. Isolates were previously identified by conventional mycological and genotypic methods. The phospholipase and proteinase activities were evaluated by agar plate methods. A total of 152 isolates were recovered from denture and underlying mucosa, including 101 Candida albicans, 18 Candida tropicalis, 14 Candida glabrata, 11 Candida guilliermondii, four Candida parapsilosis, two Saccharomyces cerevisiae and one isolate each of Candida dubliniensis and Candida krusei. Most C. albicans (97%) showed phospholipase activity; furthermore, the unique C. dubliniensis isolate showed a moderate phospholipase activity. The isolation of C. albicans (chi-square test, P = 0.0016) and phospholipase production by Candida spp. (chi-square test, P = 0.0213) was found to be significantly associated with denture stomatitis. Proteinase production was observed in <30% of isolates, and it was not related to the presence of denture stomatitis (P = 0.7675). Candida albicans isolates may produce both virulence factors, although the proteinase production was only observed in <30% of the isolates. Phospholipase production was exclusive of C. albicans and C. dubliniensis.

  17. Stimulation of Phospholipase A2 by Toxic Main Group Heavy Metals: Partly Dependent on G-proteins?

    PubMed Central

    Krug, H. F.

    1995-01-01

    Organometals induce platelet aggregation and inorganic metal ions such as Cd2+ or Pb2+ sensitise human blood platelets to aggregating agents and this action is associated with the liberation of arachidonic acid and eicosanoid formation. The same mechanism is observed using human leukaemia cells (HL-60) when treated with MeHgCl or Et3PbCl. The fatty acid liberation within human platelets and HL-60 cells could only be inhibited with phospholipase A2 inhibitors of different specificity. Preincubation of the cells with pertussis toxin reduces the activation induced by Et3PbCl to a great extent. The non-catalytic B subunit, that only mediates the binding of the toxin to the cell membranes, has no effect at all. When summarised, these results suggest that one possible mechanism for the stimulation of phospholipase A2 by Et3PbCl functions via a G-protein dependent pathway. PMID:18472750

  18. Variable substrate preference among phospholipase D toxins from sicariid spiders

    DOE PAGES

    Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; ...

    2015-03-09

    Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

  19. Therapeutic inhibition of phospholipase D1 suppresses hepatocellular carcinoma.

    PubMed

    Xiao, Junjie; Sun, Qi; Bei, Yihua; Zhang, Ling; Dimitrova-Shumkovska, Jasmina; Lv, Dongchao; Yang, Yuefeng; Cao, Yan; Zhao, Yingying; Song, Meiyi; Song, Yang; Wang, Fei; Yang, Changqing

    2016-07-01

    Hepatocellular carcinoma (HCC) represents a leading cause of deaths worldwide. Novel therapeutic targets for HCC are needed. Phospholipase D (PD) is involved in cell proliferation and migration, but its role in HCC remains unclear. In the present study, we show that PLD1, but not PLD2, was overexpressed in HCC cell lines (HepG2, Bel-7402 and Bel-7404) compared with the normal human L-02 hepatocytes. PLD1 was required for the proliferation, migration and invasion of HCC cells without affecting apoptosis and necrosis, and PLD1 overexpression was sufficient to promote those effects. By using HCC xenograft models, we demonstrated that therapeutic inhibition of PLD1 attenuated tumour growth and epithelial-mesenchymal transition (EMT) in HCC mice. Moreover, PLD1 was found to be highly expressed in tumour tissues of HCC patients. Finally, mTOR (mechanistic target of rapamycin) and Akt (protein kinase B) were identified as critical pathways responsible for the role of PLD1 in HCC cells. Taken together, the present study indicates that PLD1 activation contributes to HCC development via regulation of the proliferation, migration and invasion of HCC cells, as well as promoting the EMT process. These observations suggest that inhibition of PLD1 represents an attractive and novel therapeutic modality for HCC.

  20. Acinetobacter baumannii Virulence Is Mediated by the Concerted Action of Three Phospholipases D

    PubMed Central

    Stahl, Julia; Bergmann, Holger; Göttig, Stephan; Ebersberger, Ingo; Averhoff, Beate

    2015-01-01

    Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion. PMID:26379240

  1. The Phospholipase D1 Pathway Modulates Macroautophagy

    PubMed Central

    Dall’Armi, Claudia; Hurtado-Lorenzo, Andres; Tian, Huasong; Morel, Etienne; Nezu, Akiko; Chan, Robin B.; Yu, W. Haung; Robinson, Kimberly S.; Yeku, Oladapo; Small, Scott A.; Duff, Karen; Frohman, Michael A.; Wenk, Markus R.; Yamamoto, Akitsugu; Di Paolo, Gilbert

    2012-01-01

    While macroautophagy is known to be an essential degradative process whereby autophagosomes mediate the engulfment and delivery of cytoplasmic components into lysosomes, the lipid changes underlying autophagosomal membrane dynamics are undetermined. Here we show that phospholipase D1 (PLD1), which is primarily associated with the endosomal system, partially relocalizes to the outer membrane of autophagosome-like structures upon nutrient starvation. The localization of PLD1, as well as the starvation-induced increase in PLD activity, are altered by wortmannin, a phosphatidylinositol 3-kinase inhibitor, suggesting PLD1 may act downstream of Vps34. Pharmacological inhibition of PLD and genetic ablation of PLD1 in the mouse decrease the starvation-induced expansion of LC3-positive compartments, consistent with a role of PLD1 in the regulation of autophagy. Furthermore, inhibition of PLD results in higher levels of tau and p62 aggregates in organotypic brain slices. Our in vitro and in vivo findings establish a novel role for PLD1 in autophagy. PMID:21266992

  2. Secreted phospholipase A2 and mast cells.

    PubMed

    Murakami, Makoto; Taketomi, Yoshitaka

    2015-01-01

    Phospholipase A2s (PLA2s) are a group of enzymes that hydrolyze the sn-2 position of phospholipids to release (typically unsaturated) fatty acids and lysophospholipids, which serve as precursors for a variety of bioactive lipid mediators. Among the PLA2 superfamily, secreted PLA2 (sPLA2) enzymes comprise the largest subfamily that includes 11 isoforms with a conserved His-Asp catalytic dyad. Individual sPLA2 enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. Recent studies using transgenic and knockout mice for individual sPLA2 isofoms have revealed their involvement in various pathophysiological events. Here, we overview the current state of knowledge about sPLA2s, specifically their roles in mast cells (MCs) in the context of allergology. In particular, we highlight group III sPLA2 (PLA2G3) as an "anaphylactic sPLA2" that promotes MC maturation and thereby anaphylaxis through a previously unrecognized lipid-orchestrated circuit.

  3. Phosphatidylinositol-specific phospholipase C of Bacillus cereus: cloning, sequencing, and relationship to other phospholipases.

    PubMed Central

    Kuppe, A; Evans, L M; McMillen, D A; Griffith, O H

    1989-01-01

    The phosphatidylinositol (PI)-specific phospholipase C (PLC) of Bacillus cereus was cloned into Escherichia coli by using monoclonal antibody probes raised against the purified protein. The enzyme is specific for hydrolysis of the membrane lipid PI and PI-glycan-containing membrane anchors, which are important structural components of one class of membrane proteins. The protein expressed in E. coli comigrated with B. cereus PI-PLC in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as detected by immunoblotting, and conferred PI-PLC activity on the host. This enzyme activity was inhibited by PI-PLC-specific monoclonal antibodies. The nucleotide sequence of the PI-PLC gene suggests that this secreted bacterial protein is synthesized as a larger precursor with a 31-amino-acid N-terminal extension to the mature enzyme of 298 amino acids. From analysis of coding and flanking sequences of the gene, we conclude that the PI-PLC gene does not reside next to the gene cluster of the other two secreted phospholipases C on the bacterial chromosome. The deduced amino acid sequence of the B. cereus PI-PLC contains a stretch of significant similarity to the glycosylphosphatidylinositol-specific PLC of Trypanosoma brucei. The conserved peptide is proposed to play a role in the function of these enzymes. Images PMID:2509427

  4. Stalling autophagy: a new function for Listeria phospholipases

    PubMed Central

    Tattoli, Ivan; Sorbara, Matthew T.; Philpott, Dana J.; Girardin, Stephen E.

    2014-01-01

    Listeria monocytogenes is a Gram-positive bacterial pathogen that induces its own uptake in non-phagocytic cells. Following invasion, Listeria escapes from the entry vacuole through the secretion of a pore-forming toxin, listeriolysin O (LLO) that acts to damage and disrupt the vacuole membrane. Listeria then replicates in the cytosol and is able to spread from cell-to-cell using actin-based motility. In addition to LLO, Listeria produces two phospholipase toxins, a phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB) and a broad-range phospholipase C (PC-PLC, encoded by plcA), which contribute to bacterial virulence. It has long been recognized that secretion of PI- and PC-PLC enables the disruption of the double membrane vacuole during cell-to-cell spread, and those phospholipases have also been shown to augment LLO-dependent escape from the entry endosome. However, a specific role for Listeria phospholipases during the cytosolic stage of infection has not been previously reported. In a recent study, we demonstrated that Listeria PI-PLC and PC-PLC contribute to the bacterial escape from autophagy through a mechanism that involves direct inhibition of the autophagic flux in the infected cells [Tattoli et al. EMBO J (2013), 32, 3066-3078].

  5. How does fluoroaluminate activate human platelets?

    PubMed

    Rendu, F; Lebret, M; Tenza, D; Levy-Toledano, S

    1990-01-15

    Platelet activation induced by NaF or fluoroaluminate (AlF4-) was studied. The latter has been described to substitute for the gamma-phosphate group of the GTP molecule. With 10 mM-NaF, a concentration unable to induce any measurable Ca2+ mobilization (as measured with Indo 1), addition of AlCl3 potentiated platelet aggregation, thromboxane synthesis, diacylglycerol formation and p43 phosphorylation, without any increase in intracellular Ca2+. Neither phosphoinositide hydrolysis nor phosphatidic acid formation could be detected. AlF4- induced the release through a granule centralization within a microtubule bundle, although no myosin light-chain phosphorylation could be detected. Addition of flurbiprofen (10 microM) resulted in only partial inhibition of diacylglycerol formation, with no effect on the release reaction or on p43 phosphorylation. The present results suggest that AlF4- does not stimulate a G-protein governing the phosphoinositide-specific phospholipase C. The AlF4(-)-induced diacylglycerol formation is discussed. Moreover, these results bring evidence that there is no correlation between granule centralization and myosin light-chain phosphorylation.

  6. How does fluoroaluminate activate human platelets?

    PubMed Central

    Rendu, F; Lebret, M; Tenza, D; Levy-Toledano, S

    1990-01-01

    Platelet activation induced by NaF or fluoroaluminate (AlF4-) was studied. The latter has been described to substitute for the gamma-phosphate group of the GTP molecule. With 10 mM-NaF, a concentration unable to induce any measurable Ca2+ mobilization (as measured with Indo 1), addition of AlCl3 potentiated platelet aggregation, thromboxane synthesis, diacylglycerol formation and p43 phosphorylation, without any increase in intracellular Ca2+. Neither phosphoinositide hydrolysis nor phosphatidic acid formation could be detected. AlF4- induced the release through a granule centralization within a microtubule bundle, although no myosin light-chain phosphorylation could be detected. Addition of flurbiprofen (10 microM) resulted in only partial inhibition of diacylglycerol formation, with no effect on the release reaction or on p43 phosphorylation. The present results suggest that AlF4- does not stimulate a G-protein governing the phosphoinositide-specific phospholipase C. The AlF4(-)-induced diacylglycerol formation is discussed. Moreover, these results bring evidence that there is no correlation between granule centralization and myosin light-chain phosphorylation. Images Fig. 1. Fig. 4. Fig. 5. PMID:2302176

  7. Enzymatic action of phospholipase A₂ on liposomal drug delivery systems.

    PubMed

    Hansen, Anders H; Mouritsen, Ole G; Arouri, Ahmad

    2015-08-01

    The overexpression of secretory phospholipase A2 (sPLA2) in tumors has opened new avenues for enzyme-triggered active unloading of liposomal antitumor drug carriers selectively at the target tumor. However, the effects of the liposome composition, drug encapsulation, and tumor microenvironment on the activity of sPLA2 are still not well understood. We carried out a physico-chemical study to characterize the sPLA2-assisted breakdown of liposomes using dye-release assays in the context of drug delivery and under physiologically relevant conditions. The influence of temperature, lipid concentration, enzyme concentration, and drug loading on the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm=42°C) liposomes with snake venom sPLA2 was investigated. The sensitivity of human sPLA2 to the liposome composition was checked using binary lipid mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) phospholipids with C14 and C16 acyl chains. Increasing temperature (36-41°C) was found to mainly shorten the enzyme lag-time, whereas the effect on lipid hydrolysis rate was modest. The enzyme lag-time was also found to be inversely dependent on the lipid-to-enzyme ratio. Drug encapsulation can alter the hydrolysis profile of the carrier liposomes. The activity of human sPLA2 was highly sensitive to the phospholipid acyl-chain length and negative surface charge density of the liposomes. We believe our work will prove useful for the optimization of sPLA2-susceptible liposomal formulations as well as will provide a solid ground for predicting the hydrolysis profile of the liposomes in vivo at the target site.

  8. Rapid activation of specific phospholipase(s) D by cytokinin in Amaranthus assay system.

    PubMed

    Kravets, Volodymir S; Kolesnikov, Yaroslav S; Kretynin, Sergey V; Getman, Irina A; Romanov, Georgy A

    2010-03-01

    The suggested link between intracellular cytokinin signaling and phospholipase D (PLD, EC 3.1.4.4.) activity (Romanov et al. 2000, 2002) was investigated. The activity of PLD in the early period of cytokinin action was studied in vivo in derooted Amaranthus caudatus seedlings, using the level of phosphatidylbutanol production as a measure of PLD activity. Rapid activation of phosphatidylbutanol synthesis was demonstrated as early as within 5 min of cytokinin administration. Neomycin, a known phosphatidylinositol-4,5-bisphosphate (PIP(2)) antagonist, strongly repressed both physiological cytokinin effect and cytokinin-dependent PLD activation. N-acylethanolamine (NAE 12), an inhibitor of alpha-class PLD, did not influence significantly cytokinin effect on Amaranthus seedlings. Together, results suggest the involvement of PIP(2)-dependent non-class alpha-PLD in the molecular mechanism of cytokinin action.

  9. Variable substrate preference among phospholipase D toxins from sicariid spiders

    SciTech Connect

    Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; Delahaye, Jared L.; Bandarian, Vahe; Binford, Greta J.; Cordes, Matthew H. J.

    2015-03-09

    Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.

  10. Generation of choline for acetylcholine synthesis by phospholipase D isoforms

    PubMed Central

    Zhao, Di; Frohman, Michael A; Blusztajn, Jan Krzysztof

    2001-01-01

    Dedication This article is dedicated to the memory of Sue Kim Hanson, a graduate student in the department of Pathology and Laboratory Medicine at Boston University School of Medicine, who perished in the terrorist attacks of September 11, 2001. Abstract Background In cholinergic neurons, the hydrolysis of phosphatidylcholine (PC) by a phospholipase D (PLD)-type enzyme generates some of the precursor choline used for the synthesis of the neurotransmitter acetylcholine (ACh). We sought to determine the molecular identity of the relevant PLD using murine basal forebrain cholinergic SN56 cells in which the expression and activity of the two PLD isoforms, PLD1 and PLD2, were experimentally modified. ACh levels were examined in cells incubated in a choline-free medium, to ensure that their ACh was synthesized entirely from intracellular choline. Results PLD2, but not PLD1, mRNA and protein were detected in these cells and endogenous PLD activity and ACh synthesis were stimulated by phorbol 12-myristate 13-acetate (PMA). Introduction of a PLD2 antisense oligonucleotide into the cells reduced PLD2 mRNA and protein expression by approximately 30%. The PLD2 antisense oligomer similarly reduced basal- and PMA-stimulated PLD activity and ACh levels. Overexpression of mouse PLD2 by transient transfection increased basal- (by 74%) and PMA-stimulated (by 3.2-fold) PLD activity. Moreover, PLD2 transfection increased ACh levels by 26% in the absence of PMA and by 2.1-fold in the presence of PMA. Overexpression of human PLD1 by transient transfection increased PLD activity by 4.6-fold and ACh synthesis by 2.3-fold in the presence of PMA as compared to controls. Conclusions These data identify PLD2 as the endogenous enzyme that hydrolyzes PC to generate choline for ACh synthesis in cholinergic cells, and indicate that in a model system choline generated by PLD1 may also be used for this purpose. PMID:11734063

  11. The first report on coagulation and phospholipase A2 activities of Persian Gulf lionfish, Pterois russelli, an Iranian venomous fish.

    PubMed

    Memar, Bahareh; Jamili, Shahla; Shahbazzadeh, Delavar; Bagheri, Kamran Pooshang

    2016-04-01

    Pterois russelli is a venomous fish belonging to scorpionidae family. Regarding to high significance value for tracing potential therapeutic molecules and special agents from venomous marine creatures, the present study was aimed to characterization of the Persian Gulf lionfish venom. Proteolytic, phospholipase, hemolytic, coagulation, edematogenic and dermonecrotic activities were determined for extracted venom. The LD50 of P. russelli venom was determined by intravenous injection in white Balb/c mice. Phospholipase A2 activity was recorded at 20 μg of total venom. Coagulation activity on human plasma was shown by Prothrombin Time (PT) and activated Partial Thromboplastin Time (APTT) assays and coagulation visualized after 7 and 14 s respectively for 60 μg of crude venom. LD50 was calculated as 10.5 mg/kg. SDS-PAGE revealed the presence of major and minor protein bands between 6 and 205 kDa. Different amounts of crude venom ranged from 1.87 to 30 μg showed proteolytic activity on casein. The highest edematic activity was detected at 20 μg. Our findings showed that the edematic activity was dose dependent and persisted for 48 h after injection. The crude venom did not induce dermonecrotic activity on rabbit skin and showed no hemolytic activity on human, mouse and rabbit erythrocytes. This is the first report for phospholipase A2 and coagulation activity in venomous fish and venomous marine animals respectively. Proteolytic activity of P. russelli venom is in accordance with the other genara of scorpionidae family. According to venom activity on intrinsic and extrinsic coagulation pathways, lionfish venom would be contained an interesting pharmaceutical agent. This study is pending to further characterization of phospholipase A2, coagulation, and protease activities and also in vivo activity on animal model of surface and internal bleeding.

  12. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase

    NASA Astrophysics Data System (ADS)

    Glukhova, Alisa; Hinkovska-Galcheva, Vania; Kelly, Robert; Abe, Akira; Shayman, James A.; Tesmer, John J. G.

    2015-03-01

    Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

  13. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase

    PubMed Central

    Glukhova, Alisa; Hinkovska-Galcheva, Vania; Kelly, Robert; Abe, Akira; Shayman, James A; Tesmer, John JG

    2015-01-01

    Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high resolution crystal structures of human LPLA2 and a low resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome. PMID:25727495

  14. Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope.

    PubMed

    Muller, Vanessa Danielle; Soares, Ricardo Oliveira; dos Santos, Nilton Nascimento; Trabuco, Amanda Cristina; Cintra, Adelia Cristina; Figueiredo, Luiz Tadeu; Caliri, Antonio; Sampaio, Suely Vilela; Aquino, Victor Hugo

    2014-01-01

    The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.

  15. Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    PubMed Central

    Muller, Vanessa Danielle; Soares, Ricardo Oliveira; dos Santos-Junior, Nilton Nascimento; Trabuco, Amanda Cristina; Cintra, Adelia Cristina; Figueiredo, Luiz Tadeu; Caliri, Antonio; Sampaio, Suely Vilela; Aquino, Victor Hugo

    2014-01-01

    The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs. PMID:25383618

  16. Effects of Estradiol and Progesterone on Rat Intestinal and Hepatic Phospholipase A Activity

    DTIC Science & Technology

    1988-12-01

    intestine and liver play Major roles in lipid and lipoprotein metabolism. Both rgans contain high activities of phospholipase A, but little is known... Phospholipase A is involved in the initial step in prostaglandin synthesis (1) and may also play a role in the metabolism of plasma lipoproteins (2...and subcellular fraction. Phospholipase A (EC 3.1.1.32 and 3.1.1.4, A, - A2 ) activity was determined in these acetone powders by the method of

  17. Signalling through phospholipase C interferes with clathrin-mediated endocytosis.

    PubMed

    Carvou, Nicolas; Norden, Anthony G W; Unwin, Robert J; Cockcroft, Shamshad

    2007-01-01

    We investigated if phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) hydrolysis by phospholipase C activation through cell surface receptors would interfere with clathrin-mediated endocytosis as recruitment of clathrin assembly proteins is PtdIns(4,5)P2-dependent. In the WKPT renal epithelial cell line, endocytosed insulin and beta2-glycoprotein I (beta2gpI) were observed in separate compartments, although endocytosis of both ligands was clathrin-dependent as demonstrated by expression of the clathrin-binding C-terminal domain of AP180 (AP180-C). The two uptake mechanisms were different as only insulin uptake was reduced when the mu2-subunit of the adaptor complex AP-2 was silenced by RNA interference. ATP receptors are expressed at the apical surface of renal cells and, thus, we examined the effect of extracellular ATP on insulin and beta2gpI uptake. ATP stimulated phospholipase C activity, and also suppressed uptake of insulin, but not beta2gpI. This effect was reversed by the PLC inhibitor U-73122. In polarized cell cultures, insulin uptake was apical, whereas beta2gpI uptake was through the basolateral membrane, thus providing an explanation for selective inhibition of insulin endocytosis by ATP. Taken together, these results demonstrate that stimulation of apical G-protein-coupled P2Y receptors, which are coupled to phospholipase C activation diminishes clathrin-mediated endocytosis without interfering with basolateral endocytic mechanisms.

  18. 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

  19. Endogenous phospholipase A2 inhibitors in snakes: a brief overview.

    PubMed

    Campos, Patrícia Cota; de Melo, Lutiana Amaral; Dias, Gabriel Latorre Fortes; Fortes-Dias, Consuelo Latorre

    2016-01-01

    The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes - namely sbαPLI, sbβPLI or sbγPLI - depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbαPLIs and sbγPLIs, whereas sbβPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbαPLIs and sbγPLIs from two Old World - Gloydius brevicaudus and Malayopython reticulatus - and two New World - Bothrops alternatus and Crotalus durissus terrificus - snake species will be emphasized.

  20. Phospholipase D toxins of brown spider venom convert lysophosphatidylcholine and sphingomyelin to cyclic phosphates.

    PubMed

    Lajoie, Daniel M; Zobel-Thropp, Pamela A; Kumirov, Vlad K; Bandarian, Vahe; Binford, Greta J; Cordes, Matthew H J

    2013-01-01

    Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group. The other products of substrate cleavage have previously been reported to be monoester phospholipids, which would result from substrate hydrolysis. Using (31)P NMR and mass spectrometry we demonstrate that recombinant toxins, as well as whole venoms from diverse Loxosceles species, exclusively catalyze transphosphatidylation rather than hydrolysis, forming cyclic phosphate products from both major substrates. Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.

  1. Kinetic disruption of lipid rafts is a mechanosensor for phospholipase D

    PubMed Central

    Petersen, E. Nicholas; Chung, Hae-Won; Nayebosadri, Arman; Hansen, Scott B.

    2016-01-01

    The sensing of physical force, mechanosensation, underlies two of five human senses—touch and hearing. How transduction of force in a membrane occurs remains unclear. We asked if a biological membrane could employ kinetic energy to transduce a signal absent tension. Here we show that lipid rafts are dynamic compartments that inactivate the signalling enzyme phospholipase D2 (PLD2) by sequestering the enzyme from its substrate. Mechanical disruption of the lipid rafts activates PLD2 by mixing the enzyme with its substrate to produce the signalling lipid phosphatidic acid (PA). We calculate a latency time of <650 μs for PLD activation by mixing. Our results establish a fast, non-tension mechanism for mechanotransduction where disruption of ordered lipids initiates a mechanosensitive signal for cell growth through mechanical mixing. PMID:27976674

  2. The direct interaction of phospholipase C-gamma 1 with phospholipase D2 is important for epidermal growth factor signaling.

    PubMed

    Jang, Il Ho; Lee, Sukmook; Park, Jong Bae; Kim, Jong Hyun; Lee, Chang Sup; Hur, Eun-Mi; Kim, Il Shin; Kim, Kyong-Tai; Yagisawa, Hitoshi; Suh, Pann-Ghill; Ryu, Sung Ho

    2003-05-16

    The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-gamma1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2). PLC-gamma1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-gamma1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-gamma1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-gamma1 and PLD2 and fails to potentiate EGF-induced IP(3) formation and Ca(2+) increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-gamma1. These findings suggest that, upon EGF stimulation, PLC-gamma1 directly interacts with PLD2 and this interaction is important for PLC-gamma1 activity.

  3. An Autoinhibitory Helix in the C-Terminal Region of Phospholipase C-β Mediates Gαq Activation

    PubMed Central

    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.

    2011-01-01

    Phospholipase C-β (PLCβ) is a key regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to Gq. We have determined atomic structures of two invertebrate homologs of PLCβ (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 PLCβ3 dramatically increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ. PMID:21822282

  4. Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

    PubMed Central

    Burgess, W H; Dionne, C A; Kaplow, J; Mudd, R; Friesel, R; Zilberstein, A; Schlessinger, J; Jaye, M

    1990-01-01

    Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma. Images PMID:2167438

  5. Inhibition of purified lysosomal phospholipase A1 by beta-adrenoceptor blockers.

    PubMed

    Pappu, A S; Yazaki, P J; Hostetler, K Y

    1985-02-15

    Inhibition of rat liver lysosomal phospholipases is one of the main events that leads to accumulation of tissue phospholipids during drug-induced phospholipidosis. Drug inhibition of lysosomal phospholipase A may occur by direct effects of drugs on the enzyme (or substrate) or by drug-induced increases in intralysosomal pH. Although beta-adrenoceptor blockers have not been reported to cause lipid storage, they do inhibit lysosomal phospholipase A. To investigate the structural requirements for drug inhibition, we studied the effects of six beta-adrenoceptor blockers on purified rat liver lysosomal phospholipase A1. The agents studied include: propranolol, timolol, metoprolol, practolol, atenolol and the combined alpha and beta adrenoceptor blocking agent, labetalol. The drugs varied by two logs in their abilities to inhibit phospholipase A1 activity. The relative inhibitory potencies were propranolol greater than labetalol much greater than timolol greater than metoprolol much greater than practolol greater than atenolol. Our studies identify drug hydrophobicity as a key determinant for phospholipase A1 inhibition. A strong negative correlation was noted between the octanol/water partition coefficients and IC50 for phospholipase inhibition (r = -0.91). The ability of propranolol to inhibit phospholipase A1 was identical for the d, l and the d and l stereoisomers.

  6. Therapeutic application of natural inhibitors against snake venom phospholipase A2

    PubMed Central

    Perumal Samy, Ramar; Gopalakrishnakone, Ponnampalam; Chow, Vincent TK

    2012-01-01

    Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A2 (svPLA2). The implication of this review to update the latest research work which has been accomplished with svPLA2 inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones ­2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A2 (PLA2) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA2. Besides marine natural products that inhibit PLA2 are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA2 in inflammation that provides a rationale for seeking inhibitors of PLA2 as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA2 toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed. PMID:22359435

  7. Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite derived phospholipase

    PubMed Central

    Jarrett, Rachael; Salio, Mariolina; Lloyd-Lavery, Antonia; Subramaniam, Sumithra; Bourgeois, Elvire; Archer, Charles; Cheung, Ka Lun; Hardman, Clare; Chandler, David; Salimi, Maryam; Gutowska-Owsiak, Danuta; de la Serna, Jorge Bernardino; Fallon, Padraic G.; Jolin, Helen; Mckenzie, Andrew; Dziembowski, Andrzej; Podobas, Ewa Izabela; Bal, Wojciech; Johnson, David; Moody, D Branch

    2016-01-01

    Atopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation play a role in pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and while CD1a-expressing Langerhans cells are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. Here we observed that house dust mite (HDM) generates neolipid antigens for presentation by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth and showed rapid effector function, consistent with antigen-driven maturation. To define the underlying mechanisms, we analyzed HDM-challenged human skin and observed allergen-derived phospholipase (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2 and such cells infiltrated the skin after allergen challenge. Filaggrin insufficiency is associated with atopic dermatitis, and we observed that filaggrin inhibits PLA2 activity and inhibits CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity, such as Gell and Coombs are predicated on the idea that non-peptide stimulants of T cells act as haptens that modify peptides or proteins. However our results point to a broader model that does not posit haptenation, but instead shows that HDM proteins generate neolipid antigens which directly activate T cells. Specifically, the data identify a pathway of atopic skin inflammation, in which house dust mite-derived phospholipase A2 generates antigenic neolipids for presentation to CD1a-reactive T cells, and define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach. PMID:26865566

  8. Iron-Regulated Phospholipase C Activity Contributes to the Cytolytic Activity and Virulence of Acinetobacter baumannii

    PubMed Central

    Fiester, Steven E.; Schmidt, Robert E.; Beckett, Amber C.; Ticak, Tomislav; Carrier, Mary V.; Ghosh, Rajarshi; Ohneck, Emily J.; Metz, Maeva L.; Sellin Jeffries, Marlo K.; Actis, Luis A.

    2016-01-01

    Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during

  9. Histochemical demonstration of phospholipase B (lysolecithinase) activity in rat tissues.

    PubMed

    Ottolenghi, A; Pickett, J P; Greene, W B

    1966-12-01

    A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 micro frozen sections, fixed in cold calcium-formol and incubated at 37 degrees C in Tris buffered medium at pH 6.6 containing 2.2 X 10(-3) M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a black-brown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60 degrees C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation, corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical

  10. Messenger molecules of the phospholipase signaling system have dual effects on vascular smooth muscle contraction.

    PubMed

    Vidulescu, Cristina; Mironneau, J.; Mironneau, Chantal; Popescu, L. M.

    2000-01-01

    Background and methods. In order to investigate the role of phospholipases and their immediately derived messengers in agonist-induced contraction of portal vein smooth muscle, we used the addition in the organ bath of exogenous molecules such as: phospholipases C, A(2), and D, diacylglycerol, arachidonic acid, phosphatidic acid, choline. We also used substances modulating activity of downstream molecules like protein kinase C, phosphatidic acid phosphohydrolase, or cyclooxygenase. Results. a) Exogenous phospholipases C or A(2), respectively, induced small agonist-like contractions, while exogenous phospholipase D did not. Moreover, phospholipase D inhibited spontaneous contractions. However, when added during noradrenaline-induced plateau, phospholipase D shortly potentiated it. b) The protein kinase C activator, phorbol dibutyrate potentiated both the exogenous phospholipase C-induced contraction and the noradrenaline-induced plateau, while the protein kinase C inhibitor 1-(-5-isoquinolinesulfonyl)-2-methyl-piperazine relaxed the plateau. c) When added before noradrenaline, indomethacin inhibited both phasic and tonic contractions, but when added during the tonic contraction shortly potentiated it. Arachidonic acid strongly potentiated both spontaneous and noradrenaline-induced contractions, irrespective of the moment of its addition. d) In contrast, phosphatidic acid inhibited spontaneous contractile activity, nevertheless it was occasionally capable of inducing small contractions, and when repetitively added during the agonist-induced tonic contraction, produced short potentiations of the plateau. Pretreatment with propranolol inhibited noradrenaline-induced contractions and further addition of phosphatidic acid augmented this inhibition. Choline augmented the duration and amplitude of noradrenaline-induced tonic contraction and final contractile oscillations. Conclusions. These data suggest that messengers produced by phospholipase C and phospholipase A(2

  11. Purification and characterization of a membrane-associated phospholipase A2 from rat spleen. Its comparison with a cytosolic phospholipase A2 S-1.

    PubMed

    Ono, T; Tojo, H; Kuramitsu, S; Kagamiyama, H; Okamoto, M

    1988-04-25

    A membrane-associated phospholipase A2 was purified from rat spleen. The phospholipase A2 was solubilized from the 108,000 x g pellet fraction with 0.3% lithium dodecyl sulfate and then purified to homogeneity by successive DEAE-Cellulofine AM, octyl-Sepharose, Cellulofine GCL 300-m, S-Sepharose, and Bio-Gel P-30 chromatographies in the presence of 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate. The apparent Mr of the enzyme, estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, was about 13,600. The purified enzyme had a pH optimum in the range of pH 8.0-9.5 and required the presence of Ca2+ (4 mM) for its maximal activity. The enzyme preferentially hydrolyzed the 2-acyl ester bonds of phosphatidylglycerol in the presence and absence of sodium cholate or sodium deoxycholate. Unlike the phospholipase A2 of rat spleen supernatant, no immunocross-reactivity was observed between the purified enzyme and anti-rat pancreatic phospholipase A2 antibody. The N-terminal amino acid sequence of the enzyme was determined and found to be homologous to that of viperid and crotalid venom phospholipases A2. The results in this and the preceding report (Tojo, H., Ono, T., Kuramitsu, S., Kagamiyama, H., and Okamoto, M. (1988) J. Biol. Chem. 263, 5724-5731) demonstrate that rat spleen contains two genetically distinct phospholipase A2 isoenzymes.

  12. Ostrich pancreatic phospholipase A(2): purification and biochemical characterization.

    PubMed

    Ben Bacha, Abir; Gargouri, Youssef; Bezzine, Sofiane; Mosbah, Habib; Mejdoub, Hafedh

    2007-09-15

    Ostrich pancreatic phospholipase A(2) (OPLA(2)) was purified from delipidated pancreases. Pure protein was obtained after heat treatment (70 degrees C), precipitation by ammonium sulphate and ethanol, respectively followed by sequential column chromatography on MonoQ Sepharose and size exclusion HPLC column. Purified OPLA(2), which is not a glycosylated protein, was found to be monomeric protein with a molecular mass of 13773.93 Da. A specific activity of 840U/mg for purified OPLA(2) was measured at optimal conditions (pH 8.2 and 37 degrees C) in the presence of 4 mM NaTDC and 10 mM CaCl(2) using PC as substrate. This enzyme was also found to be able to hydrolyze, at low surface pressure, 1,2-dilauroyl-sn-glycero-3 phosphocholine (di C(12)-PC) monolayers. Maximal activity was measured at 5-8 mNm(-1). The sequence of the first 22 amino-acid residues at the N-terminal extremity of purified bird PLA(2) was determined by automatic Edman degradation and showed a high sequence homology with known mammal pancreatic secreted phospholipases A(2).

  13. Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

    PubMed Central

    Yui, Daishi; Nishida, Yoichiro; Nishina, Tomoko; Mogushi, Kaoru; Tajiri, Mio; Ishibashi, Satoru; Ajioka, Itsuki; Ishikawa, Kinya; Mizusawa, Hidehiro; Murayama, Shigeo; Yokota, Takanori

    2015-01-01

    Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD. PMID:26637123

  14. Phospholipase C beta 4 in the medial septum controls cholinergic theta oscillations and anxiety behaviors.

    PubMed

    Shin, Jonghan; Gireesh, Gangadharan; Kim, Seong-Wook; Kim, Duk-Soo; Lee, Sukyung; Kim, Yeon-Soo; Watanabe, Masahiko; Shin, Hee-Sup

    2009-12-09

    Anxiety is among the most prevalent and costly diseases of the CNS, but its underlying mechanisms are not fully understood. Although attenuated theta rhythms have been observed in human subjects with increased anxiety, no study has been done on the possible physiological link between these two manifestations. We found that the mutant mouse for phospholipase C beta 4 (PLC-beta 4(-/-)) showed attenuated theta rhythm and increased anxiety, presenting the first animal model for the human condition. PLC-beta 4 is abundantly expressed in the medial septum, a region implicated in anxiety behavior. RNA interference-mediated PLC-beta 4 knockdown in the medial septum produced a phenotype similar to that of PLC-beta 4(-/-) mice. Furthermore, increasing cholinergic signaling by administering an acetylcholinesterase inhibitor cured the anomalies in both cholinergic theta rhythm and anxiety behavior observed in PLC-beta 4(-/-) mice. These findings suggest that (1) PLC-beta 4 in the medial septum is involved in controlling cholinergic theta oscillation and (2) cholinergic theta rhythm plays a critical role in suppressing anxiety. We propose that defining the cholinergic theta rhythm profile may provide guidance in subtyping anxiety disorders in humans for more effective diagnosis and treatments.

  15. Disintegration of lysosomes mediated by GTPgammaS-treated cytosol: possible involvement of phospholipases.

    PubMed

    Sai, Y; Matsuda, T; Arai, K; Ohkuma, S

    1998-04-01

    We showed previously that cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS) disintegrated lysosomes in vitro [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877] in time-, temperature-, and dose-dependent manners. This also requires ATP, however, the latter can be substituted with deoxy-ATP, ADP, or ATPgammaS, suggesting no requirement of ATP hydrolysis. The lysis was inhibited by several chemical modifiers, including N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and by various phospholipase inhibitors (trifluoperazine, p-bromophenacyl bromide, nordihydroguaiaretic acid, W-7, primaquine, compound 48/80, neomycin, and gentamicin), but not by ONO-RS-082, an inhibitor of phospholipase A2. The reaction was also inhibited by phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidic acid, and phosphatidylcholine) and diacylglycerol. Among the phospholipase A2 hydrolysis products of phospholipids, unsaturated fatty acids (oleate, linoleate, and arachidonate) and lysophospholipid (lysophosphatidylcholine) by themselves broke lysosomes down directly, whereas saturated fatty acids (palmitate and stearate) had little effect. We found that GTPgammaS-stimulated cytosolic phospholipase A2 activity was highly sensitive to ONO-RS-082. These results suggest the participation of phospholipase(s), though not cytosolic phospholipase A2, in the GTPgammaS-dependent lysis of lysosomes.

  16. Histamine H1 and endothelin ETB receptors mediate phospholipase D stimulation in rat brain hippocampal slices.

    PubMed

    Sarri, E; Picatoste, F; Claro, E

    1995-08-01

    Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans-(1S,3R)-aminocyclopentyl-1,3-dicarboxylic acid (trans-ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [32P]Pi-prelabeled rat brain cortical and hippocampal slices. The accumulation of [32P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans-ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC50 18 microM) was inhibited by the H1 receptor antagonist mepyramine with a Ki constant of 0.7 nM and was resistant to H2 and H3 receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC50 44 nM) was not blocked by BQ-123, a specific antagonist of the ETA receptor. Endothelin-3 and the specific ETB receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC50 values of 16 and 3 nM, respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H1 and ETB receptors, respectively.

  17. Modulation of radiation induced lipid peroxidation by phospholipase A 2 and calmodulin antagonists: Relevance to detoxification

    NASA Astrophysics Data System (ADS)

    Varshney, Rajeev; Kale, R. K.

    1995-04-01

    Ghost membranes prepared from erythrocytes of Swiss albino mice were irradiated with 0.9 Gy s -1. Lipid peroxidation initiated by ionizing radiation was enhanced by phospholipase A 2, and required both phospholipase A 2 and GSH-peroxidase for consecutive action to convert fatty acid peroxides into corresponding alcohols. The ability of phospholipase A 2 to enhance lipid peroxidation was increased in presence of Ca 2+. However, in combination, phospholipase A 2 and GSH-peroxidase were effective in inhibiting lipid peroxidation. These findings show that free fatty acid peroxides considerably increase the peroxidation. Calmodulin antagonists inhibit lipid peroxidation and decrease the radiation induced release of Ca 2+ from the membranes. Our results suggest the importance of Ca 2+ dependent phospholipase A 2 in detoxification of fatty acid peroxides in the membranes. It is quite possible that scavenging of free radicals by calmodulin antagonists lower the formation of hydroperoxides, resulting in the decrease in activity of phospholipase A 2. Alternatively, decrease in Ca 2+ release due to the calmodulin antagonists might have affected the activity of phospholipase A 2. Our observations might be of considerable significance in the understanding of post irradiation effect on biological membranes.

  18. Hydrolysis of erythrocyte membrane phospholipids by a preparation of phospholipase C from Clostridium Welchii. Deactivation of (Ca-2+, Mg-2+)-ATPase and its reactivation by added lipids.

    PubMed

    Coleman, R; Bramley, T A

    1975-04-08

    1. Haemoglobin-free erythrocyte ghosts were prepared in 40 imosM bicarbonate buffer, pH 7.4, containing 1 mM EDTA (40 imosM/l mM EDTA). The ghost preparation was highly permeable on preparation but partially resealed on incubation in media containing Ca-2+. 2. A partially purified preparation of phospholipase C from Clostridum welchii caused an increase in observed Mg-2+-ATPase activity, reflecting a change in the permeability of the ghost to substrate. The phospholipase did not decrease Mg-2+-ATPase even at the highest levels tested. Mg-2+-ATPase activity could therefore be used as a permeability indicatior in these experiments. 3. Both (Ca-2+, Mg-2+)-ATPase activities of the ghosts were progressively lost as a result of the phospholipid hydrolysis induced by phospholipase C. 4. When a haemolysin in the commercial preparation was destroyed by heat-treatment, deactivation of the (Ca-2+, Mg-2+)-ATPase and (Na+, K+, Mg-2+)-ATPases were still observed but permeability changes were greatly reduced. 5. The products of phospholipase action were not inhibitory to the Ca-2+, Mg-2+)-ATPase. 6. Lysolecithin brought about a reactivation of the (Ca-2+, Mg-2+)-ATPase which was superimposed upon permeability changes in the preparation. 7. Reactivation of the (Ca-2+, Mg-2+)-ATPase was brought about by a nonlytic, mixed lipid preparation without significant effect upon permeability. 8. Human erythrocyte (Ca-2+, Mg-2+)-ATPase therefore appears to be an enzyme which responds to perturbation of the lipid environment in the membrane and is a "lipid-dependant" enzyme.

  19. Phospholipase C-β1 and schizophrenia-related behaviors.

    PubMed

    Koh, Hae-Young

    2013-09-01

    Abnormal expression patterns of phospholipase C-β1(PLC-β1) in specific brain areas of patients with schizophrenia, and its high genetic linkage to the disorder implicated a pathogenetical involvement of PLC-β1 signaling system. The schizophrenia-related behavioral phenotypes displayed in the mutant mice lacking PLC-β1 (PLC-β1 KO) suggested that PLCβ1-linked signaling pathways may be involved in the neural system whose function is disrupted in the pathogenesis of schizophrenia. In the brain, PLC-β1 is known to be linked to muscarinic acetylcholine receptors, metabotropic glutamatergic, serotonergic, and oxytocinergic systems. The objective of this review is to provide an overview of the current knowledge regarding these schizophrenia-related behaviors and discuss the probable ways in which PLC-β1signalling can be involved in the neural mechanisms for each behavior, which may help suggest future directions for research in this area.

  20. 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.

  1. 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.

  2. Molecular cloning and characterization of a novel phospholipase C, PLC-eta.

    PubMed

    Hwang, Jong-Ik; Oh, Yong-Seok; Shin, Kum-Joo; Kim, Hyun; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-07-01

    PLC (phospholipase C) plays an important role in intracellular signal transduction by hydrolysing phosphatidylinositol 4,5-bisphosphate, a membrane phospholipid. To date, 12 members of the mammalian PLC isoforms have been identified and classified into five isotypes beta, gamma, delta, epsilon and zeta, which are regulated by distinct mechanisms. In the present study, we describe the identification of a novel PLC isoform in the brains of human and mouse, named PLC-eta, which contains the conserved pleckstrin homology domain, X and Y domains for catalytic activity and the C2 domain. The first identified gene encoded 1002 (human) or 1003 (mouse) amino acids with an estimated molecular mass of 115 kDa. The purified recombinant PLC-eta exhibited Ca2+-dependent catalytic activity on phosphatidylinositol 4,5-bisphosphate. Furthermore, molecular biological analysis revealed that the PLC-eta gene was transcribed to several splicing variants. Although some transcripts were detected in most of the tissues we examined, the transcript encoding 115 kDa was restricted to the brain and lung. In addition, the expression of the 115 kDa protein was defined in only nerve tissues such as the brain and spinal cord. In situ hybridization analysis with brain revealed that PLC-eta was abundantly expressed in various regions including cerebral cortex, hippocampus, zona incerta and cerebellar Purkinje cell layer, which are neuronal cell-enriched regions. These results suggest that PLC-eta may perform fundamental roles in the brain.

  3. Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A2

    PubMed Central

    Nagano, Joseph M. G.; Hsu, Ku-Lung; Whitby, Landon R.; Niphakis, Micah J.; Speers, Anna E.; Brown, Steven J.; Spicer, Timothy; Fernandez-Vega, Virneliz; Ferguson, Jill; Hodder, Peter; Srinivasan, Prabhavathi; Gonzalez, Tara D.; Rosen, Hugh; Bahnson, Brian J.

    2013-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2 or PLA2G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA2 has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA2 have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA2 inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA2 and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA2 in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA2 function in biological systems. PMID:23260346

  4. PX-52, A novel inhibitor of 14 kDa secretory and 85 kDa cytosolic phospholipases A2.

    PubMed

    Franson, R C; Rosenthal, M D

    1997-01-01

    Previously we reported that PGBx, a prostaglandin oligomer with anti-inflammatory activity, inhibited 14 kDa phospholipase A2 (PLA2) activity and blocked arachidonic acid mobilization in prelabeled human neutrophils (Biochim. Biophys. Acta 1006:272-277, 278-286, 1989) This study describes a new inhibitor of phospholipase A2, PX-52, that also blocks agonist induced arachidonic acid mobilization in prelabeled cells. PX-52, a fatty acid polymer, inhibited hydrolysis of 14C-oleate labeled E.coli by a variety of 14 kDa PLA2s including human PMN, sperm, synovial fluid and disc, as well as porcine pancreas, N. naja, and bee venom in a dose-dependent manner with IC50s ranging from 1.0-3.7 uM. Inhibition of activity was comparable at different Ca2+ concentrations, but was relieved by increasing substrate concentration or by methylation of PX-52. Hydrolysis of [14C]-arachidonyl phosphatidylcholine by 85 kDa, cytosolic PLA2 from U937 cells was similarly inhibited by PX-52, the IC50 = 5 uM. Arachidonic acid mobilization induced by A23187 in prelabeled human PMNs was blocked by PX-52; IC50 = 10-15 uM while concentrations of up to 80 uM oleate had no effect. These results demonstrate that PX-52 inhibits the in vitro activity of secretory and cytosolic PLA2s and agonist-induced arachidonic acid release from human cells. Given its ability to block the arachidonic acid cascade, PX-52 may be useful in the control of inflammation.

  5. Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2.

    PubMed

    Cavazzini, Davide; Meschi, Francesca; Corsini, Romina; Bolchi, Angelo; Rossi, Gian Luigi; Einsle, Oliver; Ottonello, Simone

    2013-01-18

    Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.

  6. Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2*

    PubMed Central

    Cavazzini, Davide; Meschi, Francesca; Corsini, Romina; Bolchi, Angelo; Rossi, Gian Luigi; Einsle, Oliver; Ottonello, Simone

    2013-01-01

    Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A2 (sPLA2s). TbSP1, the sPLA2 primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A2, whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA2 overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation. PMID:23192346

  7. Phospholipases of Mineralization Competent Cells and Matrix Vesicles: Roles in Physiological and Pathological Mineralizations

    PubMed Central

    Mebarek, Saida; Abousalham, Abdelkarim; Magne, David; Do, Le Duy; Bandorowicz-Pikula, Joanna; Pikula, Slawomir; Buchet, René

    2013-01-01

    The present review aims to systematically and critically analyze the current knowledge on phospholipases and their role in physiological and pathological mineralization undertaken by mineralization competent cells. Cellular lipid metabolism plays an important role in biological mineralization. The physiological mechanisms of mineralization are likely to take place in tissues other than in bones and teeth under specific pathological conditions. For instance, vascular calcification in arteries of patients with renal failure, diabetes mellitus or atherosclerosis recapitulates the mechanisms of bone formation. Osteoporosis—a bone resorbing disease—and rheumatoid arthritis originating from the inflammation in the synovium are also affected by cellular lipid metabolism. The focus is on the lipid metabolism due to the effects of dietary lipids on bone health. These and other phenomena indicate that phospholipases may participate in bone remodelling as evidenced by their expression in smooth muscle cells, in bone forming osteoblasts, chondrocytes and in bone resorbing osteoclasts. Among various enzymes involved, phospholipases A1 or A2, phospholipase C, phospholipase D, autotaxin and sphingomyelinase are engaged in membrane lipid remodelling during early stages of mineralization and cell maturation in mineralization-competent cells. Numerous experimental evidences suggested that phospholipases exert their action at various stages of mineralization by affecting intracellular signaling and cell differentiation. The lipid metabolites—such as arachidonic acid, lysophospholipids, and sphingosine-1-phosphate are involved in cell signaling and inflammation reactions. Phospholipases are also important members of the cellular machinery engaged in matrix vesicle (MV) biogenesis and exocytosis. They may favour mineral formation inside MVs, may catalyse MV membrane breakdown necessary for the release of mineral deposits into extracellular matrix (ECM), or participate in

  8. Inhibition of (/sup 3/H)nitrendipine binding by phospholipase A/sub 2/

    SciTech Connect

    Goldman, M.E.; Pisano, J.J.

    1985-10-07

    Phospholipase A/sub 2/ from several sources inhibited (/sup 3/H)nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC/sub 50/ values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A/sub 2/ was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A/sub 2/ enzymatic activity, shifted the bee venom phospholipase A/sub 2/ dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A/sub 2/ (10 ng/ml) for 15 min caused a 2-fold increase in the K/sub d/ without changing the B/sub max/ compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the K/sub d/ but significantly decreased the B/sub max/ to 71% the value for untreated membranes. (/sup 3/H)Nitrendipine, preincubated with bee venom phospholipase A/sub 2/, was recovered and found to be fully active, indicating that the phospholipase A/sub 2/ did not modify the ligand. It is concluded that phospholipase A/sub 2/ acts on the membrane at or near the (/sup 3/H)nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site. 33 references, 3 figures, 1 table.

  9. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications.

    PubMed

    Borrelli, Grazia M; Trono, Daniela

    2015-09-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes.

  10. Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

    PubMed Central

    Bruntz, Ronald C.; Lindsley, Craig W.

    2014-01-01

    Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein–coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. PMID:25244928

  11. Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer.

    PubMed

    Bruntz, Ronald C; Lindsley, Craig W; Brown, H Alex

    2014-10-01

    Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein-coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions.

  12. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

    PubMed Central

    Borrelli, Grazia M.; Trono, Daniela

    2015-01-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. PMID:26340621

  13. Effects of glycosaminoglycans and glycosphingolipids on cytosolic phospholipases A2 from bovine brain.

    PubMed Central

    Yang, H C; Farooqui, A A; Horrocks, L A

    1994-01-01

    Two forms of Ca(2+)-independent cytosolic phospholipase A2 activity (110 kDa and 39 kDa) were found in bovine brain. They were separated by Sephadex G-75 column chromatography. The 110 kDa phospholipase A2 was much more active with phosphatidylethanolamine and was not affected by glycosaminoglycans, whereas the 39 kDa phospholipase A2 was much more active with ethanolamine plasmalogen and was markedly inhibited by glycosaminoglycans. Heparan sulphate was the most potent inhibitor, followed by chondroitin sulphate, hyaluronic acid and heparin. Gangliosides, especially the GM3 ganglioside, but not other glycosphingolipids, inhibited the activity of the 39 kDa phospholipase A2 in a dose-dependent manner. The heat-inactivation profiles of the 110 kDa and 39 kDa phospholipases A2 provide further evidence for the differences between these cytosolic enzymes. Interactions between glycosaminoglycans, gangliosides and phospholipases A2 may be involved in the maintenance of membrane function. PMID:8166664

  14. Lung mast cells are a source of secreted phospholipases A2

    PubMed Central

    Triggiani, Massimo; Giannattasio, Giorgio; Calabrese, Cecilia; Loffredo, Stefania; Granata, Francescopaolo; Fiorello, Alfonso; Santini, Mario; Gelb, Michael H.; Marone, Gianni

    2009-01-01

    Background Secreted phospholipases A2 (sPLA2s) are released in plasma and other biologic fluids of patients with inflammatory, autoimmune, and allergic diseases. Objective We sought to evaluate sPLA2 activity in the bronchoalveolar lavage fluid (BALF) of asthmatic patients and to examine the expression and release of sPLA2s from primary human lung mast cells (HLMCs). Methods sPLA2 activity was measured in BALF and supernatants of either unstimulated or anti-IgE–activated HLMCs as hydrolysis of oleic acid from radiolabeled Escherichia coli membranes. Expression of sPLA2s was examined by using RT-PCR. The release of cysteinyl leukotriene (LT) C4 was measured by means of enzyme immunoassay. Results Phospholipase A2 (PLA2) activity was higher in the BALF of asthmatic patients than in the control group. BALF PLA2 activity was blocked by the sPLA2 inhibitors dithiothreitol and Me-Indoxam but not by the cytosolic PLA2 inhibitor AZ-1. HLMCs spontaneously released a PLA2 activity that was increased on stimulation with anti-IgE. This PLA2 activity was blocked by dithiothreitol and Me-Indoxam but not by AZ-1. HLMCs constitutively express mRNA for group IB, IIA, IID, IIE, IIF, III, V, X, XIIA, and XIIB sPLA2s. Anti-IgE did not modify the expression of sPLA2s. The cell-impermeable inhibitor Me-Indoxam significantly reduced (up to 40%) the production of LTC4 from anti-IgE–stimulated HLMCs. Conclusions sPLA2 activity is increased in the airways of asthmatic patients. HLMCs express multiple sPLA2s and release 1 or more of them when activated by anti-IgE. The sPLA2s released by mast cells contribute to LTC4 production by acting in an autocrine fashion. Mast cells can be a source of sPLA2s in the airways of asthmatic patients. PMID:19541351

  15. Stimulation of phosphatidylcholine breakdown by thrombin and carbachol but not by tyrosine kinase receptor ligands in cells transfected with M1 muscarinic receptors. Rapid desensitization of phosphocholine-specific (PC) phospholipase D but sustained activity of PC-phospholipase C.

    PubMed

    McKenzie, F R; Seuwen, K; Pouysségur, J

    1992-11-15

    In order to evaluate the possible contribution of phospholipase D (PLD) stimulation to the mitogenic response, a screening of a variety of different compounds, some of which are known to be potent mitogens, was performed using the well characterized Chinese hamster lung fibroblast (CCL39) cell line. In wild type CCL39 cells, or derivatives expressing high levels of either the human M1 muscarinic receptor (Hm1) or the human epidermal growth factor (EGF) receptor (39M1-81 and 39ER22 clones, respectively), thrombin, a potent mitogen for all three cell types, elicited the rapid activation of PLD (t1/2 activation, 30 s). Carbachol-mediated activation of the Hm1 receptor in the 39M1-81 clone, which is not a mitogenic signal, produced a similarly rapid although greater activation of PLD. Addition of EGF to the 39ER22 clone was able to provoke both a mitogenic response and stimulate PLD, albeit a comparatively small effect. In each case, the stimulation of PLD correlated closely with the ability to stimulate inositol phospholipid breakdown and was entirely dependent on the activation of protein kinase C. Moreover, the ability of both thrombin and carbachol to stimulate PLD was found to be rapidly desensitized, with a similar time course of desensitization (t1/2 desensitization, 90 s). It has recently been reported that an increase in phospholipase C (PLC)-mediated phosphocholine (PC) hydrolysis by either addition of agonist or by extracellular addition of PC-specific PLC enzyme constitutes a mitogenic signal. In this regard, in addition to stimulation of PLD, thrombin and carbachol were both able to stimulate the activity of a phosphocholine-specific phospholipase C (PC-PLC), which did not appear to desensitize within the time course employed. By contrast, EGF was unable to elicit the stimulation of PC-PLC. Ligands such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), which bind to and activate receptors with intrinsic tyrosine kinase activity

  16. Natural phospholipase A(2) myotoxin inhibitor proteins from snakes, mammals and plants.

    PubMed

    Lizano, Sergio; Domont, Gilberto; Perales, Jonas

    2003-12-15

    A renewed interest in the phenomenon of inter- and intra-species resistance towards the toxicity of snake venoms, coupled with the search for new strategies for treatment of snake envenomations, has prompted the discovery of proteins which neutralize the major toxic components of these venoms. Among these emerging groups of proteins are inhibitors of toxic phospholipases A2 (PLA2s), many of which exhibit a wide range of toxic effects including muscle-tissue damage, neurotoxicity, and inflammation. These proteins have been isolated from both venomous and non-venomous snakes, mammals, and most recently from medicinal plant extracts. The snake blood-derived inhibitors have been grouped into three major classes, alpha, beta, and gamma, based on common structural motifs found in other proteins with diverse physiological properties. In mammals, DM64, an anti-myotoxic protein isolated from opossum serum, belongs to the immunoglobulin super gene family and is homologous to human alpha1B-glycoprotein and DM43, a metalloproteinase inhibitor from the same organism. In plants, a short note is made of WSG, a newly described anti-toxic-PLA2 glycoprotein isolated from Withania somnifera (Ashwaganda), a medicinal plant whose aqueous extracts neutralize the PLA2 activity of the Naja naja venom. The implications of these new groups of PLA2 toxin inhibitors in the context of our current understanding of snake biology as well as in the development of novel therapeutic reagents in the treatment of snake envenomations worldwide are discussed.

  17. Phospholipase A2 from bovine seminal plasma is a platelet-activating factor acetylhydrolase.

    PubMed Central

    Soubeyrand, S; Lazure, C; Manjunath, P

    1998-01-01

    The major phospholipase A2 activity from bovine seminal plasma was recently purified [Soubeyrand, Khadir, Brindle and Manjunath (1997) J. Biol. Chem. 272, 222-227]. We here show that the 60 kDa enzyme is in fact a platelet-activating factor acetylhydrolase (PAF-AH). Sequences of the N-terminus as well as of internal fragments showed 100% identity with the cDNA-deduced sequences of bovine plasma PAF-AH. The enzyme has kinetic properties similar to those of the human serum PAF-AH. Although capable of hydrolysing long-chained phosphatidylcholine, it displayed a highly preferential activity towards PAF. The enzyme activity towards phosphatidylcholine, but not PAF, was Ca2+-dependent. Biochemical characterization revealed that the enzyme is extensively N-glycosylated and that it exists predominantly as a dimer in solution. Western blot analysis revealed that the enzyme is highly heterogeneous in charge, with a maximal distribution at an isoelectric point of approx. 5.7. The enzyme was expressed exclusively in the seminal vesicles and the ampulla. No association of the enzyme with either epididymal or ejaculated spermatozoa could be detected. PMID:9405273

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

    PubMed

    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

    1998-04-01

    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.

  19. Phospholipase D Mediates Nutrient Input to Mammalian Target of Rapamycin Complex 1 (mTORC1)*

    PubMed Central

    Xu, Limei; Salloum, Darin; Medlin, Phil S.; Saqcena, Mahesh; Yellen, Paige; Perrella, Benjamin; Foster, David A.

    2011-01-01

    The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid- and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid- and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD. PMID:21622984

  20. Purification and inhibitory profile of phospholipase A2 inhibitors from Australian elapid sera.

    PubMed Central

    Hains, P G; Broady, K W

    2000-01-01

    Although the resistance of snakes to their own venom is well known, until now no investigators have examined the serum of Australian snakes. Here we describe the identification and purification of a range of phospholipase A(2) (PLA(2)) inhibitors from the serum of Australian elapids. All PLA(2) inhibitors were composed of two protein chains, an alpha-chain and a beta-chain. The alpha-chains were approx. 22.5 kDa in size and variably glycosylated, whereas the beta-chains were approx. 19.8 kDa in size and not glycosylated. Identification of isoforms of the two subunit chains was significant because three of the six sera examined were from single snake specimens. In addition, the glycosylation patterns of the alpha-chains were thoroughly investigated in these unpooled sera. The functional and structural properties of the purified inhibitors were studied. Uniquely, a snake PLA(2) inhibitor was found to inhibit human type II PLA(2) enzyme, which has implications for the treatment of the many diseases in which PLA(2) enzymes have been implicated. Further, we demonstrate that the inhibitor forms a non-covalent association with a purified PLA(2) enzyme. Finally, the purified PLA(2) inhibitor was shown to protect in vivo against the lethal affects of a homologous PLA(2) enzyme, suggesting a role for PLA(2) inhibitors in the treatment of snake bite victims. PMID:10657250

  1. The controversial role of phospholipase C epsilon (PLCε) in cancer development and progression

    PubMed Central

    Tyutyunnykova, Anna; Telegeev, Gennady; Dubrovska, Anna

    2017-01-01

    The phospholipase C (PLC) enzymes are important regulators of membrane phospholipid metabolism. PLC proteins can be activated by the receptor tyrosine kinases (RTK) or G-protein coupled receptors (GPCR) in response to the different extracellular stimuli including hormones and growth factors. Activated PLC enzymes hydrolyze phosphoinositides to increase the intracellular level of Ca2+ and produce diacylglycerol, which are important mediators of the intracellular signaling transduction. PLC family includes 13 isozymes belonging to 6 subfamilies according to their domain structures and functions. Although importance of PLC enzymes for key cellular functions is well established, the PLC proteins belonging to the ε, ζ and η subfamilies were identified and characterized only during the last decade. As a largest known PLC protein, PLCε is involved in a variety of signaling pathways and controls different cellular properties. Nevertheless, its role in carcinogenesis remains elusive. The aim of this review is to provide a comprehensive and up-to-date overview of the experimental and clinical data about the role of PLCε in the development and progression of the different types of human and experimental tumors. PMID:28382133

  2. 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

  3. Phospholipase D1 decreases type I collagen levels in hepatic stellate cells via induction of autophagy.

    PubMed

    Seo, H-Y; Jang, B-K; Jung, Y-A; Lee, E-J; Kim, H-S; Jeon, J-H; Kim, J-G; Lee, I-K; Kim, M-K; Park, K-G

    2014-06-20

    Hepatic stellate cells (HSCs) are major players in liver fibrogenesis. Accumulating evidence shows that suppression of autophagy plays an important role in the development and progression of liver disease. Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA) and choline, was recently shown to modulate autophagy. However, little is known about the effects of PLD1 on the production of type I collagen that characterizes liver fibrosis. Here, we examined whether PLD1 regulates type I collagen levels in HSCs through induction of autophagy. Adenovirus-mediated overexpression of PLD-1 (Ad-PLD1) reduced type I collagen levels in the activated human HSC lines, hTERT and LX2. Overexpression of PLD1 in HSCs led to induction of autophagy as demonstrated by increased LC3-II conversion and formation of LC3 puncta, and decreased p62 abundance. Moreover, inhibiting the induction of autophagy by treating cells with bafilomycin or a small interfering (si)RNA for ATG7 rescued Ad-PLD1-induced suppression of type I collagen accumulation in HSCs. The effects of PLD on type I collagen levels were not related to TGF-β/Smad signaling. Furthermore, treatment of cells with PA induced autophagy and inhibited type I collagen accumulation. The present study indicates that PLD1 plays a role in regulating type I collagen accumulation through induction of autophagy.

  4. 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

    2015-11-25

    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.

  5. Minimal oxidation and storage of low density lipoproteins result in an increased susceptibility to phospholipid hydrolysis by phospholipase A2.

    PubMed

    Eckey, R; Menschikowski, M; Lattke, P; Jaross, W

    1997-07-25

    In vitro-studies have shown that phospholipid hydrolysis of low density lipoproteins (LDL) by bee venom or porcine pancreatic phospholipase A2 (PLA2) leads to an increased uptake of these lipoproteins by macrophages transforming them into foam cells. Recently, a secretory phospholipase A2, group II, was detected in human atherosclerotic plaques. In order to investigate the role of this enzyme in the pathogenesis of atherosclerosis, a structurally identical human secretory PLA2 was purified from the medium of HepG2 cells stimulated with interleukin-6 and tumor necrosis factor-alpha. The activity of the purified enzyme towards the phospholipids of native and modified low density lipoproteins was compared with the activity towards Escherichia coli-membranes and other phospholipid substrates. Compared to E. coli-membranes, native LDL proved to be a poor substrate for group II PLA2. After mild oxidation induced by copper ions or by 2,2-azobis(2-amidinopropane) (AAPH), the susceptibility of LDL to phospholipid hydrolysis was found to be increased by 25 and 23%, respectively, whereas extensive copper-mediated oxidation caused a decreased hydrolysis. Aging of LDL at 6 degrees C for weeks or at 37 degrees C for hours resulted in an increase in PLA2-catalyzed phospholipid hydrolysis of up to 26-fold. LDL protected from oxidation by probucol during aging showed a lesser increase in susceptibility to phospholipid hydrolysis. Our results suggest that PLA2, group II, can increase the atherogenicity of LDL by its ability to hydrolyze the phospholipids of these lipoproteins, especially after modifications that are likely to occur in vivo.

  6. Phospholipase cbeta is critical for T cell chemotaxis.

    PubMed

    Bach, Tami L; Chen, Qing-Min; Kerr, Wesley T; Wang, Yanfeng; Lian, Lurong; Choi, John K; Wu, Dianqing; Kazanietz, Marcelo G; Koretzky, Gary A; Zigmond, Sally; Abrams, Charles S

    2007-08-15

    Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in G(alphai) protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCbeta is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCbeta isoforms (PLCbeta2 and PLCbeta3), and demonstrate that loss of PLCbeta2 and PLCbeta3 significantly impaired T cell migration. Because second messengers generated by PLCbeta lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCbeta-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1alpha was undetectable in PLCbeta2beta3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCbeta play a critical role in T lymphocyte chemotaxis.

  7. 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.

  8. Activation of Phospholipase A by Plant Defense Elicitors.

    PubMed Central

    Chandra, S.; Heinstein, P. F.; Low, P. S.

    1996-01-01

    Participation of phospholipase A (PLase A) in plant signal transduction has been documented for auxin stimulation of growth but not for elicitation of any plant defense response. In this paper, we report two independent assays for monitoring PLase A induction in plant cells and have used these assays to evaluate whether transduction of defense-related signals might require PLase A activation. Oligogalacturonic acid, a potent elicitor of the soybean (Glycine max) H2O2 burst, was unable to stimulate endogenous PLase A, suggesting that PLase A activation is not an obligate intermediate in the oligogalacturonic acid-induced burst pathway. In contrast, harpin and an extract from the pathogenic fungus Verticillium dahliae both stimulated the oxidative burst and promoted a rapid increase in PLase A activity. To evaluate the possible role of this inducible PLase A activity in transducing the oxidative burst, we tested the effect of chlorpromazine-HCl, a PLase A inhibitor on elicitor-stimulated burst activity. Pretreatment with chloropromazine was found to inhibit the H2O2 burst triggered by V. dahliae extract at the same concentration at which it blocked PLase A activation. In contrast, neither the harpin- nor oligogalacturonic acid-induced burst was altered by addition of chlorpromazine. These data suggest that PLase A stimulation may be important in certain elicitor-induced oxidative bursts (e.g. V. dahliae) and that other elicitors such as oligogalacturonic acid and harpin must operate through independent signaling intermediates to activate the same defense response. PMID:12226235

  9. Impairment of kindling development in phospholipase Cγ1 heterozygous mice

    PubMed Central

    He, Xiao Ping; Wen, Renren; McNamara, James O

    2014-01-01

    Summary Objective Elucidating molecular mechanisms underlying limbic epileptogenesis may reveal novel targets for preventive therapy. Studies of TrkB mutant mice led us to hypothesize that signaling through a specific phospholipase (PLC), PLCγ1, promoted development of kindling. Methods To test this hypothesis, we examined the development of kindling in PLCγ1 heterozygous mice. We also examined the cellular and subcellular location of PLCγ1 in adult wild type mice. Results The development of kindling was impaired in PLCγ1 heterozygous mice compared to wild type controls. PLCγ1 immunoreactivity was localized to the soma and dendrites of both excitatory and inhibitory neurons in hippocampus of adult mice. Significance This study implicates PLCγ1 signaling as the dominant pathway by which TrkB activation promotes limbic epileptogenesis. Its cellular localization places PLCγ1 in a position to modify the efficacy of both excitatory and inhibitory synaptic transmission. These findings advance PLCγ1 as a novel target for therapies aimed at preventing temporal lobe epilepsy induced by status epilepticus. PMID:24502564

  10. Intracellular signaling by phospholipase D as a therapeutic target.

    PubMed

    Steed, P M; Chow, A H

    2001-09-01

    The pharmaceutical industry has recently focused on intracellular signaling as a means to integrate the multiple facets of complex disease states, such as inflammation, because these pathways respond to numerous extracellular signals and coordinate a collection of cell responses contributing to pathology. One critical aspect of intracellular signaling is regulation of key cell functions by lipid mediators, in particular the generation of a key mediator, phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine by phospholipase D (PLD). Research in this field has intensified, due in part to the recent cloning and partial characterization of the two PLD isoforms in mammalian cells, and this work has contributed significantly to our understanding of events downstream of PA generation. It is these effector functions of PLD activity that make this pathway attractive as a therapeutic target while the biochemical properties of the PLD isozymes make them amenable to small molecule intervention. Recent studies indicate that PA, and its immediate metabolites diacylglycerol and lyso-PA, affect numerous cellular pathways including ligand-mediated secretion, cytoskeletal reorganisations, respiratory burst, prostaglandin release, cell migration, cytokine release, and mitogenesis. This review summarises the data implicating signaling via PLD in these cell functions, obtained from: (i) molecular analyses of PLD/effector interactions, (ii) correlation between PA production and cell responses, (iii) experimental manipulation of PA levels, (iv) inhibition of PLD regulators, and (v) direct inhibition of PA production. The utility of targeting PLD signaling for the treatment of acute/chronic inflammation and other indications is discussed in light of these data.

  11. Targeting NADPH oxidase and phospholipases A2 in Alzheimer's disease.

    PubMed

    Simonyi, Agnes; He, Yan; Sheng, Wenwen; Sun, Albert Y; Wood, W Gibson; Weisman, Gary A; Sun, Grace Y

    2010-06-01

    Alzheimer's disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Abeta) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Abeta. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Abeta in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A(2) (PLA(2)) and secretory PLA(2). In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Abeta NADPH oxidase and PLA(2) can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.

  12. Phospholipase C activation is required for cardioprotection by ethanol consumption

    PubMed Central

    Miyamae, Masami; Domae, Naochika; Zhou, Hui-Zhong; Sugioka, Shingo; Diamond, Ivan; Figueredo, Vincent M

    2003-01-01

    Regular alcohol consumption decreases the incidence of myocardial infarction (MI) and improves post-MI survival. It has previously been reported that chronic ethanol exposure induces long-term protection against cardiac ischemia/reperfusion injury, which improves myocardial recovery after MI. Chronic cardioprotection by ethanol requires the activation of myocyte adenosine A1 receptors and sustained intramyocyte translocation of epsilon protein kinase C. A1 receptors activate phospholipase C (PLC). In the present paper, the role of PLC in mediating ethanol’s protective effect against ischemia/reperfusion injury is investigated. Isolated hearts from guinea pigs fed 2.5% ethanol in their water for four months were subjected to ischemia/reperfusion. Hearts from ethanol-treated animals showed improved recovery of left ventricular developed pressure compared with controls (61% versus 38% of baseline, respectively; P<0.05) and decreased necrosis, assessed by the release of creatine kinase (263±18 U/mL × g dry weight versus 360±24 U/mL × g dry weight, respectively; P<0.05). Ethanol protection was abolished by the PLC antagonist, U-73122 (50 nM). These findings suggest that PLC activation is required for ethanol cardioprotection against ischemia/reperfusion injury. PMID:19649218

  13. The galactolipase activity of Fusarium solani (phospho)lipase.

    PubMed

    Jallouli, Raida; Othman, Houcemeddine; Amara, Sawsan; Parsiegla, Goetz; Carriere, Frédéric; Srairi-Abid, Najet; Gargouri, Youssef; Bezzine, Sofiane

    2015-03-01

    The purified (phospho)lipase of Fusarium solani (FSL), was known to be active on both triglycerides and phospholipids. This study aimed at assessing the potential of this enzyme in hydrolyzing galactolipids. FSL was found to hydrolyze at high rates of synthetic medium chains monogalactosyldiacylglycerol (4658±146U/mg on DiC8-MGDG) and digalactosyldiacylglycerol (3785±83U/mg on DiC8-DGDG) and natural long chain monogalactosyldiacylglycerol extracted from leek leaves (991±85U/mg). It is the microbial enzyme with the highest activity on galactolipids identified so far with a level of activity comparable to that of pancreatic lipase-related protein 2. FSL maximum activity on galactolipids was measured at pH8. The analysis of the hydrolysis product of natural MGDG from leek showed that FSL hydrolyzes preferentially the ester bond at the sn-1 position of galactolipids. To investigate the structure-activity relationships of FSL, a 3D model of this enzyme was built. In silico docking of medium chains MGDG and DGDG and phospholipid in the active site of FSL reveals structural solutions which are in concordance with in vitro tests.

  14. Acidification in the epidermis and the role of secretory phospholipases

    PubMed Central

    Chan, Aegean

    2011-01-01

    The function of the epidermis is to form an effective barrier between the dry, external environment and the interior of the body. The barrier specifically resides in the extracellular lipid membranes of the stratum corneum (SC) and an acidic pH is necessary to maintain its competency against various insults. The purpose of this review is to explore the mechanisms which are postulated to contribute to the acidification of the stratum corneum, including both exogenous and endogenous sources. However, recent research as pointed to several endogenous mechanisms as the major source of acidification, including a sodium/proton pump (NHE1) and free fatty acid conversion from phospholipids by secretory phospholipase A2 (sPLA2). sPLA2 has been shown to play a central role in the formation of the SC “acid mantle” in the early maturation of the epidermis postnatally. Many aspects of this enzyme family are complex and still being elucidated in research and the most recent findings on the localization and functions of sPL A2-IB, -IIA, -IIC, -IID, -IIE, -IIF, -III, -V, -X and -XII in the epidermis are presented here. Given their role in inflammatory dermatoses, such as psoriasis and atopic dermatitis, understanding this complex enzyme family can lead to novel, life-changing therapies. PMID:21695017

  15. Interfacial Catalysis: The Mechanism of Phospholipase A2

    PubMed Central

    Scott, David L.; White, Steven P.; Otwinowski, Zbyszek; Yuan, Wei; Gelb, Michael H.; Sigler, Paul B.

    2012-01-01

    A chemical description of the action of phospholipase A2 (PLA2) can now be inferred with confidence from three high-resolution x-ray crystal structures. The first is the structure of the PLA2 from the venom of the Chinese cobra (Naja naja atra) in a complex with a phosphonate transition-state analogue. This enzyme is typical of a large, well-studied homologous family of PLA2s. The second is a similar complex with the evolutionarily distant bee-venom PLA2. The third structure is the uninhibited PLA2 from Chinese cobra venom. Despite the different molecular architectures of the cobra and bee-venom PLA2s, the transition-state analogue interacts in a nearly identical way with the catalytic machinery of both enzymes. The disposition of the fatty-acid side chains suggests a common access route of the substrate from its position in the lipid aggregate to its productive interaction with the active site. Comparison of the cobra-venom complex with the uninhibited enzyme indicates that optimal binding and catalysis at the lipid-water interface is due to facilitated substrate diffusion from the interfacial binding surface to the catalytic site rather than an allosteric change in the enzyme’s structure. However, a second bound calcium ion changes its position upon the binding of the transition-state analogue, suggesting a mechanism for augmenting the critical electrophile. PMID:2274785

  16. Identification of a new phospholipase D in Carica papaya latex.

    PubMed

    Abdelkafi, Slim; Abousalham, Abdelkarim; Fendri, Imen; Ogata, Hiroyuki; Barouh, Nathalie; Fouquet, Benjamin; Scheirlinckx, Frantz; Villeneuve, Pierre; Carrière, Frédéric

    2012-05-15

    Phospholipase D (PLD) is a lipolytic enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. It catalyzes the hydrolysis and transphosphatidylation of glycerophospholipids at the terminal phosphodiester bond. The presence of a PLD in the latex of Carica papaya (CpPLD1) was demonstrated by transphosphatidylation of phosphatidylcholine (PtdCho) in the presence of 2% ethanol. Although the protein could not be purified to homogeneity due to its presence in high molecular mass aggregates, a protein band was separated by SDS-PAGE after SDS/chloroform-methanol/TCA-acetone extraction of the latex insoluble fraction. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by micro-LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (723 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 2424 bp encoding a protein of 808 amino acid residues, with a theoretical molecular mass of 92.05 kDa. From sequence analysis, CpPLD1 was identified as a PLD belonging to the plant phosphatidylcholine phosphatidohydrolase family.

  17. Role of phospholipase A(2) in retrograde transport of ricin.

    PubMed

    Klokk, Tove Irene; Lingelem, Anne Berit Dyve; Myrann, Anne-Grethe; Sandvig, Kirsten

    2011-09-01

    Ricin is a protein toxin classified as a bioterror agent, for which there are no known treatment options available after intoxication. It is composed of an enzymatically active A-chain connected by a disulfide bond to a cell binding B-chain. After internalization by endocytosis, ricin is transported retrogradely to the Golgi and ER, from where the ricin A-chain is translocated to the cytosol where it inhibits protein synthesis and thus induces cell death. We have identified cytoplasmic phospholipase A(2) (PLA(2)) as an important factor in ricin retrograde transport. Inhibition of PLA(2) protects against ricin challenge, however the toxin can still be endocytosed and transported to the Golgi. Interestingly, ricin transport from the Golgi to the ER is strongly impaired in response to PLA(2) inhibition. Confocal microscopy analysis shows that ricin is still colocalized with the trans-Golgi marker TGN46 in the presence of PLA(2) inhibitor, but less is colocalized with the cis-Golgi marker GM130. We propose that PLA(2) inhibition results in impaired ricin transport through the Golgi stack, thus preventing it from reaching the ER. Consequently, ricin cannot be translocated to the cytosol to exert its toxic action.

  18. Pyrimidinoceptor-mediated activation of phospholipase C and phospholipase A2 in RAW 264.7 macrophages.

    PubMed Central

    Lin, W. W.; Lee, Y. T.

    1996-01-01

    1. As well as the presence of P2Z purinoceptors previously found in macrophages, we identified pyrimidinoceptors in RAW 264.7 cells, which activate phospholipase C (PLC) and phospholipase A2 (PLA2). 2. The relative potency of agonists to stimulate inositol phosphate (IP) formation and arachidonic acid (AA) release was UTP = UDP > > ATP, ATP gamma S, 2MeSATP. For both signalling pathways, the EC50 values for UTP and UDP (3 microM) were significantly lower than that for ATP and all other analogues tested (> 100 microM). 3. UTP and UDP displayed no additivity in terms of IP formation and AA release at maximally effective concentrations. 4. UTP-, but not ATP-, evoked AA release was 60% inhibited by pertussis toxin (PTX), while stimulation of IP formation by both agonists was unaffected. Short-term treatment with phorbol 12-myristate 13-acetate (PMA) led to a dose-dependent inhibition of IP responses to UTP and UDP, but failed to affect the AA responses. Removal of extracellular Ca2+ inhibited the PI response to UTP, but abolished its AA response. 5. ATP-induction of these two transmembrane signal pathways was decreased in high Mg(2+)-containing medium but potentiated by the removal of extracellular Mg2+. 6. Suramin and reactive blue displayed equal potency to inhibit the IP responses of UTP and ATP. 7. Both UTP and UDP (0.1-100 microM) induced a sustained increase in [Ca2+]i which lasted for more than 10 min. 8. Taken together, these results indicate that in mouse RAW 264.7 macrophages, pyrimidinoceptors with specificity for UTP and UDP mediate the activation of PLC and cytosolic (c) PLA2. The activation of PLC is via a PTX-insensitive G protein, whereas that of cPLA2 is via a PTX-sensitive G protein-dependent pathway. The sustained Ca2+ influx caused by UTP contributes to the activation of cPLA2. RAW 264.7 cells also possess P2z purinoceptors which mediate ATP(4-)-induced PLC and PLA2 activation. Images Figure 3 PMID:8886407

  19. In silico-guided target identification of a scaffold-focused library: 1,3,5-triazepan-2,6-diones as novel phospholipase A2 inhibitors.

    PubMed

    Muller, Pascal; Lena, Gersande; Boilard, Eric; Bezzine, Sofiane; Lambeau, Gérard; Guichard, Gilles; Rognan, Didier

    2006-11-16

    A collection of 2150 druggable active sites from the Protein Data Bank was screened by high-throughput docking to identify putative targets for five representative molecules of a combinatorial library sharing a 1,3,5-triazepan-2,6-dione scaffold. Five targets were prioritized for experimental evaluation by computing enrichment in individual protein entries among the top 2% scoring targets. Out of the five proposed proteins, secreted phospholipase A2 (sPLA2) was shown to be a true target for a panel of 1,3,5-triazepan-2,6-diones which exhibited micromolar affinities toward two human sPLA2 members.

  20. Recombinant Phospholipase A1 of the Outer Membrane of Psychrotrophic Yersinia pseudotuberculosis: Expression, Purification, and Characterization.

    PubMed

    Bakholdina, S I; Tischenko, N M; Sidorin, E V; Isaeva, M P; Likhatskaya, G N; Dmitrenok, P S; Kim, N Yu; Chernikov, O V; Solov'eva, T F

    2016-01-01

    The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.

  1. Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

    NASA Technical Reports Server (NTRS)

    Vandenburgh, H. H.; Shansky, J.; Karlisch, P.; Solerssi, R. L.

    1993-01-01

    Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E2 and F2 alpha which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of 3H-arachidonic acid labelled phospholipids, releasing free 3H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases 3H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-3H]inositol labelled phospholipids. Phospholipase A2 (PLA2), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, 3H-arachidonic acid labelled phospholipid breakdown, and 3H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-[2-3H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

  2. 1-Butanol interferes with phospholipase D1 and protein kinase Calpha association and inhibits phospholipase D1 basal activity.

    PubMed

    Hu, Tianhui; Exton, John H

    2005-02-25

    1-Butanol is commonly used as a substrate for phospholipase D (PLD) activity measurement. Surprisingly we found that, in the presence of 30 mM 1-butanol (standard PLD assay conditions), PLD1 activity in COS-7 cells was lost after incubation for 2 min. In contrast, in the presence of the protein kinase C (PKC) inhibitor staurosporine or dominant negative PKCalpha D481E, the activity was sustained for at least 30min. The binding between PLD1 and PKCalpha was also lost after 2 min incubation with 30 mM 1-butanol while staurosporine and D481E maintained the binding. 1-Butanol at 2 mM did not inhibit PLD1 basal activity or PLD1 binding to PKCalpha, and staurosporine and PKCalpha D481E produced a constant increase in PLD1 basal activity of 2-fold. These results indicate that 1-butanol is inhibitory to PLD1 activity by reducing its association with PKCalpha, and that the concentration of 1-butanol is an important consideration in assaying basal PLD1 activity.

  3. M-Type Phospholipase A2 Receptor as Target Antigen in Idiopathic Membranous Nephropathy

    PubMed Central

    Beck, Laurence H.; Bonegio, Ramon G.B.; Lambeau, Gérard; Beck, David M.; Powell, David W.; Cummins, Timothy D.; Klein, Jon B.; Salant, David J.

    2009-01-01

    BACKGROUND Idiopathic membranous nephropathy, a common form of the nephrotic syndrome, is an antibody-mediated autoimmune glomerular disease. Serologic diagnosis has been elusive because the target antigen is unknown. METHODS We performed Western blotting of protein extracts from normal human glomeruli with serum samples from patients with idiopathic or secondary membranous nephropathy or other proteinuric or autoimmune diseases and from normal controls. We used mass spectrometry to analyze the reactive protein bands and confirmed the identity and location of the target antigen with a monospecific antibody. RESULTS Serum samples from 26 of 37 patients (70%) with idiopathic but not secondary membranous nephropathy specifically identified a 185-kD glycoprotein in non-reduced glomerular extract. Mass spectrometry of the reactive protein band detected the M-type phospholipase A2 receptor (PLA2R). Reactive serum specimens recognized recombinant PLA2R and bound the same 185-kD glomerular protein as did the monospecific anti-PLA2R antibody. Anti-PLA2R autoantibodies in serum samples from patients with membranous nephropathy were mainly IgG4, the predominant immunoglobulin subclass in glomerular deposits. PLA2R was expressed in podocytes in normal human glomeruli and colocalized with IgG4 in immune deposits in glomeruli of patients with membranous nephropathy. IgG eluted from such deposits in patients with idiopathic membranous nephropathy, but not in those with lupus membranous or IgA nephropathy, recognized PLA2R. CONCLUSIONS A majority of patients with idiopathic membranous nephropathy have antibodies against a conformation-dependent epitope in PLA2R. PLA2R is present in normal podocytes and in immune deposits in patients with idiopathic membranous nephropathy, indicating that PLA2R is a major antigen in this disease. PMID:19571279

  4. Phospholipase D signaling in serotonin-induced mitogenesis of pulmonary artery smooth muscle cells.

    PubMed

    Liu, Y; Fanburg, B L

    2008-09-01

    We have previously reported the participation of mitogen-activated protein, Rho, and phosphoinositide-3 (PI3) kinases in separate pathways in serotonin (5-HT)-induced proliferation of pulmonary artery smooth muscle cells (SMCs). In this study, we investigated the possible participation of phospholipase D (PLD) and phosphatidic acid (PA) in this growth process. 5-HT stimulated a time-dependent increase in [(3)H]phosphatidylbutanol and PA generation. Exposure of SMCs to 1-butanol or overexpression of an inactive mutant of human PLD1R898R blocked 5-HT-induced proliferation. Furthermore, 1-butanol inhibited 5-HT activation of S6K1 and S6 protein, downstream effectors of mammalian target of rapamycin (mTOR), by 80 and 72%, respectively, and partially blocked activation of extracellular signal-regulated kinase (ERK) by 30% but had no effect on other associated signaling pathways. Exogenous PA caused cellular proliferation and revitalized cyclin D1 expression by 5-HT of the 1-butanol-treated cells. PA also reproduced activations by 5-HT of mTOR, S6K1, and ERK. Transfection with inactive human PLD1 reduced 5-HT-induced activation of S6K1 by approximately 50%. Inhibition of 5-HT receptor 2A (R 2A) with ketaserin blocked PLD activation by 5-HT. Inhibition with PI3-kinase inhibitor failed to block either activation of PLD by 5-HT or PA-dependent S6K1 phosphorylation. Taken together, these results indicate that ligation of the 5-HTR 2A by 5-HT initiates PLD activation in SMCs, and that its product, PA, is an early signaling molecule in 5-HT-induced pulmonary artery SMC proliferation. Signaling by PA produces its downstream effects primarily through the mTOR/S6K1 pathway and to a lesser extent through the ERK pathway. Hydrolysis of cell membrane lipid may be important in vascular effects of 5-HT.

  5. Cholesterol regulates HERG K+ channel activation by increasing phospholipase C β1 expression.

    PubMed

    Chun, Yoon Sun; Oh, Hyun Geun; Park, Myoung Kyu; Cho, Hana; Chung, Sungkwon

    2013-01-01

    Human ether-a-go-go-related gene (HERG) K(+) channel underlies the rapidly activating delayed rectifier K(+) conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of β1 and β3 isoforms of PLC (PLCβ1, PLCβ3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCβ1 expression, we conclude that increased PLCβ1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.

  6. Forty five years with membrane phospholipids, phospholipases and lipid mediators: A historical perspective.

    PubMed

    Chap, Hugues

    2016-06-01

    Phospholipases play a key role in the metabolism of phospholipids and in cell signaling. They are also a very useful tool to explore phospholipid structure and metabolism as well as membrane organization. They are at the center of this review, covering a period starting in 1971 and focused on a number of subjects in which my colleagues and I have been involved. Those include determination of phospholipid asymmetry in the blood platelet membrane, biosynthesis of lysophosphatidic acid, biochemistry of platelet-activating factor, first attempts to define the role of phosphoinositides in cell signaling, and identification of novel digestive (phospho)lipases such as pancreatic lipase-related protein 2 (PLRP2) or phospholipase B. Besides recalling some of our contributions to those various fields, this review makes an appraisal of the impressive and often unexpected evolution of those various aspects of membrane phospholipids and lipid mediators. It is also the occasion to propose some new working hypotheses.

  7. 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

    2001-11-09

    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.

  8. Sphingosine induces phospholipase D and mitogen activated protein kinase in vascular smooth muscle cells.

    PubMed

    Taher, M M; Abd-Elfattah, A S; Sholley, M M

    1998-12-01

    The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen-activated protein kinase in vascular smooth muscle cells.

  9. Preliminary crystallographic study of an acidic phospholipase A2 from Ophiophagus hannah (king cobra).

    PubMed

    Xu, Sujuan; Gu, Lichuan; Wang, Qiuyan; Shu, Yuyan; Lin, Zhengjiong

    2002-10-01

    An acidic phospholipase A(2) (OH APLA(2)-II) with an isoelectric point (pI) of 4.0 was recently isolated from Ophiophagus hannah (king cobra) from Guangxi province, China. Comparison of this enzyme to a previously reported homologous phospholipase A(2) from the same venom shows that it lacks toxicity and exhibits a greater phospholipase activity. OH APLA(2)-II has been crystallized by the hanging-drop vapour-diffusion method using 1,6-hexanediol and magnesium chloride as precipitants. The crystal belongs to space group P6(3), with unit-cell parameters a = b = 98.06, c = 132.39 A. The diffraction data were collected under cryoconditions (100 K) and reduced to 2.1 A resolution. A molecular-replacement solution has been determined and shows that there are six molecules in one asymmetric unit.

  10. Phospholipase A2 from sheep erythrocyte membranes. Ca2+ dependence and localization.

    PubMed

    Frei, E; Zahler, P

    1979-02-02

    The calcium dependence and the time course of phosphatidylethanolamine and phosphatidylcholine degradation by sheep erythrocyte membrane suspensions in presence of Triton X-100 were investigated. One enzyme with phospholipase A2 specificity was found to be responsible for both phosphatidyl-ethanolamine and phosphatidylcholine degradation. The localization of this enzyme in the membrane of the sheep erythrocyte was investigated by proteolytic treatment of sealed erythrocyte ghosts from the outside and of ghosts which had both sides of the membrane exposed to chymotrypsin. The inability of sealed ghosts to take up chymotrypsin was followed by flux measurements of [14C]dextran carboxyl previously trapped in the ghosts. No efflux of the marker was found during the proteolytic treatment. By comparing the residual phospholipase activities in the membranes from both ghost preparations, we concluded that the phospholipase is oriented to the exterior of the sheep erythrocyte.

  11. Pathophysiological role and clinical significance of lipoprotein-associated phospholipase A₂ (Lp-PLA₂) bound to LDL and HDL.

    PubMed

    Tellis, Constantinos C; Tselepis, Alexandros D

    2014-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2), also named as platelet-activating factor (PAF)-acetylhydrolase, exhibits a Ca2+-independent phospholipase A2 activity and catalyzes the hydrolysis of the ester bond at the sn-2 position of PAF and oxidized phospholipids (oxPL). These phospholipids are formed under oxidative and inflammatory conditions, and may play important roles in atherogenesis. The vast majority of plasma Lp-PLA2 mass binds to low-density lipoprotein (LDL) while a smaller amount is associated with high-density lipoprotein (HDL). Lp-PLA2 is also bound to lipoprotein (a) [Lp(a)], very low-density lipoprotein (VLDL) and remnant lipoproteins. Several lines of evidence suggest that the role of plasma Lp-PLA2 in atherosclerosis may depend on the type of lipoprotein particle with which this enzyme is associated. Data from large Caucasian population studies have supported plasma Lp-PLA2 (primarily LDL-associated Lp-PLA2) as a cardiovascular risk marker independent of, and additive to, traditional risk factors. On the contrary, the HDL-associated Lp-PLA2 may express antiatherogenic activities and is also independently associated with lower risk for cardiac death. The present review presents data on the biochemical and enzymatic properties of Lp-PLA2 as well as its structural characteristics that determine the association with LDL and HDL. We also critically discuss the possible pathophysiological and clinical significance of the Lp- PLA2 distribution between LDL and HDL in human plasma, in view of the results of prospective epidemiologic studies on the association of Lp-PLA2 with future cardiovascular events as well the recent studies that evaluate the possible effectiveness of specific Lp-PLA2 inhibitors in reducing residual cardiovascular risk.

  12. Group IVA phospholipase A2 regulates testosterone biosynthesis by murine Leydig cells and is required for timely sexual maturation

    PubMed Central

    Kurusu, Shiro; Sapirstein, Adam; Sawada, Harumi; Kawaminami, Mitsumori; Bonventre, Joseph V.

    2015-01-01

    In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a−/− ) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a−/− mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a−/− mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors. PMID:21762109

  13. Group X Secreted Phospholipase A2 Releases ω3 Polyunsaturated Fatty Acids, Suppresses Colitis, and Promotes Sperm Fertility*

    PubMed Central

    Murase, Remi; Sato, Hiroyasu; Yamamoto, Kei; Ushida, Ayako; Nishito, Yasumasa; Ikeda, Kazutaka; Kobayashi, Tetsuyuki; Yamamoto, Toshinori; Taketomi, Yoshitaka; Murakami, Makoto

    2016-01-01

    Within the secreted phospholipase A2 (sPLA2) family, group X sPLA2 (sPLA2-X) has the highest capacity to hydrolyze cellular membranes and has long been thought to promote inflammation by releasing arachidonic acid, a precursor of pro-inflammatory eicosanoids. Unexpectedly, we found that transgenic mice globally overexpressing human sPLA2-X (PLA2G10-Tg) displayed striking immunosuppressive and lean phenotypes with lymphopenia and increased M2-like macrophages, accompanied by marked elevation of free ω3 polyunsaturated fatty acids (PUFAs) and their metabolites. Studies using Pla2g10-deficient mice revealed that endogenous sPLA2-X, which is highly expressed in the colon epithelium and spermatozoa, mobilized ω3 PUFAs or their metabolites to protect against dextran sulfate-induced colitis and to promote fertilization, respectively. In colitis, sPLA2-X deficiency increased colorectal expression of Th17 cytokines, and ω3 PUFAs attenuated their production by lamina propria cells partly through the fatty acid receptor GPR120. In comparison, cytosolic phospholipase A2 (cPLA2α) protects from colitis by mobilizing ω6 arachidonic acid metabolites, including prostaglandin E2. Thus, our results underscore a previously unrecognized role of sPLA2-X as an ω3 PUFA mobilizer in vivo, segregated mobilization of ω3 and ω6 PUFA metabolites by sPLA2-X and cPLA2α, respectively, in protection against colitis, and the novel role of a particular sPLA2-X-driven PUFA in fertilization. PMID:26828067

  14. Group X Secreted Phospholipase A2 Releases ω3 Polyunsaturated Fatty Acids, Suppresses Colitis, and Promotes Sperm Fertility.

    PubMed

    Murase, Remi; Sato, Hiroyasu; Yamamoto, Kei; Ushida, Ayako; Nishito, Yasumasa; Ikeda, Kazutaka; Kobayashi, Tetsuyuki; Yamamoto, Toshinori; Taketomi, Yoshitaka; Murakami, Makoto

    2016-03-25

    Within the secreted phospholipase A2(sPLA2) family, group X sPLA2(sPLA2-X) has the highest capacity to hydrolyze cellular membranes and has long been thought to promote inflammation by releasing arachidonic acid, a precursor of pro-inflammatory eicosanoids. Unexpectedly, we found that transgenic mice globally overexpressing human sPLA2-X (PLA2G10-Tg) displayed striking immunosuppressive and lean phenotypes with lymphopenia and increased M2-like macrophages, accompanied by marked elevation of free ω3 polyunsaturated fatty acids (PUFAs) and their metabolites. Studies usingPla2g10-deficient mice revealed that endogenous sPLA2-X, which is highly expressed in the colon epithelium and spermatozoa, mobilized ω3 PUFAs or their metabolites to protect against dextran sulfate-induced colitis and to promote fertilization, respectively. In colitis, sPLA2-X deficiency increased colorectal expression of Th17 cytokines, and ω3 PUFAs attenuated their production by lamina propria cells partly through the fatty acid receptor GPR120. In comparison, cytosolic phospholipase A2(cPLA2α) protects from colitis by mobilizing ω6 arachidonic acid metabolites, including prostaglandin E2 Thus, our results underscore a previously unrecognized role of sPLA2-X as an ω3 PUFA mobilizerin vivo, segregated mobilization of ω3 and ω6 PUFA metabolites by sPLA2-X and cPLA2α, respectively, in protection against colitis, and the novel role of a particular sPLA2-X-driven PUFA in fertilization.

  15. 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.

  16. A novel class of microbial phosphocholine-specific phospholipases C.

    PubMed

    Stonehouse, Martin J; Cota-Gomez, Adela; Parker, Sarah K; Martin, Wesley E; Hankin, Joseph A; Murphy, Robert C; Chen, Weibin; Lim, Kheng B; Hackett, Murray; Vasil, Adriana I; Vasil, Michael L

    2002-11-01

    In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.

  17. Cytosolic phospholipase A2α regulates G1 progression through modulating FOXO1 activity

    PubMed Central

    Naini, Said Movahedi; Choukroun, Gabriel J.; Ryan, James R.; Hentschel, Dirk M.; Shah, Jagesh V.; Bonventre, Joseph V.

    2016-01-01

    Group IVA phospholipase A2 [cytosolic phospholipase A2α (cPLA2α)] is a key mediator of inflammation and tumorigenesis. In this study, by using a combination of chemical inhibition and genetic approaches in zebrafish and murine cells, we identify a mechanism by which cPLA2α promotes cell proliferation. We identified 2 cpla2α genes in zebrafish, cpla2αa and cpla2αb, with conserved phospholipase activity. In zebrafish, loss of cpla2α expression or inhibition of cpla2α activity diminished G1 progression through the cell cycle. This phenotype was also seen in both mouse embryonic fibroblasts and mesangial cells. G1 progression was rescued by the addition of arachidonic acid or prostaglandin E2 (PGE2), indicating a phospholipase-dependent mechanism. We further show that PGE2, through PI3K/AKT activation, promoted Forkhead box protein O1 (FOXO1) phosphorylation and FOXO1 nuclear export. This led to up-regulation of cyclin D1 and down-regulation of p27Kip1, thus promoting G1 progression. Finally, using pharmacologic inhibitors, we show that cPLA2α, rapidly accelerated fibrosarcoma (RAF)/MEK/ERK, and PI3K/AKT signaling pathways cooperatively regulate G1 progression in response to platelet-derived growth factor stimulation. In summary, these data indicate that cPLA2α, through its phospholipase activity, is a critical effector of G1 phase progression through the cell cycle and suggest that pharmacological targeting of this enzyme may have important therapeutic benefits in disease mechanisms that involve excessive cell proliferation, in particular, cancer and proliferative glomerulopathies.—Naini, S. M., Choukroun, G. J., Ryan, J. R., Hentschel, D. M., Shah, J. V., Bonventre, J. V. Cytosolic phospholipase A2α regulates G1 progression through modulating FOXO1 activity. PMID:26644349

  18. Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

    PubMed

    De Wolf, M; Lagrou, A; Hilderson, H J; Dierick, W

    1978-12-01

    In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.

  19. Asthenozoospermia and membrane remodeling enzymes: a new role for phospholipase A2.

    PubMed

    Anfuso, C D; Olivieri, M; Bellanca, S; Salmeri, M; Motta, C; Scalia, M; Satriano, C; La Vignera, S; Burrello, N; Caporarello, N; Lupo, G; Calogero, A E

    2015-11-01

    Phosholipase A2 (PLA2 ) activity in the seminal plasma and in sperm heads is closely related to sperm motility and male fertility. Therefore, the purpose of this study was to investigate the possible involvement of different isoforms of phospholipase in asthenozoospermia. To accomplish this, cPLA2 , phospho-cPLA2 , iPLA2 , and sPLA2 were evaluated by immunofluorescence and immunoblot analyses in spermatozoa obtained from 22 normozoospermic men and 28 asthenozoospermic patients. We found significant differences in cPLA2 and its phosphorylated/activated form, iPLA2 , and sPLA2 content and distribution in normal and asthenozoospermic patients. cPLA2 was localized in heads, midpieces, and tails of all spermatozoa as constitutive enzyme, less expressed in the tail of spermatozoa with low progressive motility. While active phospho-cPLA2 distribution was homogeneous throughout the cell body of control-donor spermatozoa, lower levels were detected in the tails of asthenozoospermic patients, as opposed to its strong presence in heads. Low immunofluorescence signal for iPLA2 was found in astenozoospermic patients, whereas sPLA2 was significantly lower in the heads of asthenozoospermic patients. Spermatozoa with low progressive motility showed differences both in terms of total specific activity and of intracellular distribution. cPLA2 , iPLA2 , and sPLA2 specific activities correlated positively and in a significantly manner with sperm progressive motility both in normozoospermic men and asthenozoospermic patients. In conclusion, PLA2 s are expressed in different areas of human spermatozoa. Spermatozoa with low motility showed differences in total specific activity and enzyme distributions. We speculated that PLA2 expression and/or different distribution could be potential biomarkers of asthenozoospermia, one of the major causes of male factor infertility.

  20. 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.

  1. Overexpression of porcine lipoprotein-associated phospholipase A2 in swine.

    PubMed

    Tang, Xiaochun; Wang, Gangqi; Liu, Xingxing; Han, Xiaolei; Li, Zhuang; Ran, Guangyao; Li, Zhanjun; Song, Qi; Ji, Yuan; Wang, Haijun; Wang, Yuhui; Ouyang, Hongsheng; Pang, Daxin

    2015-09-25

    Lipoprotein-associated phospholipase A 2 (Lp-PLA2) is associated with the risk of vascular disease. It circulates in human blood predominantly in association with low-density lipoprotein cholesterol (LDL-C) and hydrolyses oxidized phospholipids into pro-inflammatory products. However, in the mouse circulation, it predominantly binds to high-density lipoprotein cholesterol (HDL-C) and exhibits anti-inflammatory properties. To further investigate the effects of Lp-PLA2 in the circulation, we generated over-expressed Lp-PLA2 transgenic swine. The eukaryotic expression plasmid of porcine Lp-PLA2 which driven by EF1α promoter was constructed and generate transgenic swine via SCNT. The expression and activity of Lp-PLA2 in transgenic swine were evaluated, and the total cholesterol (TC), HDL-C, LDL-C and triglyceride (TG) levels in the fasting and fed states were also assessed. Compared with wild-type swine controls, the transgenic swine exhibited elevated Lp-PLA2 mRNA levels and activities, and the activity did not depend on the feeding state. The TC, HDL-C and LDL-C levels were not significantly increased. There was no change in the TG levels in the fasting state between transgenic and control pigs. However, in the fed state, the TG levels of transgenic swine were slightly increased compared with the control pigs and were significantly elevated compared with the fasting state. In addition, inflammatory gene (interleukin [IL]-6, monocyte chemotactic protein [MCP]-1 and tumor necrosis factor [TNF]-α) mRNA levels in peripheral blood mononuclear cells (PBMCs) were significantly increased. The results demonstrated that Lp-PLA2 is associated with triglycerides which may be helpful for understanding the relationship of this protein with cardiovascular disease.

  2. Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity

    PubMed Central

    Wu, Jun; Nilsson, Åke; Jönsson, Bo A. G.; Stenstad, Hanna; Agace, William; Cheng, Yajun; Duan, Rui-Dong

    2005-01-01

    Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophosphatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide in the intestinal tract. The enzyme may protect the intestinal mucosa from inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase can hydrolyse and inactivate PAF. [3H]Octadecyl-labelled PAF was incubated with purified rat intestinal alk-SMase or recombinant human alk-SMase expressed in COS-7 cells. The hydrolytic products were assayed with TLC and MS. We found that alkSMase cleaved the phosphocholine head group from PAF and generated 1-O-alkyl-2-acetyl-sn-glycerol. Differing from the activity against SM, the activity against PAF was optimal at pH 7.5, inhibited by EDTA and stimulated by 0.1–0.25 mM Zn2+. The activity was abolished by site mutation of the predicted metal-binding sites that are conserved in all NPP members. Similar to the activity against SM, the activity against PAF was dependent on bile salt, particularly taurocholate and taurochenodeoxycholate. The Vmax for PAF hydrolysis was 374 μmol·h−1·(mg of protein)−1. The hydrolysis of PAF and SM could be inhibited by the presence of SM and PAF respectively, the inhibition of PAF hydrolysis by SM being stronger. The PAF-induced MAPK (mitogen-activated protein kinase) activation and IL-8 (interleukin 8) release in HT-29 cells, and chemotaxis in leucocytes were abolished by alk-SMase treatment. In conclusion, alk-SMase hydrolyses and inactivates PAF by a phospholipase C activity. The finding reveals a novel function, by which alk-SMase may counteract the development of intestinal inflammation and colon cancer. PMID:16255717

  3. Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2

    PubMed Central

    Cavigiolio, Giorgio; Jayaraman, Shobini

    2014-01-01

    In the acute phase of the inflammatory response, secretory phospholipase A2 (sPLA2) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA2 in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA2-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA2 reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA2 reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA2 by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA2 with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA2 with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA2 was Ca2+-independent, implicating a different mechanism from the Ca2+-dependent PLA2-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA2 proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA2 that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis. PMID:24523407

  4. Expression of phospholipase C isozymes by murine B lymphocytes.

    PubMed

    Hempel, W M; DeFranco, A L

    1991-06-01

    Cross-linking of membrane (m) Ig, the B cell receptor for Ag, activates protein tyrosine phosphorylation and hydrolysis of phosphotidylinositol 4,5-bisphosphate. The latter signal transduction pathway is an important mediator of antigen receptor engagement. The initial event in this pathway is the activation of phospholipase C (PLC). The identity of the isozyme of PLC used in B cells and the mechanism by which it becomes activated are currently unknown. The cDNA encoding five different isozymes have been cloned. As a first step in identifying the isozyme of PLC that is coupled to mIgM, murine cDNA fragments for the five cloned PLC isozymes were generated by the polymerase chain reaction (PCR), cloned, and used to screen a panel of B cell lines representing different stages of development for PLC mRNA expression. All the B cell lines tested expressed high levels of PLC alpha and PLC gamma 2 mRNA, whereas PLC beta and PLC delta mRNA expression were undetectable by both Northern blot and PCR analysis. PLC gamma 1 had a more complicated pattern of mRNA expression. PLC gamma 1 mRNA expression was lower than that observed for PLC alpha or PLC gamma 2 mRNA and varied widely among different cell lines. The pattern of PLC gamma 1 mRNA expression did not correlate with the developmental stage of the cell lines. The pattern of PLC gamma 1 protein expression in the panel of B cell lines correlated with the pattern of PLC gamma 1 mRNA expression. PLC gamma 1 expression was very low in several B cell lines, despite the fact that these cell lines show mIgM-stimulatable PLC activity. The variable and in some cases very low expression of PLC gamma 1 suggests that it may not be the form of PLC that is activated by mIgM. In contrast, PLC alpha and PLC gamma 2 were abundantly expressed in all B cell lines tested. This observation is consistent with the possibility that PLC alpha or PLC gamma 2 is activated by mIgM.

  5. A tyrosine kinase regulates alpha-adrenoceptor-stimulated contraction and phospholipase D activation in the rat aorta.

    PubMed

    Jinsi, A; Paradise, J; Deth, R C

    1996-04-29

    Since previous studies had indicated a role for tyrosine kinases in alpha 2-adrenoceptor-induced contractile responses in other blood vessels, as well as in the activation of phospholipase D, we examined the sensitivity of these responses in rat aorta to the tyrosine kinase inhibitor genistein. Contractions induced by both noradrenaline and the alpha 2-adrenoceptor-selective agonist UK14304 (5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline) were fully inhibited by genistein, with the latter responses being more sensitive. Contractions induced by high K+ buffer were also inhibited, but to a lesser extent. Both agonists caused a stimulation of phospholipase D activity, which could be blocked by pretreatment with pertussis toxin, indicating involvement of either Gi or Go. Genistein completely inhibited the agonist-induced phospholipase D activity and also substantially reduced the basal level of phospholipase D activity. Pretreatment with either the alpha 1-adrenoceptor antagonist prazosin or the alpha 2-adrenoceptor antagonist rauwolscine was also effective in eliminating the agonist-induced increase of phospholipase D. These results indicate that a tyrosine kinase-regulated phospholipase D plays a critical role in alpha-adrenoceptor-induced contractions of the rat aorta and that stimulation of both alpha 1- and alpha 2-adrenoceptors is essential to allow phospholipase activation.

  6. Identification of the Elusive Mammalian Enzyme Phospatidylcholine-Specific Phospholipase C

    DTIC Science & Technology

    2015-01-01

    mammalian protein, phosphatidycholine- specific phospholipase C (PC-PLC) in the inflammatory processes involved in progression of rheumatoid arthritis (RA...serum, rheumatoid arthritis , transcriptome sequencing, HUVECs, U937 cells 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...aims at identifying novel players that are critically involved in the progression of rheumatoid arthritis (RA). The identification of these factors

  7. Activation of phospholipase A/sub 2/ by carbon tetrachloride in isolated rat hepatocytes

    SciTech Connect

    Glende, E.A. Jr.; Pushpendran, C.K.

    1986-03-05

    Freshly isolated rat hepatocytes were incubated with /sup 3/H-arachidonic acid or /sup 14/C-ethanolamine for 1 hour in order to label cellular lipids. Thin-layer chromatographic analysis indicated that of the /sup 3/H-arachidonate incorporated into lipid nearly 50% was found in phosphatidylcholine and 15% in phosphatidylethanolamine. /sup 14/C-Ethanolamine was incorporated mainly into phosphatidylethanolamine. Hepatocytes labeled as such were exposed to carbon tetrachloride (CCl/sub 4/) for periods up to 4 hours. Phospholipase A/sub 2/ of these preparations was determined by measuring either the release of /sup 4/H-arachidonic acid from cellular phospholipids prelabeled with /sup 3/H-arachidonic acid or measuring the formation of /sup 14/C-lysophosphatidylethanolamine from cellular lipids prelabeled with /sup 14/C-ethanolamine. Through the use of hexane-partition extraction and thin-layer chromatographic analysis of hepatocyte lipid extracts it was found that CCl, stimulated phospholipase A/sub 2/ activity in a dose- an time-dependent manner. Carbon tetrachloride at concentrations of 0.23 to 1.3 mM produced a 1.4- to 5.3-fold increase in phospholipase activity which was initiated within 30 to 60 minutes of incubation at 37/sup 0/. A role for phospholipase activation as a secondary mechanism of CCl/sub 4/-induced hepatocyte injury is proposed.

  8. Characteristics and Lethality of a Novel Recombinant Dermonecrotic Venom Phospholipase D from Hemiscorpius lepturus

    PubMed Central

    Torabi, Elham; Behdani, Mahdi; Hosseininejad Chafi, Mohammad; Moazzami, Reza; Sabatier, Jean-Marc; Khalaj, Vahid; Shahbazzadeh, Delavar; Pooshang Bagheri, Kamran

    2017-01-01

    Hemoscorpius lepturus is the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation are remarkably similar to those reported for brown spiders, including dermonecrosis, hematuria, renal failure and even death. The lethality and toxicity of brown spiders’ venom have been attributed to its phospholipase D activity. This study aims to identify a phospholipase D with possible lethality and dermonecrotic activity in H. lepturus venom. In this study, a cDNA library of the venom glands was generated by Illumina RNA sequencing. Phospholipase D (PLD) from H. lepturus was characterized according to its significant similarity with PLDs from brown spiders. The main chain designated as Hl-RecPLD1 (the first recombinant isoform of H. lepturus PLD) was cloned, expressed and purified. Sphingomyelinase, dermonecrotic and lethal activities were examined. Hl-PLD1 showed remarkable sequence similarity and structural homology with PLDs of brown spiders. The conformation of Hl-PLD1 was predicted as a “TIM beta/alpha-barrel”. The lethal dose 50 (LD50) and dermonecrotic activities of Hl-RecPLD1 were determined as 3.1 µg/mouse and 0.7 cm2 at 1 µg respectively. It is the first report indicating that a similar molecular evolutionary mechanism has occurred in both American brown spiders and this Iranian scorpion. In conclusion, Hl-RecPLD1 is a highly active phospholipase D, which would be considered as the lethal dermonecrotic toxin in H. lepturus venom. PMID:28335389

  9. Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.; Shansky, Janet; Karlisch, Patricia; Solerssi, Rosa Lopez

    1991-01-01

    Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins E2 and F2(alpha) which regulate protein turnover rates and muscle cell growth. Mechnical stimulation significantly increases the breakdown rate of (3)H-arachidonic acid labelled phospholipids, releasing free (3)H-arachidonic acid, and the rate-limiting precursor of prostaglandin synthesis. Mechanical stimulation also significantly increases (3)H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-2-(3)H inositol labelled phospholipids. Phospholipase A2, phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are activated by stretch. The lipase inhibitors bromophenacylbromide and RHC80267 together reduce stretch-induced prostaglandin production by 73-83 percent. The stretch-induced increases in prostaglandin production, (3)H-arachidonic acid labelled phospholipid breakdown, and (3)H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-2-(3)H inositol labelled phospholipids are dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and prostaglandins through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

  10. Role of polyamines and phospholipase D in maize (Zea mays L.) response to drought stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydroponic experiment was conducted to elucidate the role of polyamines and phospholipase D (PLD) in regulating response of maize plants to drought stress (DS). During the early stage of DS, an increase in PLD activity, independent of polyamines contents, was mainly responsible for stomatal closure...

  11. Chemoenzymatic synthesis of fluorogenic phospholipids and evaluation in assays of phospholipases A, C and D.

    PubMed

    Piel, Mathilde S; Peters, Günther H J; Brask, Jesper

    2017-01-01

    Phospholipases are ubiquitous in nature and the target of significant research aiming at both their physiological roles and technical applications in e.g. the food industry. In the search for sensitive and selective phospholipase assays, we have focused on synthetic FRET (Förster resonance energy transfer) substrates. This has led to the development of a facile, easily scalable and low cost synthesis of fluorogenic phospholipids featuring the dansyl/dabcyl fluorophore/quencher-pair on the fatty acid ω-position and on the phosphatidylethanolamine head group, respectively. Hence, the two substrates lyso-(dansyl-FA)-GPE-dabcyl (6) and (dansyl-FA)2-GPE-dabcyl (7) were synthesized by a chemoenzymatic strategy, in which preparation of (6) further included a novel selective enzymatic esterification step. As proof of concept, activity of a handful of phospholipases, one from each of the PLA1, PLA2, PLC and PLD classes, were assayed using substrates (6) and (7), and the kinetic parameter kcat/KM was determined. The PLA1 (Lecitase Ultra™) was found to be highly active on both substrates, whereas the PLD (from white cabbage) had no activity, presumably due to steric effects associated with the dabcyl-functionalization of the head group. It was further substantiated that the substrates are specific towards phospholipase activity as the tested lipase (Lipolase™) showed close to zero activity.

  12. Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

    PubMed Central

    Ishida, K.; Alviano, D.S.; Silva, B.G.; Guerra, C.R.; Costa, A.S.; Nucci, M.; Alviano, C.S.; Rozental, S.

    2012-01-01

    Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes. PMID:22415116

  13. The selective activation of the cardiac sarcolemmal sodium-calcium exchanger by plasmalogenic phosphatidic acid produced by phospholipase D.

    PubMed

    Hale, C C; Ebeling, E G; Hsu, F F; Ford, D A

    1998-01-30

    Since plasmalogens are the predominant phospholipid of cardiac sarcolemma, the activation of the sodium-calcium exchanger by either plasmenylethanolamine or plasmalogenic phosphatidic acid generated by phospholipase D was explored. Sodium-calcium exchange activity was 7-fold greater in proteoliposomes comprised of plasmenylethanolamine compared to proteoliposomes comprised of only plasmenylcholine. Phospholipase D treatment of proteoliposomes resulted in 1 mol % conversion of plasmenylcholine or phosphatidylcholine to their respective phosphatidic acid molecular species with a concomitant 8-fold or 2-fold activation of sodium-calcium exchange activity, respectfully. Thus, phospholipase D-mediated hydrolysis of plasmalogens to phosphatidic acid may be an important mechanism for the regulation of the sodium-calcium exchanger.

  14. [Simplified microdetermination of cerebral phospholipase A1, A2 and lysophopholipase].

    PubMed

    Hirashima, Y; Koshu, K; Kamiyama, K; Endo, S; Takaku, A; Honda, T; Takasaki, C

    1983-08-01

    The purpose of our study was to examine the ischemia induced enzymatic changes of decaylation-reacylation cycle of membrane phospholipids in dog brain. In this study, we developed new modified method for assay of phospholipase A1, A2 and lysophospholipase which is simpler and needs only a smaller amount of materials. For the first report, we introduced this new method and demonstrated some properties of phospholipase A1, A2 and lysophospholipase in dog brain. Crude enzyme solution for assays of phospholipase A1, A2 and lysophospholipase was gained from extraction of frozen brain with aceton, butanol and saline. The level of phosphorus in the enzyme extract was determined and only those extracts which had a level of phosphorus within a certain range were used. The substrates for assays were L-alpha-[beta-palmitoyl-1-14C] phosphatidylcholine, dipalmitoyl for phospholipase A1 and A2 and L-lysophosphatidylcholine-1-[1-14C] palmitoyl for lysophospolipase respectively. Each radioactive substrates was diluted with cold carrier lipid to give the proper specific activity. Reaction system including substrate, buffer [pH 7.0] and enzyme extract was incubated for 10 hours at 38 degrees C. But for the assay of phospholipase A1 and A2, enzyme solution was pre-incubated at 70 degrees C for 5 minutes. In our new method, reaction mixture was directly separated by TLC without extracting lipids. Enzyme activities were calculated from radio thin-layer chromatograms. Furthermore, we made a comparison between our method and the former one. The value of each enzyme activity was slightly higher in our method than in the former one. However, it was revealed that the results were reproducible in both methods.

  15. Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection.

    PubMed

    Vijay, Rahul; Hua, Xiaoyang; Meyerholz, David K; Miki, Yoshimi; Yamamoto, Kei; Gelb, Michael; Murakami, Makoto; Perlman, Stanley

    2015-10-19

    Oxidative stress and chronic low-grade inflammation in the lungs are associated with aging and may contribute to age-related immune dysfunction. To maintain lung homeostasis, chronic inflammation is countered by enhanced expression of proresolving/antiinflammatory factors. Here, we show that age-dependent increases of one such factor in the lungs, a phospholipase A2 (PLA2) group IID (PLA2G2D) with antiinflammatory properties, contributed to worse outcomes in mice infected with severe acute respiratory syndrome-coronavirus (SARS-CoV). Strikingly, infection of mice lacking PLA2G2D expression (Pla2g2d(-/-) mice) converted a uniformly lethal infection to a nonlethal one (>80% survival), subsequent to development of enhanced respiratory DC migration to the draining lymph nodes, augmented antivirus T cell responses, and diminished lung damage. We also observed similar effects in influenza A virus-infected middle-aged Pla2g2d(-/-) mice. Furthermore, oxidative stress, probably via lipid peroxidation, was found to induce PLA2G2D expression in mice and in human monocyte-derived macrophages. Thus, our results suggest that directed inhibition of a single inducible phospholipase, PLA2G2D, in the lungs of older patients with severe respiratory infections is potentially an attractive therapeutic intervention to restore immune function.

  16. Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome–CoV infection

    PubMed Central

    Vijay, Rahul; Hua, Xiaoyang; Meyerholz, David K.; Miki, Yoshimi; Yamamoto, Kei; Gelb, Michael; Murakami, Makoto

    2015-01-01

    Oxidative stress and chronic low-grade inflammation in the lungs are associated with aging and may contribute to age-related immune dysfunction. To maintain lung homeostasis, chronic inflammation is countered by enhanced expression of proresolving/antiinflammatory factors. Here, we show that age-dependent increases of one such factor in the lungs, a phospholipase A2 (PLA2) group IID (PLA2G2D) with antiinflammatory properties, contributed to worse outcomes in mice infected with severe acute respiratory syndrome-coronavirus (SARS-CoV). Strikingly, infection of mice lacking PLA2G2D expression (Pla2g2d−/− mice) converted a uniformly lethal infection to a nonlethal one (>80% survival), subsequent to development of enhanced respiratory DC migration to the draining lymph nodes, augmented antivirus T cell responses, and diminished lung damage. We also observed similar effects in influenza A virus–infected middle-aged Pla2g2d−/− mice. Furthermore, oxidative stress, probably via lipid peroxidation, was found to induce PLA2G2D expression in mice and in human monocyte–derived macrophages. Thus, our results suggest that directed inhibition of a single inducible phospholipase, PLA2G2D, in the lungs of older patients with severe respiratory infections is potentially an attractive therapeutic intervention to restore immune function. PMID:26392224

  17. The Antimicrobial Activity of an Acidic Phospholipase A₂ (NN-XIa-PLA₂) from the Venom of Naja naja naja (Indian Cobra).

    PubMed

    Sudarshan, S; Dhananjaya, B L

    2015-08-01

    Microbial resistance against antibiotics is considered as a potentially serious threat to public health. Therefore, there is much interest in developing new molecules with novel modes of action. In this study, when antimicrobial potential of an acidic protein-NN-XIa-PLA2 (Naja naja venom phospholipase A2 fraction-XIa) of N. naja venom was evaluated, it demonstrated potent bactericidal action against the human pathogenic strains. It inhibited more significantly, the gram-positive bacteria, when compared to gram-negative bacteria. The minimum inhibitory concentration (MIC) values ranged from 17 to 20 μg/ml. It was interesting to observe that the NN-XIa-PLA2 showed comparable antibacterial activity to the standard antibiotics used. It was found that there was a strong correlation between phospholipase A2 (PLA2) activities, hemolytic, and antimicrobial activity. Further, it is found that in the presence of p-bromophenacyl bromide (p-BPB), there is a significant decrease in enzymatic activity and associated antimicrobial activities, suggesting that a strong correlation exists between catalytic activity and antimicrobial effects, which thereby destabilize the membrane bilayer. However, other mechanisms cannot be completely ruled out. Thus, these studies encourage further in-depth study on molecular mechanisms of antibacterial properties and thereby help in development of this protein into a possible therapeutic lead molecule for treating bacterial infections.

  18. Cytosolic Phospholipase A2 and Lysophospholipids in Tumor Angiogenesis

    PubMed Central

    Linkous, Amanda G.

    2010-01-01

    Background Lung cancer and glioblastoma multiforme are highly angiogenic and, despite advances in treatment, remain resistant to therapy. Cytosolic phospholipase A2 (cPLA2) activation contributes to treatment resistance through transduction of prosurvival signals. We investigated cPLA2 as a novel molecular target for antiangiogenesis therapy. Methods Glioblastoma (GL261) and Lewis lung carcinoma (LLC) heterotopic tumor models were used to study the effects of cPLA2 expression on tumor growth and vascularity in C57/BL6 mice wild type for (cPLA2α+/+) or deficient in (cPLA2α−/−) cPLA2α, the predominant isoform in endothelium (n = 6–7 mice per group). The effect of inhibiting cPLA2 activity on GL261 and LLC tumor growth was studied in mice treated with the chemical cPLA2 inhibitor 4-[2-[5-chloro-1-(diphenylmethyl)-2-methyl-1H-indol-3-yl]-ethoxy]benzoic acid (CDIBA). Endothelial cell proliferation and function were evaluated by Ki-67 immunofluorescence and migration assays in primary cultures of murine pulmonary microvascular endothelial cells (MPMEC) isolated from cPLA2α+/+ and cPLA2α−/− mice. Proliferation, invasive migration, and tubule formation were assayed in mouse vascular endothelial 3B-11 cells treated with CDIBA. Effects of lysophosphatidylcholine, arachidonic acid, and lysophosphatidic acid (lipid mediators of tumorigenesis and angiogenesis) on proliferation and migration were examined in 3B-11 cells and cPLA2α−/− MPMEC. All statistical tests were two-sided. Results GL261 tumor progression proceeded normally in cPLA2α+/+ mice, whereas no GL261 tumors formed in cPLA2α−/− mice. In the LLC tumor model, spontaneous tumor regression was observed in 50% of cPLA2α−/− mice. Immunohistochemical examination of the remaining tumors from cPLA2α−/− mice revealed attenuated vascularity (P ≤ .001) compared with tumors from cPLA2α+/+ mice. Inhibition of cPLA2 activity by CDIBA resulted in a delay in tumor growth (eg, LLC model: average

  19. Cloning, expression, purification and characterization of patatin, a novel phospholipase A.

    PubMed

    Hirschberg, H J; Simons, J W; Dekker, N; Egmond, M R

    2001-10-01

    Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.

  20. Matrix Metalloproteinase‐2 Negatively Regulates Cardiac Secreted Phospholipase A2 to Modulate Inflammation and Fever

    PubMed Central

    Berry, Evan; Hernandez‐Anzaldo, Samuel; Ghomashchi, Farideh; Lehner, Richard; Murakami, Makoto; Gelb, Michael H.; Kassiri, Zamaneh; Wang, Xiang; Fernandez‐Patron, Carlos

    2015-01-01

    Background Matrix metalloproteinase (MMP)‐2 deficiency makes humans and mice susceptible to inflammation. Here, we reveal an MMP‐2–mediated mechanism that modulates the inflammatory response via secretory phospholipase A2 (sPLA2), a phospholipid hydrolase that releases fatty acids, including precursors of eicosanoids. Methods and Results Mmp2−/− (and, to a lesser extent, Mmp7−/− and Mmp9−/−) mice had between 10‐ and 1000‐fold elevated sPLA2 activity in plasma and heart, increased eicosanoids and inflammatory markers (both in the liver and heart), and exacerbated lipopolysaccharide‐induced fever, all of which were blunted by adenovirus‐mediated MMP‐2 overexpression and varespladib (pharmacological sPLA2 inhibitor). Moreover, Mmp2 deficiency caused sPLA2‐mediated dysregulation of cardiac lipid metabolic gene expression. Compared with liver, kidney, and skeletal muscle, the heart was the single major source of the Ca2+‐dependent, ≈20‐kDa, varespladib‐inhibitable sPLA2 that circulates when MMP‐2 is deficient. PLA2G5, which is a major cardiac sPLA2 isoform, was proinflammatory when Mmp2 was deficient. Treatment of wild‐type (Mmp2+/+) mice with doxycycline (to inhibit MMP‐2) recapitulated the Mmp2−/− phenotype of increased cardiac sPLA2 activity, prostaglandin E2 levels, and inflammatory gene expression. Treatment with either indomethacin (to inhibit cyclooxygenase‐dependent eicosanoid production) or varespladib (which inhibited eicosanoid production) triggered acute hypertension in Mmp2−/− mice, revealing their reliance on eicosanoids for blood pressure homeostasis. Conclusions A heart‐centric MMP‐2/sPLA2 axis may modulate blood pressure homeostasis, inflammatory and metabolic gene expression, and the severity of fever. This discovery helps researchers to understand the cardiovascular and systemic effects of MMP‐2 inhibitors and suggests a disease mechanism for human MMP‐2 gene deficiency. PMID:25820137

  1. Apolipoprotein CIII regulates lipoprotein-associated phospholipase A2 expression via the MAPK and NFκB pathways

    PubMed Central

    Han, Xiaolei; Wang, Tiedong; Zhang, Jifeng; Liu, Xingxing; Li, Zhuang; Wang, Gangqi; Song, Qi; Pang, Daxin; Ouyang, Hongsheng; Tang, Xiaochun

    2015-01-01

    Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo. PMID:25836672

  2. Bee venom phospholipase A2 ameliorates motor dysfunction and modulates microglia activation in Parkinson's disease alpha-synuclein transgenic mice

    PubMed Central

    Ye, Minsook; Chung, Hwan-Suck; Lee, Chanju; Hyun Song, Joo; Shim, Insop; Kim, Youn-Sub; Bae, Hyunsu

    2016-01-01

    α-Synuclein (α-Syn) has a critical role in microglia-mediated neuroinflammation, which leads to the development of Parkinson's disease (PD). Recent studies have shown that bee venom (BV) has beneficial effects on PD symptoms in human patients or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin-induced PD mice. This study investigated whether treatment with BV-derived phospholipase A2 (bvPLA2) would improve the motor dysfunction and pathological features of PD in human A53T α-Syn mutant transgenic (A53T Tg) mice. The motor dysfunction of A53T Tg mice was assessed using the pole test. The levels of α-Syn, microglia and the M1/M2 phenotype in the spinal cord were evaluated by immunofluorescence. bvPLA2 treatment significantly ameliorated motor dysfunction in A53T Tg mice. In addition, bvPLA2 significantly reduced the expression of α-Syn, the activation and numbers of microglia, and the ratio of M1/M2 in A53T Tg mice. These results suggest that bvPLA2 could be a promising treatment option for PD. PMID:27388550

  3. Apolipoprotein CIII regulates lipoprotein-associated phospholipase A2 expression via the MAPK and NFκB pathways.

    PubMed

    Han, Xiaolei; Wang, Tiedong; Zhang, Jifeng; Liu, Xingxing; Li, Zhuang; Wang, Gangqi; Song, Qi; Pang, Daxin; Ouyang, Hongsheng; Tang, Xiaochun

    2015-04-02

    Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo.

  4. Novel phospholipase A2 inhibitors from python serum are potent peptide antibiotics.

    PubMed

    Samy, Ramar Perumal; Thwin, Maung Maung; Stiles, Brad G; Satyanarayana-Jois, Seetharama; Chinnathambi, Arunachalam; Zayed, M E; Alharbi, Sulaiman Ali; Siveen, Kodappully Sivaraman; Sikka, Sakshi; Kumar, Alan Prem; Sethi, Gautam; Lim, Lina Hsiu Kim

    2015-04-01

    Antimicrobial peptides (AMPs) play a vital role in defense against resistant bacteria. In this study, eight different AMPs synthesized from Python reticulatus serum protein were tested for bactericidal activity against various Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Burkholderia pseudomallei (KHW and TES strains), and Proteus vulgaris) using a disc-diffusion method (20 μg/disc). Among the tested peptides, phospholipase A2 inhibitory peptide (PIP)-18[59-76], β-Asp65-PIP[59-67], D-Ala66-PNT.II, and D60,65E-PIP[59-67] displayed the most potent bactericidal activity against all tested pathogens in a dose-dependent manner (100-6.8 μg/ml), with a remarkable activity noted against S. aureus at 6.8 μg/ml dose within 6 h of incubation. Determination of minimum inhibitory concentrations (MICs) by a micro-broth dilution method at 100-3.125 μg/ml revealed that PIP-18[59-76], β-Asp65-PIP[59-67] and D-Ala66-PNT.II peptides exerted a potent inhibitory effect against S. aureus and B. pseudomallei (KHW) (MICs 3.125 μg/ml), while a much less inhibitory potency (MICs 12.5 μg/ml) was noted for β-Asp65-PIP[59-67] and D-Ala66-PNT.II peptides against B. pseudomallei (TES). Higher doses of peptides had no effect on the other two strains (i.e., Klebsiella pneumoniae and Streptococcus pneumoniae). Overall, PIP-18[59-76] possessed higher antimicrobial activity than that of chloramphenicol (CHL), ceftazidime (CF) and streptomycin (ST) (30 μg/disc). When the two most active peptides, PIP-18[59-76] and β-Asp65-PIP[59-67], were applied topically at a 150 mg/kg dose for testing wound healing activity in a mouse model of S. aureus infection, the former accelerates faster wound healing than the latter peptide at 14 days post-treatment. The western blot data suggest that the topical application of peptides (PIP-18[59-67] and β-Asp65-PIP[59-67]) modulates NF-kB mediated wound repair in mice with relatively little haemolytic (100-1.56 μg/ml) and cytotoxic (1000

  5. A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.

    PubMed

    Shashidhar, M S; Volwerk, J J; Griffith, O H; Keana, J F

    1991-12-01

    A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.

  6. Phospholipase A/sub 2/ stimulation during cell secretion in rat basophilic leukemia cells

    SciTech Connect

    Garcia-Gil, M.; Siraganian, R.P.

    1986-01-01

    The bridging of IgE receptors on rat basophilic leukemia cells (RBL-2H3) results in a number of biochemical events that accompany histamine secretion. Prominent among these is the release of arachidonic acid from cellular phospholipids, which could be due to the activation of phospholipase enzymes. In the present experiments they studied the intracellular activation of phospholipase A/sub 2/ (PLA/sub 2/) during histamine release. The enzyme in the homogenates was capable of cleaving arachidonic acid from different phospholipids. The production of lysophospholipids could play a critical role in histamine release from cells. These results demonstrate the activation of PLA/sub 2/ enzyme in cellular homogenates during the secretory process.

  7. PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination.

    PubMed

    Kim, Jun-Dal; Park, Kyung-Eui; Ishida, Junji; Kako, Koichiro; Hamada, Juri; Kani, Shuichi; Takeuchi, Miki; Namiki, Kana; Fukui, Hajime; Fukuhara, Shigetomo; Hibi, Masahiko; Kobayashi, Makoto; Kanaho, Yasunori; Kasuya, Yoshitoshi; Mochizuki, Naoki; Fukamizu, Akiyoshi

    2015-12-01

    The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8 (-/-)) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8 (-/-) mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8 (-/-) mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.

  8. Antibacterial properties of intestinal phospholipase A2 from the common stingray Dasyatis pastinaca.

    PubMed

    Ben Bacha, Abir; Abid, Islem; Horchani, Habib

    2012-11-01

    Stingray phospholipase A(2) group IIA (SPLA(2)-IIA) was recently isolated and purified to homogeneity from the intestine of the common stingray Dasyatis pastinaca, suggesting that this enzyme plays an important role in systemic bactericidal defense. The present study showed that SPLA(2)-IIA was highly bactericidal against Gram-positive bacteria with inhibition zones and minimal inhibitory concentration values in the range of 13-25 mm and 2-8 μg/ml, respectively, whereas Gram-negative bacteria exhibited a much higher resistance. The bactericidal efficiency of SPLA(2)-IIA was shown to be unaffected by high protein and salt concentrations, but dependent upon the presence of calcium ions, and then correlated to the hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray phospholipase A(2) group IIA presents no cytotoxicity after its incubation with MDA-MB-231 cells. SPLA(2)-IIA may be considered as a future therapeutic agent against bacterial infections.

  9. Expression of a cytosolic phospholipase A2 by ovine endometrium on days 11-14 of a simulated oestrous cycle.

    PubMed

    Graf, G A; Burns, P D; Silvia, W J

    1999-03-01

    Oxytocin stimulates the synthesis and secretion of PGF2 alpha from uterine tissues in vivo and in vitro late in the ovine oestrous cycle. The synthesis of eicosanoids is dependent upon the availability of free arachidonic acid which is released through the activity of arachidonate releasing phospholipases. In the present study, the following hypothesis was tested: the ovine endometrium expresses a cytosolic phospholipase A2 (cPLA2) and expression or activity of cPLA2 increases as uterine secretory responsiveness to oxytocin develops late in the oestrous cycle. Endometrial tissue was collected from cyclic ewes on day 15 of the oestrous cycle for the preparation of tissue homogenates and isolation of mRNA to determine whether ovine endometrium expressed a cPLA2. A 110 kDa band was detected by western blotting, indicating the presence of a putative ovine cPLA2. A 834 bp fragment of the ovine cPLA2 shared 87% homology with human and mouse cDNA, and northern blot hybridization analysis indicated a single 3.4 kb transcript. A total of 20 ewes were ovariectomized and treated with progesterone and oestrogen to simulate the oestrous cycle to determine whether the expression or activity of ovine cPLA2 changed during the onset of uterine secretory responsiveness to oxytocin in vivo. On days 11-14 (n = 5 per day) of a simulated oestrous cycle, caruncular endometrium was evaluated for expression of ovine cPLA2 mRNA and protein and the synthesis of PGF2 alpha in response to melittin (a potent stimulator of PLA2 activity). Immunoreactive cPLA2 and cPLA2 mRNA were observed on all days and did not increase during the development of uterine responsiveness to oxytocin in vivo. Similarly, melittin increased the synthesis of PGF2 alpha irrespective of day, indicating the presence of a functional cPLA2 on all days examined. These data indicate that the ovine endometrium expresses a functional cPLA2 and that ample concentrations of cPLA2 are present by day 11 of a simulated oestrous

  10. Structural Basis for Different Phosphoinositide Specificities of the PX Domains of Sorting Nexins Regulating G-protein Signaling*

    PubMed Central

    Mas, Caroline; Norwood, Suzanne J.; Bugarcic, Andrea; Kinna, Genevieve; Leneva, Natalya; Kovtun, Oleksiy; Ghai, Rajesh; Ona Yanez, Lorena E.; Davis, Jasmine L.; Teasdale, Rohan D.; Collins, Brett M.

    2014-01-01

    Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Gαs-coupled G protein-coupled receptor signaling. Here we delineate the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids and we find that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. Our findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking. PMID:25148684

  11. Structural basis for different phosphoinositide specificities of the PX domains of sorting nexins regulating G-protein signaling.

    PubMed

    Mas, Caroline; Norwood, Suzanne J; Bugarcic, Andrea; Kinna, Genevieve; Leneva, Natalya; Kovtun, Oleksiy; Ghai, Rajesh; Ona Yanez, Lorena E; Davis, Jasmine L; Teasdale, Rohan D; Collins, Brett M

    2014-10-10

    Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Gαs-coupled G protein-coupled receptor signaling. Here we delineate the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids and we find that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. Our findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking.

  12. Identification of the Elusive Mammalian Enzyme Phosphatidylcholine-Specific Phospholipase C

    DTIC Science & Technology

    2014-07-01

    processes involved in progression of rheumatoid arthritis (RA). Thus, the main scopes of this proposal are: 1. to identify the PC-PLC gene and protein...of PC-PLC. 15. SUBJECT TERMS Phosphatidycholine-specific phospholipase C, lipopolisaccharide, oxidized lipoproteins, serum, rheumatoid arthritis ...present proposal aims at identifying novel players that are critically involved in the progression of rheumatoid arthritis (RA). The identification of

  13. Dependence of stimulus-transcription coupling on phospholipase D in agonist-stimulated pituitary cells.

    PubMed Central

    Cesnjaj, M; Zheng, L; Catt, K J; Stojilkovic, S S

    1995-01-01

    Stimulation of phospholipase D activity is frequently observed during agonist activation of Ca(2+)-mobilizing receptors, but the cellular functions of this signaling pathway are not well defined. Pituitary gonadotrophs express Ca(2+)-mobilizing receptors for gonadotropin-releasing hormone (GnRH) and endothelin (ET), activation of which stimulates luteinizing hormone secretion and transient expression of c-fos. In pituitary cells and alpha T3-1 gonadotrophs, GnRH action was associated with both initial and sustained diacylglycerol (DG) production, whereas ET-1 induced only a transient DG response. Also, phospholipase D activity, estimated by the production of phosphatidylethanol from phosphatidylcholine in the presence of ethanol, was stimulated by GnRH but not ET-1. Such formation of phosphatidylethanol at the expense of phosphatidic acid (PA) during GnRH-induced activation of phospholipase D significantly reduced the production of PA, DG, and cytidine diphosphate diacylglycerol. Inhibition of PA-phosphohydrolase activity by propranolol also decreased GnRH-induced DG production and, in contrast to ethanol, increased PA and cytidine diphosphate diacylglycerol levels. The fall in DG production caused by ethanol and propranolol was accompanied by inhibition of GnRH-induced c-fos expression, whereas agonist-induced luteinizing hormone release was not affected. In contrast to their inhibitory actions on GnRH-induced early gene expression, neither ethanol nor propranolol affected ET-1-induced c-fos expression, or GnRH- and ET-1-induced inositol trisphosphate/Ca2+ signaling. These findings demonstrate that phospholipase D participates in stimulus-transcription but not stimulus-secretion coupling, and indicate that DG is the primary signal for this action. Images PMID:7579706

  14. Quantum dot-NBD-liposome luminescent probes for monitoring phospholipase A2 activity.

    PubMed

    Kethineedi, Venkata R; Crivat, Georgeta; Tarr, Matthew A; Rosenzweig, Zeev

    2013-12-01

    In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot-fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C6-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A2 as low as 0.0075 U mL(-1) and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A2 inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).

  15. Study of the Role of Cytosolic Phospholipase A2 Alpha in Eicosanoid Generation and Thymocyte Maturation in the Thymus

    PubMed Central

    Rousseau, Matthieu; Naika, Gajendra S.; Perron, Jean; Jacques, Frederic; Gelb, Michael H.; Boilard, Eric

    2015-01-01

    The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α. PMID:25969996

  16. Study of the role of cytosolic phospholipase A2 alpha in eicosanoid generation and thymocyte maturation in the thymus.

    PubMed

    Rousseau, Matthieu; Naika, Gajendra S; Perron, Jean; Jacques, Frederic; Gelb, Michael H; Boilard, Eric

    2015-01-01

    The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.

  17. mu-opioid receptor-stimulated synthesis of reactive oxygen species is mediated via phospholipase D2.

    PubMed

    Koch, Thomas; Seifert, Anja; Wu, Dai-Fei; Rankovic, Marija; Kraus, Jürgen; Börner, Christine; Brandenburg, Lars-Ove; Schröder, Helmut; Höllt, Volker

    2009-08-01

    We have recently shown that the activation of the rat mu-opioid receptor (MOPr, also termed MOR1) by the mu-agonist [D-Ala(2), Me Phe(4), Glyol(5)]enkephalin (DAMGO) leads to an increase in phospholipase D2 (PLD2) activity and an induction of receptor endocytosis, whereas the agonist morphine which does not induce opioid receptor endocytosis fails to activate PLD2. We report here that MOPr-mediated activation of PLD2 stimulates production of reactive oxygen molecules via NADH/NADPH oxidase. Oxidative stress was measured with the fluorescent probe dichlorodihydrofluorescein diacetate and the role of PLD2 was assessed by the PLD inhibitor D-erythro-sphingosine (sphinganine) and by PLD2-small interfering RNA transfection. To determine whether NADH/NADPH oxidase contributes to opioid-induced production of reactive oxygen species, mu-agonist-stimulated cells were pre-treated with the flavoprotein inhibitor, diphenylene iodonium, or the specific NADPH oxidase inhibitor, apocynin. Our results demonstrate that receptor-internalizing agonists (like DAMGO, beta-endorphin, methadone, piritramide, fentanyl, sufentanil, and etonitazene) strongly induce NADH/NADPH-mediated ROS synthesis via PLD-dependent signaling pathways, whereas agonists that do not induce MOPr endocytosis and PLD2 activation (like morphine, buprenorphine, hydromorphone, and oxycodone) failed to activate ROS synthesis in transfected human embryonic kidney 293 cells. These findings indicate that the agonist-selective PLD2 activation plays a key role in the regulation of NADH/NADPH-mediated ROS formation by opioids.

  18. A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis.

    PubMed

    Boilard, Eric; Lai, Ying; Larabee, Katherine; Balestrieri, Barbara; Ghomashchi, Farideh; Fujioka, Daisuke; Gobezie, Reuben; Coblyn, Jonathan S; Weinblatt, Michael E; Massarotti, Elena M; Thornhill, Thomas S; Divangahi, Maziar; Remold, Heinz; Lambeau, Gérard; Gelb, Michael H; Arm, Jonathan P; Lee, David M

    2010-05-01

    Phospholipase A2 (PLA2) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the pro-inflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation.

  19. A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis

    PubMed Central

    Boilard, Eric; Lai, Ying; Larabee, Katherine; Balestrieri, Barbara; Ghomashchi, Farideh; Fujioka, Daisuke; Gobezie, Reuben; Coblyn, Jonathan S; Weinblatt, Michael E; Massarotti, Elena M; Thornhill, Thomas S; Divangahi, Maziar; Remold, Heinz; Lambeau, Gérard; Gelb, Michael H; Arm, Jonathan P; Lee, David M

    2010-01-01

    Phospholipase A2 (PLA2) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the pro-inflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation. PMID:20432503

  20. The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri.

    PubMed

    Wang, Xia; Jiang, Feng; Zheng, Jianhua; Chen, Lihong; Dong, Jie; Sun, Lilian; Zhu, Yafang; Liu, Bo; Yang, Jian; Yang, Guowei; Jin, Qi

    2016-09-01

    Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

  1. Activation of phospholipase C in SH-SY5Y neuroblastoma cells by potassium-induced calcium entry.

    PubMed Central

    Smart, D.; Wandless, A.; Lambert, D. G.

    1995-01-01

    1. We used SH-SY5Y human neuroblastoma cells to investigate whether depolarization with high K+ could stimulate inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) formation and, if so, the mechanism involved. 2. Ins(1,4,5)P3 was measured by a specific radioreceptor mass assay, whilst [Ca2+]i was measured fluorimetrically with the Ca2+ indicator dye, Fura-2. 3. Depolarization with K+ caused a time- and dose-dependent increase in [Ca2+]i (peak at 27 s, EC50 of 50.0 +/- 9.0 mM) and Ins(1,4,5)P3 formation (peak at 30 s, EC50 of 47.4 +/- 1.1 mM). 4. Both the K(+)-induced Ins(1,4,5)P3 formation and increase in [Ca2+]i were inhibited dose-dependently by the L-type voltage-sensitive Ca2+ channel closer, (R+)-BayK8644, with IC50 values of 53.4 nM and 87.9 nM respectively. 5. These data show a close temporal and dose-response relationship between Ca2+ entry via L-type voltage-sensitive Ca2+ channels and Ins(1,4,5)P3 formation following depolarization with K+, indicating that Ca2+ influx can activate phospholipase C in SH-SY5Y cells. PMID:8528562

  2. Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin.

    PubMed

    Grisanti, Laurel A; Kurada, Lalitha; Cilz, Nicholas I; Porter, James E; Lei, Saobo

    2012-08-15

    Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.

  3. Cold stress affects H(+)-ATPase and phospholipase D activity in Arabidopsis.

    PubMed

    Muzi, Carlo; Camoni, Lorenzo; Visconti, Sabina; Aducci, Patrizia

    2016-11-01

    Low temperature is an environmental stress that greatly influences plant performance and distribution. Plants exposed to cold stress exhibit modifications of plasma membrane physical properties that can affect their functionality. Here it is reported the effect of low temperature exposure of Arabidopsis plants on the activity of phospholipase D and H(+)-ATPase, the master enzyme located at the plasma membrane. The H(+)-ATPase activity was differently affected, depending on the length of cold stress imposed. In particular, an exposure to 4 °C for 6 h determined the strong inhibition of the H(+)-ATPase activity, that correlates with a reduced association with the regulatory 14-3-3 proteins. A longer exposure first caused the full recovery of the enzymatic activity followed by a significant activation, in accordance with both the increased association with 14-3-3 proteins and induction of H(+)-ATPase gene transcription. Different time lengths of cold stress treatment were also shown to strongly stimulate the phospholipase D activity and affect the phosphatidic acid levels of the plasma membranes. Our results suggest a functional correlation between the activity of phospholipase D and H(+)-ATPase mediated by phosphatidic acid release during the cold stress response.

  4. [The state of phospholipase D in solution and its catalytic activity].

    PubMed

    Rakhimov, M M; Mad'iarov, Sh R

    1977-04-01

    Functioning of water-soluble phospholipase D from cotton seeds is studied on two phases contact area (liquid-liquid, liquid-solid substance) and on the surface of mixed lecitine and sodium dodecylsulphate micelles. It is found that water-soluble phospholipase D, which normally has no catalytic activity, is capable to hydrolyse its substrates in the presence of organic solvents, solid adsorbents and sodium dodecylsulphate. The data obtained show that in all the cases studied the activation observed is due to adsorption immobilization of the enzyme. K lambda and K alpha constants are introduced, which are characteristics of immobilyzing ability of agents-matrices for immobilization. Phase transitions, which take place in heterogenous system (enzyme-activator-substrate-water solution), are found to be a necessary condition for the enzyme activation. A hypothesis, that catalytical activity of water-soluble phospholipase D is inherent of the adsorbed enzyme, is discussed on the basis of the data on comparative study of adsorbed and water-soluble enzymes.

  5. Involvement of phospholipases C and D in the defence responses of riboflavin-treated tobacco cells.

    PubMed

    Wang, Lianlian; Zhu, Xiaoping; Liu, Jinwei; Chu, Xiaojing; Jiao, Jiao; Liang, Yuancun

    2013-04-01

    Riboflavin is an activator of defence responses in plants that increases resistance against diseases caused by fungal, oomycete, bacterial and viral pathogens. However, the mechanisms driving defence activation by riboflavin are poorly understood. We investigated the signal transduction pathways of phospholipase C (PLC) and phospholipase D (PLD) in tobacco (Nicotiana tabacum) suspension cells using a pharmacological approach to confirm whether riboflavin-mediated activation of the defence response is dependent on both PLC and PLD. The expression patterns analysed by quantitative reverse transcription-polymerase chain reaction demonstrated that the tobacco PLC and PLD gene families were differentially expressed in riboflavin-treated tobacco cells. PLC and PLD expression accompanied defence responses including the expression of defence response genes (PAL, PR-1a and PR-1b), the production of hydrogen peroxide and the accumulation of the phytoalexin scopoletin in tobacco cells treated with riboflavin. These defence responses were significantly inhibited in the presence of the PLC inhibitor U73122 and the PLD inhibitor 1-butanol; however, inhibitor analogues had no effect. Moreover, treating tobacco cells with phosphatidic acid, a signalling molecule produced by phospholipase catalysis, induced the accumulation of the phytoalexin scopoletin and compensated for the suppressive effects of U73122 and 1-butanol on riboflavin-induced accumulation of the phytoalexin. These results offer pharmacological evidence that PLC and PLD play a role in riboflavin-induced defences of tobacco.

  6. Cryptococcal phospholipase B antigen is not detected in serum of patients infected with Cryptococcus neoformans using a sandwich enzyme-linked immunosorbent assay.

    PubMed

    Wu, Qi Xuan; Chen, Sharon C A; Santangelo, Rosemary T; Martin, Patricia; Malik, Richard; Sorrell, Tania C

    2007-05-01

    Extracellular phospholipase B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL(-1). PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not be detected in serum using a PLB antigen-based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitable for serodiagnosis of active cryptococcal infection is warranted.

  7. 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}.

  8. Phospholipase A2 inhibitors protect against prion and Aβ mediated synapse degeneration

    PubMed Central

    2010-01-01

    Background An early event in the neuropathology of prion and Alzheimer's diseases is the loss of synapses and a corresponding reduction in the level of synaptophysin, a pre-synaptic membrane protein essential for neurotransmission. The molecular mechanisms involved in synapse degeneration in these diseases are poorly understood. In this study the process of synapse degeneration was investigated by measuring the synaptophysin content of cultured neurones incubated with the prion derived peptide (PrP82-146) or with Aβ1-42, a peptide thought to trigger pathogenesis in Alzheimer's disease. A pharmacological approach was used to screen cell signalling pathways involved in synapse degeneration. Results Pre-treatment with phospholipase A2 inhibitors (AACOCF3, MAFP and aristolochic acids) protected against synapse degeneration in cultured cortical and hippocampal neurones incubated with PrP82-146 or Aβ1-42. Synapse degeneration was also observed following the addition of a specific phospholipase A2 activating peptide (PLAP) and the addition of PrP82-146 or Aβ1-42 activated cytoplasmic phospholipase A2 within synapses. Activation of phospholipase A2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B, Hexa-PAF and CV6029) protected against synapse degeneration induced by PrP82-146, Aβ1-42 and PLAP. PAF facilitated the production of prostaglandin E2, which also caused synapse degeneration and pre-treatment with the prostanoid E receptor antagonist AH13205 protected against PrP82-146, Aβ1-42 and PAF induced synapse degeneration. Conclusions Our results are consistent with the hypothesis that PrP82-146 and Aβ1-42trigger abnormal activation of cytoplasmic phospholipase A2 resident within synapses, resulting in elevated levels of PAF and prostaglandin E2that cause synapse degeneration. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse degeneration seen during

  9. 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)

  10. Crystal structures of brain group-VIII phospholipase A2 in nonaged complexes with the organophosphorus nerve agents soman and sarin.

    PubMed

    Epstein, Todd M; Samanta, Uttamkumar; Kirby, Stephen D; Cerasoli, Douglas M; Bahnson, Brian J

    2009-04-21

    Insecticide and nerve agent organophosphorus (OP) compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun, and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process, called "aging", which dealkylates the nerve agent adduct and results in a product that is highly resistant to reactivation by any known means. Here we report the structures of paraoxon, soman, and sarin complexes of group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a nonaged adduct; a stereoselective preference for binding of the P(S)C(S) isomer of soman and the P(S) isomer of sarin was also noted. The stability of the nonaged complexes was corroborated by trypsin digest and electrospray ionization mass spectrometry, which indicates nonaged complexes are formed with diisopropylfluorophosphate, soman, and sarin. The P(S) stereoselectivity for reaction with sarin was confirmed by reaction of racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate each stereoisomer. The P(S) stereoisomers of soman and sarin are known to be the more toxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtained for nonaged complexes of group-VIII phospholipase A2 are compared to those obtained for other serine hydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights can now be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding and hydrolysis functions.

  11. Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. III. Activation of phospholipase A2 in sperm homogenate by delta 9-tetrahydrocannabinol.

    PubMed

    Chang, M C; Berkery, D; Laychock, S G; Schuel, H

    1991-07-25

    Inhibition of the egg jelly induced acrosome reaction by delta 9-tetrahydrocannabinol (THC) is associated with the localized disruption of the nuclear envelope and the formation of lipid deposits in sea urchin sperm. This suggests that THC may activate phospholipase(s) within the sperm. We now report effects of THC on phospholipase A2 activity in homogenates of sea urchin sperm using 1-stearoyl-2-[1-14C]arachidonyl phosphatidylcholine as substrate. The release of radioactive arachidonic acid was measured after a 30-min incubation with the enzyme. In the absence of exogenous Ca2+, 100 microM THC produced a significant (P less than 0.001) increase in phospholipase A2 activity. THC activated phospholipase A2 in a concentration (1-100 microM) and time-dependent (0-30 min) manner. Exogenous calcium (10 mM) significantly augmented basal (P less than 0.001) and THC-stimulated (P less than 0.005) phospholipase A2 activity. Calcium chelators [ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)] inhibited the basal level of phospholipase A2 activity in the sperm homogenate, and prevented the activation of phospholipase A2 by THC. Submicromolar levels of free calcium ions were required for THC stimulation of phospholipase A2. Cannabinol which mimics the effects of THC on the acrosome reaction also activated phospholipase A2 in sperm homogenate. These results suggest that THC may alter lipid metabolism in sperm by activating calcium-dependent phospholipase A2. Putative metabolites derived from this process may inhibit the acrosome reaction and thereby reduce the fertilizing capacity of sea urchin sperm.

  12. The Finding of a Group IIE Phospholipase A2 Gene in a Specified Segment of Protobothrops flavoviridis Genome and Its Possible Evolutionary Relationship to Group IIA Phospholipase A2 Genes

    PubMed Central

    Yamaguchi, Kazuaki; Chijiwa, Takahito; Ikeda, Naoki; Shibata, Hiroki; Fukumaki, Yasuyuki; Oda-Ueda, Naoko; Hattori, Shosaku; Ohno, Motonori

    2014-01-01

    The genes encoding group IIE phospholipase A2, abbreviated as IIE PLA2, and its 5' and 3' flanking regions of Crotalinae snakes such as Protobothrops flavoviridis, P. tokarensis, P. elegans, and Ovophis okinavensis, were found and sequenced. The genes consisted of four exons and three introns and coded for 22 or 24 amino acid residues of the signal peptides and 134 amino acid residues of the mature proteins. These IIE PLA2s show high similarity to those from mammals and Colubridae snakes. The high expression level of IIE PLA2s in Crotalinae venom glands suggests that they should work as venomous proteins. The blast analysis indicated that the gene encoding OTUD3, which is ovarian tumor domain-containing protein 3, is located in the 3' downstream of IIE PLA2 gene. Moreover, a group IIA PLA2 gene was found in the 5' upstream of IIE PLA2 gene linked to the OTUD3 gene (OTUD3) in the P. flavoviridis genome. It became evident that the specified arrangement of IIA PLA2 gene, IIE PLA2 gene, and OTUD3 in this order is common in the genomes of humans to snakes. The present finding that the genes encoding various secretory PLA2s form a cluster in the genomes of humans to birds is closely related to the previous finding that six venom PLA2 isozyme genes are densely clustered in the so-called NIS-1 fragment of the P. flavoviridis genome. It is also suggested that venom IIA PLA2 genes may be evolutionarily derived from the IIE PLA2 gene. PMID:25529307

  13. Candida albicans isolates from a Malaysian hospital exhibit more potent phospholipase and haemolysin activities than non-albicans Candida isolates.

    PubMed

    Chin, V K; Foong, K J; Maha, A; Rusliza, B; Norhafizah, M; Ng, K P; Chong, P P

    2013-12-01

    This study was aimed at determining the phospholipase and haemolysin activity of Candida isolates in Malaysia. A total of 37 Candida clinical isolates representing seven species, Candida albicans (12), Candida tropicalis (8), Candida glabrata (4), Candida parapsilosis (1), Candida krusei (4), Candida orthopsilosis (1) and Candida rugosa (7) were tested. In vitro phospholipase activity was determined by using egg yolk plate assay whereas in vitro haemolysin activity was tested by using blood plate assay on sheep blood Sabouraud's dextrose agar (SDA) enriched with glucose. Phospholipase activity was detected in 75% (9 out of 12) of the C. albicans isolates. Among the 25 non- C. albicans Candida isolates, phospholipase activity was detected in only 24% of these isolates. The phospholipase activity of C. albicans was significantly higher than that of the non- C. albicans Candida isolates (P=0.002). Haemolysin activity was detected in 100% of the C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, and C. orthopsilosis isolates while 75% of the C. krusei isolates and 12.3% of the C. rugosa isolates showed haemolysin activity. The haemolytic activity of C. albicans was significantly higher than that of the non- C. albicans Candida isolates (P=0.0001).The findings in this study indicate that C. albicans isolates in Malaysia may possess greater virulence potential than the non-albicans species.

  14. Cloning, Sequencing, and Role in Virulence of Two Phospholipases (A1 and C) from Mesophilic Aeromonas sp. Serogroup O:34

    PubMed Central

    Merino, Susana; Aguilar, Alicia; Nogueras, Maria Mercedes; Regue, Miguel; Swift, Simon; Tomás, Juan M.

    1999-01-01

    Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5α. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process. PMID:10417167

  15. Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Starkov, Vladislav G; Osipov, Alexey V; Andreeva, Tatyana V; Filkin, Sergey Yu; Gorbacheva, Elena V; Astashev, Maxim E; Tsetlin, Victor I; Utkin, Yuri N

    2014-01-01

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

  16. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    SciTech Connect

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. )

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  17. Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom

    PubMed Central

    Fernandes, Carlos A. H.; Gartuzo, Elaine C. G.; Pagotto, Ivan; Comparetti, Edson J.; Huancahuire-Vega, Salomón; Ponce-Soto, Luis Alberto; Costa, Tássia R.; Marangoni, Sergio; Soares, Andreimar M.; Fontes, Marcos R. M.

    2012-01-01

    Two myotoxic and noncatalytic Lys49-phospholipases A2 (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A2 (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56–2.05 Å and belonged to space groups P3121 (braziliantoxin-II), P6522 (braziliantoxin-III) and P21 (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A2 (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A2 braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A2. PMID:22869126

  18. Involvement of Protein cAMP-dependent Kinase, Phospholipase A2 and Phospholipase C in Sperm Acrosome Reaction of Chinchilla lanigera.

    PubMed

    Gramajo-Bühler, M C; Zelarayán, L; Sánchez-Toranzo, G

    2016-02-01

    The mechanisms involved in fertilization are the centre of attention in order to determine the conditions required to reproduce in vitro the events that take place in vivo, with special interest in endangered species. Previous data from mouse sperm, where acrosome reaction (AR) occurs more often in the interstitium of the cumulus oophorus, contribute to strengthen the use of progesterone as a physiological inducer of this process. We studied the participation of protein kinase A (PKA), phospholipases A2 and C (PLA2 , PLC) in the AR induced by progesterone from Chinchilla epididymal spermatozoa. The addition of db-cAMP to the incubation medium caused an increase of 58% in the AR, while the use of H89 (30 μm), a PKA inhibitor, reflected a decrease of 40% in the percentage of reacted gametes. The assays conducted with arachidonic acid showed a maximum increase of 23% in the AR. When gametes were pre-incubated with PLA2 inhibitors, a dose-dependent inhibitory effect was observed. The addition of phorbol12-myristate13-acetate (10 μm) revealed higher percentages of AR induction (60%). When PLC was inhibited with neomycin and U73122, a dose-dependent decrease in AR percentages was observed. Combined inhibition of PKA, PLA2 and PLC, AR values similar to control were obtained. This work shows evidence, for the first time in Chinchilla, that progesterone activates the AC/cAMP/PKA system as well as sperm phospholipases and that these signalling pathways participate jointly and cooperatively in AR. These results contribute to the understanding of the complex regulation that is triggered in sperm after the effect of progesterone.

  19. Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) from Pseudomonas aeruginosa

    PubMed Central

    Vasil, Michael L.; Berka, Randy M.; Gray, Gregory L.; Nakai, Hiroshi

    1982-01-01

    Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa is a phosphate (Pi)-regulated extracellular protein which may be a significant virulence factor of this organism. The gene for this hemolytic enzyme was cloned on a 4.1-megadalton (Mdal) fragment from a BamHI digest of P. aeruginosa PAO1 genomic DNA and was inserted into the BamHI sites of the multicopy Escherichia coli(pBR322) and P. aeruginosa(pMW79) vectors. The E. coli and P. aeruginosa recombinant plasmids were designated pGV26 and pVB81, respectively. A restriction map of the 4.1-Mdal fragment from pGV26 was constructed, using double and single digestions with BamHI and EcoRI and several different restriction enzymes. Based on information from this map, a 2.4-Mdal BamHI/BglII fragment containing the gene for phospholipase C was subcloned to pBR322. The hybrid plasmids pGV26 and pVB81 direct the synthesis of enzymatically active phospholipase C, which is also hemolytic. The plasmid-directed synthesis of phospholipase C in E. coli or P. aeruginosa is not repressible by Pi as is the chromosomally directed synthesis in P. aeruginosa. Data are presented which suggest that the synthesis of phospholipase C from pGV26 and pVB81 is directed from the tetracycline resistance gene promoter. The level of enzyme activity produced by E. coli(pGV26) is slightly higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions. In contrast, the levels produced by P. aeruginosa(pVB81) are at least 600-fold higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions and approximately 20-fold higher than those produced by P. aeruginosa(pMW79) under derepressed conditions. The majority (85%) of the enzyme produced by E. coli(pGV26) remained cell associated, whereas >95% of the enzyme produced by P. aeruginosa(pVB81) was extracellular. Analysis of extracellular proteins from cultures of P. aeruginosa(pMW79) and P. aeruginosa(pVB81) by high-performance liquid chromotography and

  20. Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

    PubMed

    Cerminati, Sebastián; Eberhardt, Florencia; Elena, Claudia E; Peirú, Salvador; Castelli, María E; Menzella, Hugo G

    2017-02-25

    Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

  1. Loss of platelet-derived growth factor-stimulated phospholipase activity in NIH-3T3 cells expressing the EJ-ras oncogene

    SciTech Connect

    Benjamin, C.W.; Tarpley, W.G.; Gorman, R.R.

    1987-01-01

    Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. The authors now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A/sub 2/ and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E/sub 2/ (PGE/sub 2/) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ml. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells is not due to a defect in the prostaglandin cyclooxygenase enzyme, since incubation of control cells and EJ-ras-transfected cells in 0.33, 3.3, or 33 ..mu..M arachidonate resulted in identical levels of PGE/sub 2/ release. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells also does not result from the loss of functional PDGF receptors. EJ-ras-transformed cells bind 70% as much /sup 125/I-labeled PDGF as control cells and are stimulated to incorporate (/sup 3/H)thymidine and to proliferate after exposure to PDGF. Determination of total water-soluble inositolphospholipids and changes in the specific activities of phosphatidylcholine in control and EJ-ras-transfected cells demonstrated that PDGF-stimulated phospholipase C and A/sub 2/ activities are inhibited in the EJ-ras-transfected cells.

  2. Calcium-independent phospholipases A2 and their roles in biological processes and diseases

    PubMed Central

    Ramanadham, Sasanka; Ali, Tomader; Ashley, Jason W.; Bone, Robert N.; Hancock, William D.; Lei, Xiaoyong

    2015-01-01

    Among the family of phospholipases A2 (PLA2s) are the Ca2+-independent PLA2s (iPLA2s) and they are designated group VI iPLA2s. In relation to secretory and cytosolic PLA2s, the iPLA2s are more recently described and details of their expression and roles in biological functions are rapidly emerging. The iPLA2s or patatin-like phospholipases (PNPLAs) are intracellular enzymes that do not require Ca2+ for activity, and contain lipase (GXSXG) and nucleotide-binding (GXGXXG) consensus sequences. Though nine PNPLAs have been recognized, PNPLA8 (membrane-associated iPLA2γ) and PNPLA9 (cytosol-associated iPLA2β) are the most widely studied and understood. The iPLA2s manifest a variety of activities in addition to phospholipase, are ubiquitously expressed, and participate in a multitude of biological processes, including fat catabolism, cell differentiation, maintenance of mitochondrial integrity, phospholipid remodeling, cell proliferation, signal transduction, and cell death. As might be expected, increased or decreased expression of iPLA2s can have profound effects on the metabolic state, CNS function, cardiovascular performance, and cell survival; therefore, dysregulation of iPLA2s can be a critical factor in the development of many diseases. This review is aimed at providing a general framework of the current understanding of the iPLA2s and discussion of the potential mechanisms of action of the iPLA2s and related involved lipid mediators. PMID:26023050

  3. Phospholipase signaling is modified differentially by phytoregulators in Capsicum chinense J. cells.

    PubMed

    Muñoz-Sánchez, J Armando; Altúzar-Molina, Alma; Hérnandez-Sotomayor, S M Teresa

    2012-09-01

    Plant defense mechanisms respond to diverse environmental factors and play key roles in signaling pathways. The phospholipidic signaling pathway forms part of the plant response to several phytoregulators, such as salicylic acid (SA) and methyl jasmonate (MJ), which have been widely used to stimulate secondary metabolite production in cell cultures. ( 1) Furthermore, it has been reported that the levels of such phytoregulators as SA and MJ can increase in response to stressful conditions. ( 2) (,) ( 3) The phospholipidic signal transduction system involves the generation of second messengers by the hydrolysis of phospholipids. In this study, we examined how phospholipidic signaling can be modulated depending on the growth stage of the culture, and we focused on two key lipases having relevant roles in the signaling cascades in plants. An evaluation was made of the effects of SA and MJ on the phospholipase activities in Capsicum chinense Jacq. suspension cells at different phases of the culture cycle. The treatment with SA differentially modified the phospholipase C (PLC) (EC: 3.1.4.3) and phospholipase D (PLD) (EC: 3.1.4.4) activities in a dose-dependent manner that also depended on the day of the culture cycle. In contrast, the treatment with MJ resulted in a biphasic behavior of the PLC and PLD activities. We conclude that the enzymatic activities in the phospholipidic signaling pathways are modified differentially depending on the day of the culture's growth cycle; accordingly, the response capacity to such environmental factors as phytoregulators is variable at different stages of growth and the physiology of the cells.

  4. Phospholipase signaling is modified differentially by phytoregulators in Capsicum chinense J. cells

    PubMed Central

    Muñoz-Sánchez, J. Armando; Altúzar-Molina, Alma; Hérnandez-Sotomayor, S. M. Teresa

    2012-01-01

    Plant defense mechanisms respond to diverse environmental factors and play key roles in signaling pathways. The phospholipidic signaling pathway forms part of the plant response to several phytoregulators, such as salicylic acid (SA) and methyl jasmonate (MJ), which have been widely used to stimulate secondary metabolite production in cell cultures.1 Furthermore, it has been reported that the levels of such phytoregulators as SA and MJ can increase in response to stressful conditions.2,3 The phospholipidic signal transduction system involves the generation of second messengers by the hydrolysis of phospholipids. In this study, we examined how phospholipidic signaling can be modulated depending on the growth stage of the culture, and we focused on two key lipases having relevant roles in the signaling cascades in plants. An evaluation was made of the effects of SA and MJ on the phospholipase activities in Capsicum chinense Jacq. suspension cells at different phases of the culture cycle. The treatment with SA differentially modified the phospholipase C (PLC) (EC: 3.1.4.3) and phospholipase D (PLD) (EC: 3.1.4.4) activities in a dose-dependent manner that also depended on the day of the culture cycle. In contrast, the treatment with MJ resulted in a biphasic behavior of the PLC and PLD activities. We conclude that the enzymatic activities in the phospholipidic signaling pathways are modified differentially depending on the day of the culture’s growth cycle; accordingly, the response capacity to such environmental factors as phytoregulators is variable at different stages of growth and the physiology of the cells. PMID:22899070

  5. 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.

  6. Trichomonas vaginalis acidic phospholipase A2: isolation and partial amino acid sequence.

    PubMed

    Escobedo-Guajardo, Brenda L; González-Salazar, Francisco; Palacios-Corona, Rebeca; Torres de la Cruz, Víctor M; Morales-Vallarta, Mario; Mata-Cárdenas, Benito D; Garza-González, Jesús N; Rivera-Silva, Gerardo; Vargas-Villarreal, Javier

    2013-12-01

    Sexually transmitted diseases are a major cause of acute disease worldwide, and trichomoniasis is the most common and curable disease, generating more than 170 million cases annually worldwide. Trichomonas vaginalis is the causal agent of trichomoniasis and has the ability to destroy in vitro cell monolayers of the vaginal mucosa, where the phospholipases A2 (PLA2) have been reported as potential virulence factors. These enzymes have been partially characterized from the subcellular fraction S30 of pathogenic T. vaginalis strains. The main objective of this study was to purify a phospholipase A2 from T. vaginalis, make a partial characterization, obtain a partial amino acid sequence, and determine its enzymatic participation as hemolytic factor causing lysis of erythrocytes. Trichomonas S30, RF30 and UFF30 sub-fractions from GT-15 strain have the capacity to hydrolyze [2-(14)C-PA]-PC at pH 6.0. Proteins from the UFF30 sub-fraction were separated by affinity chromatography into two eluted fractions with detectable PLA A2 activity. The EDTA-eluted fraction was analyzed by HPLC using on-line HPLC-tandem mass spectrometry and two protein peaks were observed at 8.2 and 13 kDa. Peptide sequences were identified from the proteins present in the eluted EDTA UFF30 fraction; bioinformatic analysis using Protein Link Global Server charged with T. vaginalis protein database suggests that eluted peptides correspond a putative ubiquitin protein in the 8.2 kDa fraction and a phospholipase preserved in the 13 kDa fraction. The EDTA-eluted fraction hydrolyzed [2-(14)C-PA]-PC lyses erythrocytes from Sprague-Dawley in a time and dose-dependent manner. The acidic hemolytic activity decreased by 84% with the addition of 100 μM of Rosenthal's inhibitor.

  7. Proteinase and phospholipase activities and development at different temperatures of yeasts isolated from bovine milk.

    PubMed

    Melville, Priscilla A; Benites, Nilson R; Ruz-Peres, Monica; Yokoya, Eugenio

    2011-11-01

    The presence of yeasts in milk may cause physical and chemical changes limiting the durability and compromising the quality of the product. Moreover, milk and dairy products contaminated by yeasts may be a potential means of transmission of these microorganisms to man and animals causing several kinds of infections. This study aimed to determine whether different species of yeasts isolated from bovine raw milk had the ability to develop at 37°C and/or under refrigeration temperature. Proteinase and phospholipase activities resulting from these yeasts were also monitored at different temperatures. Five genera of yeasts (Aureobasidium sp., Candida spp., Geotrichum spp., Trichosporon spp. and Rhodotorula spp.) isolated from bovine raw milk samples were evaluated. All strains showed one or a combination of characteristics: growth at 37°C (99·09% of the strains), psychrotrophic behaviour (50·9%), proteinase production (16·81% of the strains at 37°C and 4·09% under refrigeration) and phospholipase production (36·36% of the isolates at 37°C and 10·9% under refrigeration), and all these factors may compromise the quality of the product. Proteinase production was similar for strains incubated at 37°C (16·81% of the isolates) and room temperature (17·27%) but there was less amount of phospholipase-producing strains at room temperature (15·45% of the isolates were positive) when compared with incubation at 37°C (36·36%). Enzymes production at 37°C by yeasts isolated from milk confirmed their pathogenic potential. The refrigeration temperature was found to be most efficient to inhibit enzymes production and consequently ensure better quality of milk. The viability of yeasts and the activity of their enzymes at different temperatures are worrying because this can compromise the quality of dairy products at all stages of production and/or storage, and represent a risk to the consumer.

  8. Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases.

    PubMed Central

    Falo, L D; Haber, S I; Herrmann, S; Benacerraf, B; Rock, K L

    1987-01-01

    To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) we analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A2 and phospholipase C. In addition it is observed with three distinct antigens--ovalbumin, bovine insulin, and poly(LGlu56LLys35LPhe9) [(GluLysPhe)n]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to controls in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or "processing independent" antigens. In parallel studies 125I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. At least some of the biotin-insulin surface sites are immunologically relevant, because the presentation of processed biotin-insulin by fixed APC is blocked by avidin. This effect is specific. Avidin binding to biotin-insulin-exposed APC does not inhibit allospecific stimulation nor the presentation of unconjugated insulin. These studies demonstrate that phospholipase effectively removes processed cell surface antigen. PMID:3467371

  9. Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Bassyouni, Rasha H; Wegdan, Ahmed Ashraf; Abdelmoneim, Abdelsamie; Said, Wessam; AboElnaga, Fatma

    2015-10-01

    Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

  10. Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans.

    PubMed

    Farnoud, Amir M; Mor, Visesato; Singh, Ashutosh; Del Poeta, Maurizio

    2014-11-03

    Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.

  11. Head group specificity of phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.).

    PubMed

    Oblozinsky, M; Ulbrich-Hofmann, R; Bezakova, L

    2005-02-01

    The biocatalytical potential of two new phospholipase D (PLD) isoenzymes from poppy seedlings (Papaver somniferum L.), PLD-A and PLD-B, was examined by comparing their activities in phospholipid transformation. Both enzymes showed the same ratio in rates of hydrolysis [phosphatidylcholine (PC):phosphatidylglycerol (PG):phosphatidylserine:phosphatidylinositol = 1:0.5:0.3:0.1] and were inactive towards phosphatidylethanolamine (PE). PLD-A did not catalyze head group exchange whereas PLD-B showed a high transphosphatidylation potential in the conversion of PC into PG and PE. This enzyme also catalyzed the transesterification of octadecylphosphocholine into octadecylphosphoglycerol or octadecylphosphoethanolamine.

  12. Comparison of total protein and phospholipase A(2) levels in individual coralsnake venoms.

    PubMed

    Kopper, Randall A; Harper, George R; Zimmerman, Sloane; Hook, Jessica

    2013-12-15

    Studies of differences or changes in venom protein levels or enzymatic activities have significance only if contrasted to the normal variations between individual snakes. This study involves the analysis and comparison of venom from 13 individual Texas coralsnakes (Micrurus tener tener) in order to detect differences in the volume, total protein concentration, electrophoretic profile, and PLA2 enzyme activity. A significant inverse correlation between venom volume and total protein concentration was found. Although the 13 venoms were indistinguishable from their electrophoretic protein profiles, phospholipase A2 enzymatic activities varied considerably.

  13. Structure of a cardiotoxic phospholipase A(2) from Ophiophagus hannah with the "pancreatic loop".

    PubMed

    Zhang, Hai-Long; Xu, Su-Juan; Wang, Qiu-Yan; Song, Shi-Ying; Shu, Yu-Yan; Lin, Zheng-Jiong

    2002-06-01

    The crystal structure of an acidic phospholipase A(2) from Ophiophagus hannah (king cobra) has been determined by molecular replacement at 2.6-A resolution to a crystallographic R factor of 20.5% (R(free)=23.3%) with reasonable stereochemistry. The venom enzyme contains an unusual "pancreatic loop." The conformation of the loop is well defined and different from those in pancreas PLA(2), showing its structural variability. This analysis provides the first structure of a PLA(2)-type cardiotoxin. The sites related to the cardiotoxic and myotoxic activities are explored and the oligomer observed in the crystalline state is described.

  14. Phospholipase A2 and Arachidonic Acid in Alzheimer’s Disease

    PubMed Central

    Sanchez-Mejia, Rene O.; Mucke, Lennart

    2011-01-01

    Essential fatty acids (EFA) play a critical role in the brain and regulate many of the processes altered in Alzheimer’s disease (AD). Technical advances are allowing for the dissection of complex lipid pathways in normal and diseased states. Arachidonic acid (AA) and specific isoforms of phospholipase A2 (PLA2) appear to play critical mediator roles in amyloid-β (Aβ) - induced pathogenesis, leading to learning, memory, and behavioral impairments in mouse models of AD. These findings and ongoing research into lipid biology in AD and related disorders promise to reveal new pharmacological targets that may lead to better treatments for these devastating conditions. PMID:20553961

  15. Clinical and biological role of secretory phospholipase A2 in acute respiratory distress syndrome infants

    PubMed Central

    2013-01-01

    Introduction Secretory phospholipase A2 is supposed to play a role in acute lung injury but no data are available for pediatric acute respiratory distress syndrome (ARDS). It is not clear which enzyme subtypes are secreted and what the relationships are between enzyme activity, biophysical and biochemical parameters, and clinical outcomes. We aimed to measure the enzyme and identify its subtypes and to study its biochemical and biophysical effect. The secondary aim was to correlate enzyme activity with clinical outcome. Methods Bronchoalveolar lavage was performed in 24 infants with ARDS and 14 controls with no lung disease. Samples were assayed for secretory phospholipase A2 and molecules related to its activity and expression. Western blotting and captive bubble surfactometry were also performed. Clinical data were real time downloaded. Results Tumor necrosis factor-α (814 (506-2,499) vs. 287 (111-1,315) pg/mL; P = 0.04), enzyme activity (430 (253-600) vs. 149 (61-387) IU/mL; P = 0.01), free fatty acids (4.3 (2.8-8.6) vs. 2 (0.8-4.6) mM; P = 0.026), and minimum surface tension (25.6 ± 6.1 vs. 18 ± 1.8 mN/m; P = 0.006) were higher in ARDS than in controls. Phospholipids are lower in ARDS than in controls (76.5 (54-100) vs. 1,094 (536-2,907) μg/mL; P = 0.0001). Three enzyme subtypes were identified (-IIA, -V, -X), although in lower quantities in controls; another subtype (-IB) was mainly detected in ARDS. Significant correlations exist between enzyme activity, free fatty acids (ρ = 0.823; P < 0.001), and surface tension (ρ = 0.55; P < 0.028). Correlations also exist with intensive care stay (ρ = 0.54; P = 0.001), PRISM-III24 (ρ = 0.79; P< 0.001), duration of ventilation (ρ = 0.53; P = 0.002), and oxygen therapy (ρ = 0.54; P = 0.001). Conclusions Secretory phospholipase A2 activity is raised in pediatric ARDS and constituted of four subtypes. Enzyme correlates with some inflammatory mediators, surface tension, and major clinical outcomes. Secretory

  16. Role of cardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom

    PubMed Central

    Chang, C. C.; Chuang, Sing-Tai; Lee, C. Y.; Wei, J. W.

    1972-01-01

    1. The effects of phospholipase A (PhA), cardiotoxin (CTX) and neurotoxin (cobrotoxin) isolated from Formosan cobra (Naja naja atra) venom on conduction of the rat phrenic nerve and membrane potential of the rat diaphragm were studied. 2. Phospholipase A, lysolecithin and cobrotoxin were without effect on the axonal conduction. Cardiotoxin was the only active agent in cobra venom, but it was less potent than the crude venom. 3. The blocking action of cardiotoxin was markedly accelerated by the simultaneous administration of phospholipase A. However, the minimum effective concentration of cardiotoxin (100 μg/ml), was not decreased by phospholipase A. Pretreatment of the nerve with phospholipase A, followed by washout, did not alter the activity of cardiotoxin. 4. Cardiotoxin (3 μg/ml) completely depolarized the membrane of superficial muscle fibres within 60 min, being 3 times more potent than the crude venom. Phospholipase A, on the other hand, needed a dose 30 times higher and a prolonged period of incubation to induce depolarization of similar extent. Cobrotoxin was without effect on membrane potentials. 5. CaCl2 (10 mM) effectively antagonized the nerve blocking as well as the depolarizing effect of the crude venom, cardiotoxin or cardiotoxin plus phospholipase A. By contrast, the slow depolarizing effect of phospholipase A was enhanced by high concentrations of calcium. 6. Cardiotoxic fractions of Indian cobra venom affected both nerve conduction and diaphragm membrane potential in exactly the same way as cardiotoxin. Toxin A of the same venom was without effect. 7. It is concluded that the active agent in cobra venoms either on axonal conduction or on muscle membrane is cardiotoxin. The synergistic effect of phospholipase A on cardiotoxin appears to be due to acceleration rather than potentiation of its action. The mechanism of action of cardiotoxin and its synergism by phospholipase A are discussed. PMID:5041453

  17. [Secreted phospholipases A2 (sPLA2): friends or foes? Are they actors in antibacterial and anti-HIV resistance?].

    PubMed

    Villarrubia, Vicente G; Costa, Luis A; Díez, Roberto A

    2004-11-27

    In this paper the authors update on the deletereous or beneficial roles of human and animal secretory phospholipases A2 (sPLA2). Although human sPLA2-IIA (inflammatory) was initially thought as a foe because its pathogenic implication in sepsis, multiorganic failure or other related syndromes, recent data indicates its role in in the antiinfectious host resistance. Thus, sPLA2-IIA exhibits potent bactericidal activities against gram-negative and gram-positive (in this case, together with other endogenous inflammatory factors) bacteria. Surprisingly, human sPLA-IIA does not show in vitro anti-human immunodeficiency virus (HIV) activity, whilst several sPLA2-IA isolated from bee and serpent venons do it: this is the case for crotoxin, a sPLA2-IA isolated from the venon of Crotalus durissus terrificus (sPLA2-Cdt). The mechanism for the in vitro anti-HIV activity of sPLA2-Cdt (inhibition of Gag p24) appears to be related to the ability of the drug to desestabilize ancorage (heparans) and fusion (cholesterol) receptors on HIV target cells.

  18. Cryptococcus neoformans phospholipase B1 activates host cell Rac1 for traversal across the blood-brain barrier.

    PubMed

    Maruvada, Ravi; Zhu, Longkun; Pearce, Donna; Zheng, Yi; Perfect, John; Kwon-Chung, Kyung J; Kim, Kwang Sik

    2012-10-01

    Cryptococcus neoformans penetration into the central nervous system (CNS) requires traversal of the blood-brain barrier that is composed of a single layer of human brain microvascular endothelial cells (HBMEC), but the underlying mechanisms of C. neoformans traversal remain incompletely understood. C. neoformans transcytosis of HBMEC monolayer involves rearrangements of the host cell actin cytoskeleton and small GTP-binding Rho family proteins such as Rac1 are shown to regulate host cell actin cytoskeleton. We, therefore, examined whether C. neoformans traversal of the blood-brain barrier involves host Rac1. While the levels of activated Rac1 (GTP-Rac1) in HBMEC increased significantly upon incubation with C. neoformans strains, pharmacological inhibition and down-modulation of Rac1 significantly decreased C. neoformans transcytosis of HBMEC monolayer. Also, Rac1 inhibition was efficient in preventing C. neoformans penetration into the brain. In addition, C. neoformans phospholipase B1 (Plb1) was shown to contribute to activating host cell Rac1, andSTAT3 was observed to associate with GTP-Rac1 in HBMEC that were incubated with C. neoformans strain but not with its Δplb1 mutant. These findings demonstrate for the first time that C. neoformans Plb1 aids fungal traversal across the blood-brain barrier by activating host cell Rac1 and its association with STAT3, and suggest that pharmacological intervention of host-microbial interaction contributing to traversal of the blood-brain barrier may prevent C. neoformans penetration into the brain.

  19. Interleukin-22-Induced Antimicrobial Phospholipase A2 Group IIA Mediates Protective Innate Immunity of Nonhematopoietic Cells against Listeria monocytogenes.

    PubMed

    Okita, Yamato; Shiono, Takeru; Yahagi, Ayano; Hamada, Satoru; Umemura, Masayuki; Matsuzaki, Goro

    2015-12-07

    Listeria monocytogenes is a bacterial pathogen which establishes intracellular parasitism in various cells, including macrophages and nonhematopoietic cells, such as hepatocytes. It has been reported that several proinflammatory cytokines have pivotal roles in innate protection against L. monocytogenes infection. We found that a proinflammatory cytokine, interleukin 22 (IL-22), was expressed by CD3(+) CD4(+) T cells at an early stage of L. monocytogenes infection in mice. To assess the influence of IL-22 on L. monocytogenes infection in hepatocytes, cells of a human hepatocellular carcinoma line, HepG2, were treated with IL-22 before L. monocytogenes infection in vitro. Gene expression analysis of the IL-22-treated HepG2 cells identified phospholipase A2 group IIA (PLA2G2A) as an upregulated antimicrobial molecule. Addition of recombinant PLA2G2A to the HepG2 culture significantly suppressed L. monocytogenes infection. Culture supernatant of the IL-22-treated HepG2 cells contained bactericidal activity against L. monocytogenes, and the activity was abrogated by a specific PLA2G2A inhibitor, demonstrating that HepG2 cells secreted PLA2G2A, which killed extracellular L. monocytogenes. Furthermore, colocalization of PLA2G2A and L. monocytogenes was detected in the IL-22-treated infected HepG2 cells, which suggests involvement of PLA2G2A in the mechanism of intracellular killing of L. monocytogenes by HepG2 cells. These results suggest that IL-22 induced at an early stage of L. monocytogenes infection enhances innate immunity against L. monocytogenes in the liver by stimulating hepatocytes to produce an antimicrobial molecule, PLA2G2A.

  20. Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

    PubMed Central

    Beck, Goichi; Shinzawa, Koei; Hayakawa, Hideki; Baba, Kousuke; Yasuda, Toru; Sumi-Akamaru, Hisae; Tsujimoto, Yoshihide; Mochizuki, Hideki

    2015-01-01

    Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA2β), which is encoded by the PLA2G6 gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA2β-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA2β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA2β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA2β-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA2β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction. PMID:26506412

  1. Expression of group IIA phospholipase A2 is an independent predictor of favorable outcome for patients with gastric cancer.

    PubMed

    Wang, Xi; Huang, Chun-Jin; Yu, Guan-Zhen; Wang, Jie-Jun; Wang, Rui; Li, Yu-Mei; Wu, Qiong

    2013-10-01

    Growing evidence suggests that phospholipase A2 (PLA2) plays a pivotal role in tumorigenesis in human gastrointestinal cancer. One of the well-studied isoforms of PLA2, group IIA PLA2 (PLA2G2A), appears to exert its protumorigenic or antitumorigenic effects in a tissue-specific manner. The present study was designed to determine the expression profile and prognostic value of PLA2G2A in gastric cancer in a large Chinese cohort. By using real-time polymerase chain reaction, the amount of PLA2G2A messenger RNA in 60 pairs of fresh gastric tumors and adjacent noncancerous mucosa was measured. The immunostaining of PLA2G2A in 866 gastric cancers with paired noncancerous tissues was assayed. No expression of PLA2G2A was found in normal gastric mucosa, and focal expression of PLA2G2A was noticed in intestinal metaplasia, whereas significantly increased expression of PLA2G2A was observed in the cytoplasm of gastric cancer cells. Furthermore, the extent of PLA2G2A expression was associated with tumor size (P < .001), tumor differentiation (P = .001), T class (P < .001), N class (P < .001), and TNM stage (P < .001) of gastric cancer. Multivariate analysis showed that PLA2G2A expression was an independent predictor of survival for patients with gastric cancer (P = .024). Expression of PLA2G2A seems to be protective for patients with gastric cancer (hazard ratio, 1.423; 95% confidence interval, 1.047-1.935), and it may be a target for achieving better treatment outcomes.

  2. Group X Phospholipase A2 Stimulates the Proliferation of Colon Cancer Cells by Producing Various Lipid Mediators

    PubMed Central

    Surrel, Fanny; Jemel, Ikram; Boilard, Eric; Bollinger, James G.; Payré, Christine; Mounier, Carine M.; Talvinen, Kati A.; Laine, Veli J. O.; Nevalainen, Timo J.; Gelb, Michael H.

    2009-01-01

    Among mammalian secreted phospholipases A2 (sPLA2s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA2 [mouse (m)GX] is one of the most highly expressed PLA2 in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA2s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA2 inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA2α and M-type sPLA2 receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA2 mitogenic effects. Together, our results indicate that group X sPLA2 may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression. PMID:19602573

  3. Group X phospholipase A2 stimulates the proliferation of colon cancer cells by producing various lipid mediators.

    PubMed

    Surrel, Fanny; Jemel, Ikram; Boilard, Eric; Bollinger, James G; Payré, Christine; Mounier, Carine M; Talvinen, Kati A; Laine, Veli J O; Nevalainen, Timo J; Gelb, Michael H; Lambeau, Gérard

    2009-10-01

    Among mammalian secreted phospholipases A2 (sPLA(2)s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA(2) [mouse (m)GX] is one of the most highly expressed PLA(2) in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA(2)s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA(2) inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA(2)alpha and M-type sPLA(2) receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA(2) mitogenic effects. Together, our results indicate that group X sPLA(2) may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression.

  4. Extracellular phospholipase A2 secretion is a common effector pathway of interleukin-1 and tumour necrosis factor action.

    PubMed

    Vadas, P; Pruzanski, W; Stefanski, E; Ellies, L G; Aubin, J E; Sos, A; Melcher, A

    1991-06-01

    Inflammatory processes are characterized by increased levels of extracellular phospholipase A2 (PLA2) and cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF). IL-1, TNF and PLA2 share a number of proinflammatory, arthritogenic effects. The sequential induction, first of the cytokines followed by PLA2, suggests that these cytokines may regulate synthesis and secretion of PLA2. To test this postulate, foetal rat calvarial bone-forming cells (FRCC) were treated with recombinant human IL-1 and TNF and extracellular PLA2 release was quantitated. Both IL-1 and TNF induced the de novo synthesis of PLA2 in a concentration-dependent manner. Continuous exposure of FRCC in primary culture to IL-1 (50 units/ml) over 15 days resulted in as much as 100-fold increase in PLA2 secretion. IL-1 (50 units/ml) added to post-confluent cultures for a 48-h pulse increased PLA2 activity 9.4-fold. The combination of IL-1 (50 units/ml) and TNF (500 units/ml) was synergistic with an observed increase in extracellular PLA2 secretion of 146-fold following a 48-h pulse. Interleukin-6, alone or in combination with IL-1 or TNF, did not further enhance PLA2 synthesis of secretion. Cytokine-induced synthesis of PLA2 was inhibited 80% by 10 microM cycloheximide but not by dexamethasone over the range of 10(-6) to 10(-8) M. FRCC-derived PLA2 was neutral-active with a pH optimum of 6-7.5 and was calcium-dependent with optimal activity in the presence of 2-7 mM calcium. It had absolute 2-acyl specificity using micellar phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin.

    PubMed

    Chiou, Yi-Ling; Lin, Shinne-Ren; Hu, Wan-Ping; Chang, Long-Sen

    2014-12-15

    Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity.

  6. SKF-83959 is not a highly-biased functionally selective D1 dopamine receptor ligand with activity at phospholipase C.

    PubMed

    Lee, Sang-Min; Kant, Andrew; Blake, Daniel; Murthy, Vishakantha; Boyd, Kevin; Wyrick, Steven J; Mailman, Richard B

    2014-11-01

    SKF-83959 [6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine] is reported to be a functionally selective dopamine D1 receptor ligand with high bias for D1-mediated phospholipase C (PLC) versus D1-coupled adenylate cyclase signaling. This signaling bias is proposed to explain behavioral activity in both rat and primate Parkinson's disease models, and a D1-D2 heterodimer has been proposed as the underlying mechanism. We have conducted an in-depth pharmacological characterization of this compound in dopamine D1 and D2 receptors in both rat brain and heterologous systems expressing human D1 or D2 receptors. Contrary to common assumptions, SKF-83959 is similar to the classical, well-characterized partial agonist SKF38393 in all systems. It is a partial agonist (not an antagonist) at adenylate cyclase in vitro and ex vivo, and is a partial agonist in D1-mediated β-arrestin recruitment. Contrary to earlier reports, it does not have D1-mediated effects on PLC signaling in heterologous systems. Because drug metabolites can also contribute, its 3-N-demethylated analog also was synthesized and tested. As expected from the known structure-activity relationships of the benzazepines, this compound also had high affinity for the D1 receptor and somewhat higher intrinsic activity than the parent ligand, and also might contribute to in vivo effects of SKF-83959. Together, these data demonstrate that SKF-83959 is not a highly-biased functionally selective D1 ligand, and that its reported behavioral data can be explained solely by its partial D1 agonism in canonical signaling pathway(s). Mechanisms that have been proposed based on the purported signaling novelty of SKF-83959 at PLC should be reconsidered.

  7. IgE antibodies to bee venom, phospholipase A, melittin and wasp venom.

    PubMed

    Jarisch, R; Yman, L; Boltz, A; Sandor, I; Janitsch, A

    1979-09-01

    Specific IgE antibodies against bee venom, phospholipase A, melittin and wasp venom have been examined in fifty patients with an unusually severe reaction after bee or wasp sting. Two thirds of the bee venom-sensitive patients also have detectable IgE antibodies to wasp venom. More than 50% of the wasp venom-sensitive patients are also allergic to bee venom. Phospholipase A and melittin IgE antibodies were found, respectively, in two thirds and one third of the bee venom-sensitive cases. Specific IgE antibody determinations by the Radioallergosorbent test play an essential role in the diagnostic work. After a reaction to hymenoptera stings both bee and wasp venom tests are necessary due to the high incidence of a false or incomplete identification of the stinging insect. Melittin, known for its potent pharmacological activity and possibly responsible for most of the side effects in bee venom immunotherapy, can probably not be excluded from therapeutic venom preparations since IgE antibodies to the melittin preparation were detected in one third of the cases.

  8. Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize.

    PubMed

    Gilles, Laurine M; Khaled, Abdelsabour; Laffaire, Jean-Baptiste; Chaignon, Sandrine; Gendrot, Ghislaine; Laplaige, Jérôme; Bergès, Hélène; Beydon, Genséric; Bayle, Vincent; Barret, Pierre; Comadran, Jordi; Martinant, Jean-Pierre; Rogowsky, Peter M; Widiez, Thomas

    2017-03-15

    Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.

  9. Lipoprotein-associated phospholipase A2 prognostic role in atherosclerotic complications

    PubMed Central

    Maiolino, Giuseppe; Bisogni, Valeria; Rossitto, Giacomo; Rossi, Gian Paolo

    2015-01-01

    Atherosclerosis manifests itself clinically at advanced stages when plaques undergo hemorrhage and/or rupture with superimposed thrombosis, thus abruptly stopping blood supply. Identification of markers of plaque destabilization at a pre-clinical stage is, therefore, a major goal of cardiovascular research. Promising results along this line were provided by studies investigating the lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of phospholipase A2 proteins family that plays a key role in the metabolism of pro-inflammatory phospholipids, as oxidized low-density lipoproteins, and in the generation of pro-atherogenic metabolites, including lysophosphatidylcholine and oxidized free fatty acids. We herein review the experimental and clinical studies supporting use of Lp-PLA2 activity for predicting cardiovascular events. To his end we considered not only Lp-PLA2 activity and mass, but also Lp-PLA2 gene variations and their association with incident coronary artery disease, stroke, and cardiovascular mortality. Based on these evidences the major scientific societies have included in their guidelines the measurement of Lp-PLA2 activity among the biomarkers that are useful in risk stratification of adult asymptomatic patients at intermediate cardiovascular risk. The results of two recently published major clinical trials with the Lp-PLA2 inhibitor darapladib, which seem to challenge the pathogenic role of Lp-PLA2, will also be discussed. PMID:26516415

  10. Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade.

    PubMed

    Kalachova, Tetiana; Iakovenko, Oksana; Kretinin, Sergii; Kravets, Volodymyr

    2013-05-01

    Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway.

  11. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities[S

    PubMed Central

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Popowycz, Florence; Khatib, Abdallah; Queneau, Yves; Abousalham, Abdelkarim

    2016-01-01

    A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase®, guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement. PMID:27194811

  12. A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.

    PubMed

    Jiang, Feng; Waterfield, Nicholas R; Yang, Jian; Yang, Guowei; Jin, Qi

    2014-05-14

    Widely found in animal and plant-associated proteobacteria, type VI secretion systems (T6SSs) are potentially capable of facilitating diverse interactions with eukaryotes and/or other bacteria. Pseudomonas aeruginosa encodes three distinct T6SS haemolysin coregulated protein (Hcp) secretion islands (H1, H2, and H3-T6SS), each involved in different aspects of the bacterium's interaction with other organisms. Here we describe the characterization of a P. aeruginosa H3-T6SS-dependent phospholipase D effector, PldB, and its three tightly linked cognate immunity proteins. PldB targets the periplasm of prokaryotic cells and exerts an antibacterial activity. Surprisingly, PldB also facilitates intracellular invasion of host eukaryotic cells by activation of the PI3K/Akt pathway, revealing it to be a trans-kingdom effector. Our findings imply a potentially widespread T6SS-mediated mechanism, which deploys a single phospholipase effector to influence both prokaryotic cells and eukaryotic hosts.

  13. Phospholipase D1 is involved in α1-adrenergic contraction of murine vascular smooth muscle.

    PubMed

    Wegener, Jörg W; Loga, Florian; Stegner, David; Nieswandt, Bernhard; Hofmann, Franz

    2014-03-01

    α1-Adrenergic stimulation increases blood vessel tone in mammals. This process involves a number of intracellular signaling pathways that include signaling via phospholipase C, diacylglycerol (DAG), and protein kinase C. So far, it is not certain whether signaling via phospholipase D (PLD) and PLD-derived DAG is involved in this process. We asked whether PLD participates in the α1-adrenergic-mediated signaling in vascular smooth muscle. α1-Adrenergic-induced contraction was assessed by myography of isolated aortic rings and by pressure recordings using the hindlimb perfusion model in mice. The effects of the PLD inhibitor 1-butanol (IC50 0.15 vol%) and the inactive congener 2-butanol were comparatively studied. Inhibition of PLD by 1-butanol reduced specifically the α1-adrenergic-induced contraction and the α1-adrenergic-induced pressure increase by 10 and 40% of the maximum, respectively. 1-Butanol did not influence the aortic contractions induced by high extracellular potassium, by the thromboxane analog U46619, or by a phorbol ester. The effects of 1-butanol were absent in mice that lack PLD1 (Pld1(-/-) mice) or that selectively lack the CaV1.2 channel in smooth muscle (sm-CaV1.2(-/-) mice) but still present in the heterozygous control mice. α1-Adrenergic contraction of vascular smooth muscle involves activation of PLD1, which controls a portion of the α1-adrenergic-induced CaV1.2 channel activity.

  14. 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.

  15. Divalent cations increase lipid order in erythrocytes and susceptibility to secretory phospholipase A2.

    PubMed

    Vest, Rebekah S; Gonzales, Laurie J; Permann, Seth A; Spencer, Emily; Hansen, Lee D; Judd, Allan M; Bell, John D

    2004-04-01

    Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree to which ions affected membrane properties correlated with the ionic radius and electronegativity of the ions. Lastly, erythrocytes became more susceptible to enzyme hydrolysis in the presence of elevated intracellular levels of nickel and manganese, but not magnesium. These differences appeared related to the ability of the ions to induce a transition in erythrocyte shape.

  16. Cyclin A2 modulates EMT via β-catenin and phospholipase C pathways.

    PubMed

    Cheung, Caroline T; Bendris, Nawal; Paul, Conception; Hamieh, Abdallah; Anouar, Youssef; Hahne, Michael; Blanchard, Jean-Marie; Lemmers, Bénédicte

    2015-08-01

    We have previously demonstrated that Cyclin A2 is involved in cytoskeletal dynamics, epithelial-mesenchymal transition (EMT) and metastasis. This phenotype was potentiated by activated oncogenic H-Ras. However, the mechanisms governing EMT in these cells have not yet been elucidated. Here, we dissected the pathways that are responsible for EMT in cells deficient for Cyclin A2. In Cyclin A2-depleted normal murine mammary gland (NMuMG) cells expressing RasV12, we found that β-catenin was liberated from the cell membrane and cell-cell junctions and underwent nuclear translocation and activation. Components of the canonical wingless (WNT) pathway, including WNT8b, WNT10a, WNT10b, frizzled 1 and 2 and TCF4 were upregulated at the messenger RNA and protein levels following Cyclin A2 depletion. However, suppression of the WNT pathway using the acetyltransferase porcupine inhibitor C59 did not reverse EMT whereas a dominant negative form of TCF4 as well as inhibition of phospholipase C using U73122 were able to do so. This suggests that a WNT-independent mechanism of β-catenin activation via phospholipase C is involved in the EMT induced by Cyclin A2 depletion. Our findings will broaden our knowledge on how Cyclin A2 contributes to EMT and metastasis.

  17. Lipoprotein-associated phospholipase A2 prognostic role in atherosclerotic complications.

    PubMed

    Maiolino, Giuseppe; Bisogni, Valeria; Rossitto, Giacomo; Rossi, Gian Paolo

    2015-10-26

    Atherosclerosis manifests itself clinically at advanced stages when plaques undergo hemorrhage and/or rupture with superimposed thrombosis, thus abruptly stopping blood supply. Identification of markers of plaque destabilization at a pre-clinical stage is, therefore, a major goal of cardiovascular research. Promising results along this line were provided by studies investigating the lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of phospholipase A2 proteins family that plays a key role in the metabolism of pro-inflammatory phospholipids, as oxidized low-density lipoproteins, and in the generation of pro-atherogenic metabolites, including lysophosphatidylcholine and oxidized free fatty acids. We herein review the experimental and clinical studies supporting use of Lp-PLA2 activity for predicting cardiovascular events. To his end we considered not only Lp-PLA2 activity and mass, but also Lp-PLA2 gene variations and their association with incident coronary artery disease, stroke, and cardiovascular mortality. Based on these evidences the major scientific societies have included in their guidelines the measurement of Lp-PLA2 activity among the biomarkers that are useful in risk stratification of adult asymptomatic patients at intermediate cardiovascular risk. The results of two recently published major clinical trials with the Lp-PLA2 inhibitor darapladib, which seem to challenge the pathogenic role of Lp-PLA2, will also be discussed.

  18. OKT3-induced nephrotoxicity is associated with release of group II secretory phospholipase A2.

    PubMed

    Wever, P C; Roest, R W; Wolbink-Kamp, A M; Wolbink, G J; Weening, J J; Hack, C E; ten Berge, J M

    1996-10-01

    Administration of the murine IgG2a CD3 monoclonal antibody OKT3 exerts a transient nephrotoxic effect. Increased levels of group II secretory phospholipase A2 (sPLA2-II) might account for this nephrotoxicity as sPLA2-II induces the biosynthesis of prostaglandins, vasoactive lipid mediators that influence glomerular haemodynamics and renal function. Furthermore, extracellular phospholipases seem to be involved in proximal tubular cell injury. We studied plasma sPLA2-II levels in relation to circulating creatinine, tumour necrosis factor alpha, interleukin 6 and C-reactive protein levels in 15 renal allograft recipients receiving rejection treatment with OKT3. As a control group, we studied 15 renal allograft recipients receiving rejection treatment with methylprednisolone. A maximal fourfold increase in sPLA2-II levels was observed 48 h after the first OKT3 administration, preceded by increased tumour necrosis factor alpha and interleukin 6 levels and accompanied by increased C-reactive protein levels. Creatinine levels reached a maximal increase 72 h after initiation of treatment. During methylprednisolone treatment no increase in any of the studied parameters was observed. Thus, administration of OKT3 induces increased sPLA2-II levels, presumably via generation of cytokines. We hypothesize that sPLA2-II may contribute to the nephrotoxic effect of OKT3 by inducing vasoconstrictive prostaglandins and renal tubular cell injury.

  19. 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.

  20. Phospholipase D δ knock-out mutants are tolerant to severe drought stress

    PubMed Central

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance. PMID:26340512

  1. Phospholipase D δ knock-out mutants are tolerant to severe drought stress.

    PubMed

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; Ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance.

  2. Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria, and open their transition pores.

    PubMed

    Rigoni, Michela; Paoli, Marco; Milanesi, Eva; Caccin, Paola; Rasola, Andrea; Bernardi, Paolo; Montecucco, Cesare

    2008-12-05

    Snake presynaptic neurotoxins with phospholipase A(2) activity are potent inducers of paralysis through inhibition of the neuromuscular junction. These neurotoxins were recently shown to induce exocytosis of synaptic vesicles following the production of lysophospholipids and fatty acids and a sustained influx of Ca(2+) from the medium. Here, we show that these toxins are able to penetrate spinal cord motor neurons and cerebellar granule neurons and selectively bind to mitochondria. As a result of this interaction, mitochondria depolarize and undergo a profound shape change from elongated and spaghetti-like to round and swollen. We show that snake presynaptic phospholipase A(2) neurotoxins facilitate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. The relative potency of the snake neurotoxins was similar for the permeability transition pore opening and for the phospholipid hydrolysis activities, suggesting a causal relationship, which is also supported by the effect of phospholipid hydrolysis products, lysophospholipids and fatty acids, on mitochondrial pore opening. These findings contribute to define the cellular events that lead to intoxication of nerve terminals by these snake neurotoxins and suggest that mitochondrial impairment is an important determinant of their toxicity.

  3. Phosphatidylinositol-Glycan-Phospholipase D Is Involved in Neurodegeneration in Prion Disease

    PubMed Central

    Jin, Jae-Kwang; Jang, Byungki; Jin, Hyoung Tae; Choi, Eun-Kyoung; Jung, Cha-Gyun; Akatsu, Hiroyasu; Kim, Jae-Il; Carp, Richard I.; Kim, Yong-Sun

    2015-01-01

    PrPSc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrPC) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrPSc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer’s disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases. PMID:25867459

  4. Cytotoxic activities of [Ser⁴⁹]phospholipase A₂ from the venom of the saw-scaled vipers Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus.

    PubMed

    Conlon, J Michael; Attoub, Samir; Arafat, Hama; Mechkarska, Milena; Casewell, Nicholas R; Harrison, Robert A; Calvete, Juan J

    2013-09-01

    Fractionation by reversed-phase HPLC of venom from four species of saw-scaled viper: Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus led to identification in each sample of an abundant protein with cytotoxic activity against human non-small cell lung adenocarcinoma A549 cells. The active component in each case was identified by MALDI-TOF mass fingerprinting of tryptic digests as [Ser⁴⁹]phospholipase A₂ ([Ser⁴⁹]PLA₂). An isoform of [Ser⁴⁹]PLA₂ containing the single Ala¹⁸→ Val substitution and a partially characterized [Asp⁴⁹]PLA₂ were also present in the E. coloratus venom. LC₅₀ values against A549 cells for the purified [Ser⁴⁹]PLA₂ proteins from the four species are in the range 2.9-8.5 μM. This range is not significantly different from the range of LC₅₀ values against human umbilical vein endothelial HUVEC cells (2.5-12.2 μM) indicating that the [Ser⁴⁹]PLA₂ proteins show no differential anti-tumor activity. The LC₅₀ value for [Ser⁴⁹]PLA₂ from E. ocellatus against human erythrocytes is >100 μM and the MIC values against Escherichia coli and Staphylococcus aureus are >100 μM. It is suggested that the [Ser⁴⁹]PLA₂ proteins play a major role in producing local tissue necrosis and hemorrhage at the site of envenomation.

  5. 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…

  6. The effect of centrally injected CDP-choline on respiratory system; involvement of phospholipase to thromboxane signaling pathway.

    PubMed

    Topuz, Bora B; Altinbas, Burcin; Yilmaz, Mustafa S; Saha, Sikha; Batten, Trevor F; Savci, Vahide; Yalcin, Murat

    2014-05-01

    CDP-choline is an endogenous metabolite in phosphatidylcholine biosynthesis. Exogenous administration of CDP-choline has been shown to affect brain metabolism and to exhibit cardiovascular, neuroendocrine neuroprotective actions. On the other hand, little is known regarding its respiratory actions and/or central mechanism of its respiratory effect. Therefore the current study was designed to investigate the possible effects of centrally injected CDP-choline on respiratory system and the mediation of the central cholinergic receptors and phospholipase to thromboxane signaling pathway on CDP-choline-induced respiratory effects in anaesthetized rats. Intracerebroventricularly (i.c.v.) administration of CDP-choline induced dose- and time-dependent increased respiratory rates, tidal volume and minute ventilation of male anaesthetized Spraque Dawley rats. İ.c.v. pretreatment with atropine failed to alter the hyperventilation responses to CDP-choline whereas mecamylamine, cholinergic nicotinic receptor antagonist, mepacrine, phospholipase A2 inhibitor, and neomycin phospholipase C inhibitor, blocked completely the hyperventilation induced by CDP-choline. In addition, central pretreatment with furegrelate, thromboxane A2 synthesis inhibitor, also partially blocked CDP-choline-evoked hyperventilation effects. These data show that centrally administered CDP-choline induces hyperventilation which is mediated by activation of central nicotinic receptors and phospholipase to thromboxane signaling pathway.

  7. Draft Genome Sequence of Caenibacillus caldisaponilyticus B157T, a Thermophilic and Phospholipase-Producing Bacterium Isolated from Acidulocompost

    PubMed Central

    Tsujimoto, Yoshiyuki; Saito, Ryo; Sahara, Takehiko; Kimura, Nobutada; Tsuruoka, Naoki; Shigeri, Yasushi

    2017-01-01

    ABSTRACT Caenibacillus caldisaponilyticus B157T (= NBRC 111400T = DSM 101100T), in the family Sporolactobacillaceae, was isolated from acidulocompost as a thermophilic and phospholipid-degrading bacterium. Here, we report the 3.36-Mb draft genome sequence, with a G+C content of 51.8%, to provide the genetic information coding for phospholipases. PMID:28360164

  8. A role for phospholipase D (Pld1p) in growth, secretion, and regulation of membrane lipid synthesis in yeast.

    PubMed

    Sreenivas, A; Patton-Vogt, J L; Bruno, V; Griac, P; Henry, S A

    1998-07-03

    The SEC14 gene encodes a phosphatidylinositol/phosphatidylcholine transfer protein essential for secretion and growth in yeast (1). Mutations (cki1, cct1, and cpt1) in the CDP-choline pathway for phosphatidylcholine synthesis suppress the sec14 growth defect (2), permitting sec14(ts) cki1, sec14(ts) cct1, and sec14(ts) cpt1 strains to grow at the sec14(ts) restrictive temperature. Previously, we reported that these double mutant strains also excrete the phospholipid metabolites, choline and inositol (3). We now report that these choline and inositol excretion phenotypes are eliminated when the SPO14 (PLD1) gene encoding phospholipase D1 is deleted. In contrast to sec14(ts) cki1 strains, sec14(ts) cki1 pld1 strains are not viable at the sec14(ts) restrictive temperature and exhibit a pattern of invertase secretion comparable with sec14(ts) strains. Thus, the PLD1 gene product appears to play an essential role in the suppression of the sec14(ts) defect by CDP-choline pathway mutations, indicating a role for phospholipase D1 in growth and secretion. Furthermore, sec14(ts) strains exhibit elevated Ca2+-independent, phophatidylinositol 4,5-bisphosphate-stimulated phospholipase D activity. We also propose that phospholipase D1-mediated phosphatidylcholine turnover generates a signal that activates transcription of INO1, the structural gene for inositol 1-phosphate synthase.

  9. Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2.

    PubMed

    Fuentes, Lucía; Pérez, Rebeca; Nieto, María L; Balsinde, Jesús; Balboa, María A

    2003-11-07

    Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.

  10. Polymerized soluble venom--human serum albumin

    SciTech Connect

    Patterson, R.; Suszko, I.M.; Grammer, L.C.

    1985-03-01

    Extensive previous studies have demonstrated that attempts to produce polymers of Hymenoptera venoms for human immunotherapy resulted in insoluble precipitates that could be injected with safety but with very limited immunogenicity in allergic patients. We now report soluble polymers prepared by conjugating bee venom with human serum albumin with glutaraldehyde. The bee venom-albumin polymer (BVAP) preparation was fractionated on Sephacryl S-300 to have a molecular weight range higher than catalase. /sup 125/I-labeled bee venom phospholipase A was almost completely incorporated into BVAP. Rabbit antibody responses to bee venom and bee venom phospholipase A were induced by BVAP. Human antisera against bee venom were absorbed by BVAP. No new antigenic determinants on BVAP were present as evidenced by absorption of antisera against BVAP by bee venom and albumin. BVAP has potential immunotherapeutic value in patients with anaphylactic sensitivity to bee venom.

  11. The correlation between anti phospholipase A2 specific IgE and clinical symptoms after a bee sting in beekeepers

    PubMed Central

    Matysiak, Joanna; Bręborowicz, Anna; Dereziński, Paweł; Kokot, Zenon J.

    2016-01-01

    Introduction Beekeepers are a group of people with high exposure to honeybee stings and with a very high risk of allergy to bee venom. Therefore, they are a proper population to study the correlations between clinical symptoms and results of diagnostic tests. Aim The primary aim of our study was to assess the correlations between total IgE, venom- and phospholipase A2-specific IgE and clinical symptoms after a bee sting in beekeepers. The secondary aim was to compare the results of diagnostic tests in beekeepers and in individuals with standard exposure to bees. Material and methods Fifty-four individuals were divided into two groups: beekeepers and control group. The levels of total IgE (tIgE), venom-specific IgE (venom sIgE), and phospholipase A2-specific IgE (phospholipase A2 sIgE) were analyzed. Results Our study showed no statistically significant correlation between the clinical symptoms after a sting and tIgE in the entire analyzed group. There was also no correlation between venom sIgE level and clinical symptoms either in beekeepers or in the group with standard exposure to bees. We observed a statistically significant correlation between phospholipase A2 sIgE level and clinical signs after a sting in the group of beekeepers, whereas no such correlation was detected in the control group. Significantly higher venom-specific IgE levels in the beekeepers, as compared to control individuals were shown. Conclusions In beekeepers, the severity of clinical symptoms after a bee sting correlated better with phospholipase A2 sIgE than with venom sIgE levels. PMID:27512356

  12. Phospholipase A{sub 2} is involved in the mechanism of activation of neutrophils by polychlorinated biphenyls

    SciTech Connect

    Tithof, P.K.; Schiamberg, E.; Ganey, P.E.; Peters-Golden, M.

    1996-01-01

    Aroclor 1242, a mixture of polychlorinated biphenyls (PCBs), activates neutrophils to produce superoxide anion (O{sub 2}{sup {minus}}) by a mechanism that involves phospholipase C-dependent hydrolysis of membrane phosphoinositides; however, subsequent signal transduction mechanisms are unknown. This study determines whether phospholipase A{sub 2}-dependent release of arachidonic acid is involved in PCB-induced O{sub 2}{sup {minus}} production. O{sub 2}{sup {minus}} production was measured in vitro in glycogen-elicited, rat neutrophils in the presence and absence of the inhibitors of phospholipase A{sub 2}: quinacrine, 4-bromophenacyl bromide (BPB), and manoalide. All three agents significantly decreased the amount of O{sub 2}{sup {minus}} detected during stimulation of neutrophils with Aroclor 1242. Similar inhibition occurred when neutrophils were activated with the classical stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate. The effects of BPB and manoalide were not a result of cytotoxicity or other nonspecific effects. Significant release of {sup 3}H-arachidonic acid preceded O{sub 2}{sup {minus}} production in neutrophils stimulated with Aroclor 1242 or fMLP. Manoalide, at a concentration that abolished O{sub 2}{sup {minus}} production, also inhibited the release of {sup 3}H-arachidonate. Aspirin, zileuton, or WEB 2086 did not affect Aroclor 1242-induced O{sub 2}{sup {minus}} production, suggesting that eicosanoids and platelet-activating factor are not needed for neutrophil activation by PCBs. Activation of phos-pholipase A{sub 2} and O{sub 2}{sup {minus}} production do not appear to involve the Ah receptor. These data suggest that Aroclor 1242 stimulates neutrophils to produce O{sub 2}{sup {minus}} by a mechanism that involves phospholipase A{sub 2}-dependent release of arachiodonic acid. 49 refs., 6 figs., 2 tabs.

  13. The hydrophobic amino acids involved in the interdomain association of phospholipase D1 regulate the shuttling of phospholipase D1 from vesicular organelles into the nucleus.

    PubMed

    Jang, Young Hoon; Min, Do Sik

    2012-10-31

    Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1 are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-β, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the hydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.

  14. Autoregulation of phospholipase D activity is coupled to selective induction of phospholipase D1 expression to promote invasion of breast cancer cells.

    PubMed

    Kang, Dong Woo; Park, Mi Hee; Lee, Young Jun; Kim, Hyung Sik; Lindsley, Craig W; Alex Brown, H; Min, Do Sik

    2011-02-15

    Phospholipase D (PLD) is an important signaling enzyme implicated in the control of many biological processes, including cell proliferation and survival. Despite the importance of the duration and amplitude of PLD signaling in carcinogenesis, mechanisms that regulate PLD expression remain poorly understood. In our study, we define the regulatory components of the machinery that specifies selective PLD1 induction via signals propagated through PLD activity. We demonstrate for the first time that establishment of a positive feedback loop that is dependent on enzymatic activity originating from both PLD1 and PLD2 isozymes enhances selective expression of PLD1, but not PLD2. Phosphatidic acid, the product of PLD activity, leads to an increase in the Ras-ERK/PI3K-NFκB signaling cascade and enhances binding of NFκB to the PLD1 promoter, consequently inducing selective PLD1 expression in SK-BR3 breast cancer cells. Moreover, selective PLD inhibitor suppressed epidermal growth factor-induced matrix metalloproteinase upregulation and invasion by inhibiting PLD1 expression. In conclusion, we propose that autoregulation of PLD activity might be coupled to induction of PLD1 expression, and thereby play a role in carcinogenesis.

  15. Neuronal damage by secretory phospholipase A2: modulation by cytosolic phospholipase A2, platelet-activating factor, and cyclooxygenase-2 in neuronal cells in culture.

    PubMed

    Kolko, Miriam; Rodriguez de Turco, Elena B; Diemer, Nils H; Bazan, Nicolas G

    2003-02-27

    Activation of cytosolic phospholipase A(2) (cPLA(2)) is an early event in brain injury, which leads to the formation and accumulation of bioactive lipids: platelet-activating factor (PAF), free arachidonic acid, and eicosanoids. A cross-talk between secretory PLA(2) (sPLA(2)) and cPLA(2) in neural signal transduction has previously been suggested (J Biol Chem 271:32722; 1996). Here we show, using neuronal cell cultures, an up-regulation of cPLA(2) expression and an inhibition by the selective cPLA(2) inhibitor AACOCF3 after exposure to neurotoxic concentrations of sPLA(2)-OS2. Pretreatment of neuronal cultures with recombinant PAF acetylhydrolase (rPAF-AH) or the presynaptic PAF receptor antagonist, BN52021, partially blocked neuronal cell death induced by sPLA(2)-OS2. Furthermore, selective COX-2 inhibitors ameliorated sPLA(2)-OS2-induced neurotoxicity. We conclude that sPLA(2)-OS2 activates a neuronal signaling cascade that includes activation of cPLA(2), arachidonic acid release, PAF production, and induction of COX-2.

  16. Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1.

    PubMed

    Choi, Jang Hyun; Hong, Won-Pyo; Yun, Sanguk; Kim, Hyeon Soo; Lee, Jong-Ryul; Park, Jong Bae; Bae, Yun Soo; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-10-01

    Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.

  17. Combining phospholipases and a liquid lipase for one-step biodiesel production using crude oils

    PubMed Central

    2014-01-01

    Background Enzymatic biodiesel is becoming an increasingly popular topic in bioenergy literature because of its potential to overcome the problems posed by chemical processes. However, the high cost of the enzymatic process still remains the main drawback for its industrial application, mostly because of the high price of refined oils. Unfortunately, low cost substrates, such as crude soybean oil, often release a product that hardly accomplishes the final required biodiesel specifications and need an additional pretreatment for gums removal. In order to reduce costs and to make the enzymatic process more efficient, we developed an innovative system for enzymatic biodiesel production involving a combination of a lipase and two phospholipases. This allows performing the enzymatic degumming and transesterification in a single step, using crude soybean oil as feedstock, and converting part of the phospholipids into biodiesel. Since the two processes have never been studied together, an accurate analysis of the different reaction components and conditions was carried out. Results Crude soybean oil, used as low cost feedstock, is characterized by a high content of phospholipids (900 ppm of phosphorus). However, after the combined activity of different phospholipases and liquid lipase Callera Trans L, a complete transformation into fatty acid methyl esters (FAMEs >95%) and a good reduction of phosphorus (P <5 ppm) was achieved. The combination of enzymes allowed avoidance of the acid treatment required for gums removal, the consequent caustic neutralization, and the high temperature commonly used in degumming systems, making the overall process more eco-friendly and with higher yield. Once the conditions were established, the process was also tested with different vegetable oils with variable phosphorus contents. Conclusions Use of liquid lipase Callera Trans L in biodiesel production can provide numerous and sustainable benefits. Besides reducing the costs derived from

  18. Prostacyclin receptor-independent inhibition of phospholipase C activity by non-prostanoid prostacyclin mimetics

    PubMed Central

    Chow, Kevin B S; Wong, Yung H; Wise, Helen

    2001-01-01

    Chinese hamster ovary (CHO) cells were transiently transfected with the mouse prostacyclin (mIP) receptor to examine IP agonist-mediated stimulation of [3H]-cyclic AMP and [3H]-inositol phosphate production.The prostacyclin analogues, cicaprost, iloprost, carbacyclin and prostaglandin E1, stimulated adenylyl cyclase activity with EC50 values of 5, 6, 25 and 95 nM, respectively. These IP agonists also stimulated the phospholipase C pathway with 10 – 40 fold lower potency than stimulation of adenylyl cyclase.The non-prostanoid prostacyclin mimetics, octimibate, BMY 42393 and BMY 45778, also stimulated adenylyl cyclase activity, with EC50 values of 219, 166 and 398 nM, respectively, but failed to stimulate [3H]-inositol phosphate production.Octimibate, BMY 42393 and BMY 45778 inhibited iloprost-stimulated [3H]-inositol phosphate production in a non-competitive manner.Activation of the endogenously-expressed P2 purinergic receptor by ATP led to an increase in [3H]-inositol phosphate production which was inhibited by the non-prostanoid prostacyclin mimetics in non-transfected CHO cells. Prostacyclin analogues and other prostanoid receptor ligands failed to inhibit ATP-stimulated [3H]-inositol phosphate production.A comparison between the IP receptor-specific non-prostanoid ONO-1310 and the structurally-related EP3 receptor-specific agonist ONO-AP-324, indicated that the inhibitory effect of non-prostanoids was specific for those compounds known to activate IP receptors.The non-prostanoid prostacyclin mimetics also inhibited phospholipase C activity when stimulated by constitutively-active mutant GαqRC, Gα14RC and Gα16QL transiently expressed in CHO cells. These drugs did not inhibit adenylyl cyclase activity when stimulated by the constitutively-active mutant GαsQL.These results suggest that non-prostanoid prostacyclin mimetics can specifically inhibit [3H]-inositol phosphate production by targeting Gq/11 and/or phospholipase C in CHO cells, and

  19. Design, synthesis, and biological evaluation of 3-(1-Aryl-1H-indol-5-yl)propanoic acids as new indole-based cytosolic phospholipase A2α inhibitors.

    PubMed

    Tomoo, Toshiyuki; Nakatsuka, Takashi; Katayama, Toyoko; Hayashi, Yasuhiro; Fujieda, Yusuke; Terakawa, Maki; Nagahira, Kazuhiro

    2014-09-11

    This article describes the design, synthesis, and biological evaluation of new indole-based cytosolic phospholipase A2α (cPLA2α, a group IVA phospholipase A2) inhibitors. A screening-hit compound from our library, (E)-3-{4-[(4-chlorophenyl)thio]-3-nitrophenyl}acrylic acid (5), was used to design a class of 3-(1-aryl-1H-indol-5-yl)propanoic acids as new small molecule inhibitors. The resultant structure-activity relationships studied using the isolated enzyme and by cell-based assays revealed that the 1-(p-O-substituted)phenyl, 3-phenylethyl, and 5-propanoic acid groups on the indole core are essential for good inhibitory activity against cPLA2α. Optimization of the p-substituents on the N1 phenyl group led to the discovery of 56n (ASB14780), which was shown to be a potent inhibitor of cPLA2α via enzyme assay, cell-based assay, and guinea pig and human whole-blood assays. It displayed oral efficacy toward mice tetradecanoyl phorbol acetate-induced ear edema and guinea pig ovalbumin-induced asthma models.

  20. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

    PubMed

    Kumar, Dharmendra; Banerjee, Tuhina; Pratap, Chandra Bhan; Tilak, Ragini

    2015-04-15

    Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described. Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any invasive fungemia. Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-albicans candida species over C. albicans and the diversity of Candida spp causing infections.

  1. Putative roles for phospholipase Cη enzymes in neuronal Ca2+ signal modulation.

    PubMed

    Popovics, Petra; Stewart, Alan J

    2012-02-01

    The most recently identified PLC (phospholipase C) enzymes belong to the PLCη family. Their unique Ca2+-sensitivity and their specific appearance in neurons have attracted great attention since their discovery; however, their physiological role(s) in neurons are still yet to be established. PLCη enzymes are expressed in the neocortex, hippocampus and cerebellum. PLCη2 is also expressed at high levels in pituitary gland, pineal gland and in the retina. Driven by the specific localization of PLCη enzymes in different brain areas, in the present paper, we discuss the roles that they may play in neural processes, including differentiation, memory formation, circadian rhythm regulation, neurotransmitter/hormone release and the pathogenesis of neurodegenerative disorders associated with aberrant Ca2+ signalling, such as Alzheimer's disease.

  2. Amylase, lipase, pancreatic isoamylase, and phospholipase A in diagnosis of acute pancreatitis.

    PubMed

    Clavé, P; Guillaumes, S; Blanco, I; Nabau, N; Mercé, J; Farré, A; Marruecos, L; Lluís, F

    1995-08-01

    To determine the utility of serum amylase (AMY), lipase (Lp), pancreatic isoamylase (isoA), phospholipase A (PLA), and urine AMY in the diagnosis of acute pancreatitis, samples of serum and urine were obtained on admission and every day thereafter for 5 days from 384 patients with acute abdominal pain. Diagnostic accuracy, determined as the area under the receiver operating characteristic curve, was > 0.975 for serum AMY, Lp, isoA, and urine AMY. For each of these enzymes, a threshold value (twice to sixfold the upper limit of the reference values) offering diagnostic efficiency > 95% could be determined. In contrast, accuracy and efficiency of serum PLA were low. The profiles of these enzymes in acute pancreatitis decreased in a parallel fashion over 5 days except for PLA. We conclude that diagnostic utilities are similar for serum AMY, Lp, isoA, and urine AMY for acute pancreatitis, provided that an appropriate threshold is established.

  3. Phospholipase D1-regulated autophagy supplies free fatty acids to counter nutrient stress in cancer cells

    PubMed Central

    Cai, Ming; He, Jingquan; Xiong, Jian; Tay, Li Wei Rachel; Wang, Ziqing; Rog, Colin; Wang, Jingshu; Xie, Yizhao; Wang, Guobin; Banno, Yoshiko; Li, Feng; Zhu, Michael; Du, Guangwei

    2016-01-01

    Cancer cells utilize flexible metabolic programs to maintain viability and proliferation under stress conditions including nutrient deprivation. Here we report that phospholipase D1 (PLD1) participates in the regulation of metabolic plasticity in cancer cells. PLD1 activity is required for cancer cell survival during prolonged glucose deprivation. Blocking PLD1 sensitizes cancer cells to glycolysis inhibition by 2-deoxy-D-glucose (2-DG) and results in decreased autophagic flux, enlarged lysosomes, and increased lysosomal pH. Mechanistically, PLD1-regulated autophagy hydrolyzes bulk membrane phospholipids to supply fatty acids (FAs) for oxidation in mitochondria. In low glucose cultures, the blockade of fatty acid oxidation (FAO) by PLD1 inhibition suppresses adenosine triphosphate (ATP) production and increases reactive oxygen species (ROS), leading to cancer cell death. In summary, our findings reveal a novel role of PLD1 in sustaining cancer cell survival during metabolic stress, and suggest PLD1 as a potential target for anticancer metabolism therapy. PMID:27809301

  4. Isolation and characterization of actinomycetes strains that produce phospholipase D having high transphosphatidylation activity.

    PubMed

    Nakazawa, Yozo; Uchino, Masataka; Sagane, Yoshimasa; Sato, Hiroaki; Takano, Katsumi

    2009-01-01

    The present study was conducted to screen microorganisms that produce phospholipase D (PLD), and we especially focused on the strains having high transphosphatidylation activity. Eighty bacterial strains were isolated from soil samples by a screening method utilizing a preliminary selection medium with phosphatidylcholine (PC) as the sole carbon source. The culture supernatants were then assayed for PLD activity. The finding of dual PLD activities in cultures revealed that the hydrolytic and transphosphatidylation activities were correlated. Consequently, six strains were selected as stably producing PLD enzyme(s) during continuous subcultures. The culture supernatants of selected strains synthesized phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine from PC with high conversion rates. These isolated strains will be made available to carry out phospholipid modification through the efficient transphosphatidylation activity of the PLD that they produce.

  5. The bacterium Xenorhabdus nematophila inhibits phospholipases A2 from insect, prokaryote, and vertebrate sources

    NASA Astrophysics Data System (ADS)

    Park, Youngjin; Kim, Yonggyun; Stanley, David

    The bacterium, Xenorhabdus nematophila, is a virulent insect pathogen. Part of its pathogenicity is due to impairing cellular immunity by blocking biosynthesis of eicosanoids, the major recognized signal transduction system in insect cellular immunity. X. nematophila inhibits the first step in eicosanoid biosynthesis, phospholipase A2 (PLA2). Here we report that the bacterium inhibits PLA2 from two insect immune tissues, hemocytes and fat body, as well as PLA2s selected to represent a wide range of organisms, including prokaryotes, insects, reptiles, and mammals. Our finding on a bacterial inhibitor of PLA2 activity contributes new insight into the chemical ecology of microbe-host interactions, which usually involve actions rather than inhibitors of PLA2s.

  6. Molecular cloning of phospholipases A(2) from venom glands of Echis carpet vipers.

    PubMed

    Bharati, K; Hasson, S S; Oliver, J; Laing, G D; Theakston, R D G; Harrison, R A

    2003-06-01

    Venom toxin-specific antibodies offer a more rational treatment of snake envenoming than conventional antivenom. Here, we describe novel cDNAs encoding phospholipase A(2) (PLA(2)) isoforms from venom gland RNA of Echis pyramidum leakeyi (Epl), Echis sochureki (Es) and Echis ocellatus (Eo). The deduced amino acid sequences of these cDNAs encoded proteins with high overall sequence identity to the viper group II PLA(2) protein family, including the 14 cysteine residues capable of forming seven disulphide bonds that characterize this group of PLA(2) enzymes. Comparison of the PLA(2) sequences from Echis with those from related vipers failed to make significant geographic, taxonomic or PLA(2)-function distinctions between these Echis PLA(2) isoforms. However, their deduced hydrophilicity profiles revealed a conserved tertiary structure that we will exploit, by epidermal DNA immunization, to generate PLA(2)-neutralizing antibodies with polyspecific potential.

  7. Cloning and functional expression of secreted phospholipases A(2) from Bothrops diporus (Yarará Chica).

    PubMed

    Yunes Quartino, Pablo Javier; Barra, José Luis; Fidelio, Gerardo Daniel

    2012-10-19

    Bothrops diporus is a very common viper in Argentina. At present, no complete sequence of secreted phospholipase A(2) (sPLA(2)) from this snake has been reported. We have cloned two sPLA(2) isoenzymes as well as a putative sPLA(2)-like myotoxin from venom gland. The two sPLA(2) were expressed as inclusion bodies in Escherichia coli with an N-terminal tag of ubiquitin. After in vitro renaturation and cleavage step, using an ubiquitin specific peptidase, the recombinants exhibited sPLA(2) activity when analyzed by means of Langmuir dilauroylphosphatidylcholine monolayers as substrate. Both enzymes have a similar surface pressure-activity profile when compared with non-recombinant purified isoforms. To our knowledge, this is the first time that analysis of optimal lateral pressure of substrate monolayers by using the surface barostat technique is performed on recombinant sPLA(2)s.

  8. Pharmacophore-based discovery of a novel cytosolic phospholipase A2α inhibitor

    PubMed Central

    Noha, Stefan M.; Jazzar, Bianca; Kuehnl, Susanne; Rollinger, Judith M.; Stuppner, Hermann; Schaible, Anja M.; Werz, Oliver; Wolber, Gerhard; Schuster, Daniela

    2012-01-01

    The release of arachidonic acid, a precursor in the production of prostaglandins and leukotrienes, is achieved by activity of the cytosolic phospholipase A2α (cPLA2α). Signaling mediated by this class of bioactive lipids, which are collectively referred to as eicosanoids, has numerous effects in physiological and pathological processes. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the National Cancer Institute (NCI) database, leading to the identification of 4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957) as cPLA2α inhibitor in cell-free and cell-based in vitro assays. PMID:22192589

  9. Phospholipase A2 receptor positive membranous nephropathy long after living donor kidney transplantation between identical twins.

    PubMed

    Saito, Hisako; Hamasaki, Yoshifumi; Tojo, Akihiro; Shintani, Yukako; Shimizu, Akira; Nangaku, Masaomi

    2015-07-01

    Although membranous nephropathy (MN) is a commonly observed cause of post-transplant glomerulonephritis, distinguishing de novo from recurrent MN in kidney allograft is often difficult. Phospholipase A2 receptor (PLA2R) staining is useful for diagnosing recurrent MN in allografts similarly to idiopathic MN in native kidney. No specific treatment strategy has been established for MN, especially when accompanied with HCV infection in kidney transplant recipients. This report describes a 66-year-old man who was diagnosed as having PLA2R positive membranous nephropathy accompanied with already-known IgA nephropathy and HCV infection 26 years after kidney transplantation conducted between identical twins. PLA2R was detected along capillary loops, implying that this patient is affected by the same pathogenic mechanism as idiopathic MN, not secondary MN associated with other disorders such as HCV infection. The patient successfully achieved clinical remission after steroid therapy.

  10. Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment

    PubMed Central

    Balietti, Marta; Giuli, Cinzia; Fattoretti, Patrizia; Fabbietti, Paolo; Postacchini, Demetrio; Conti, Fiorenzo

    2016-01-01

    We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A. PMID:26836161

  11. A Role for Phospholipase A2 Activity in Membrane Tubule Formation and TGN Trafficking

    PubMed Central

    Schmidt, John A.; Kalkofen, Danielle N.; Donovan, Kirk W.; Brown, William J.

    2015-01-01

    We have investigated the role of phospholipase A2 (PLA2) enzymes in generating membrane tubules at the trans-Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over-expressing kinase dead protein kinase D were inhibited by the PLA2 inhibitors ONO-RS-082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes. Finally, use of the reversible antagonist ONO and time-lapse imaging revealed for the first time that PLA2 antagonists inhibit the initiation of membrane tubule formation at the TGN. Thus, PLA2 enzymes appear to have an important role in the earliest steps of membrane tubule formation at the TGN, which are utilized for membrane trafficking. PMID:20874826

  12. Cytoplasmic Phospholipase A2 Antagonists Inhibit Multiple Endocytic Membrane Trafficking Pathways

    PubMed Central

    Doody, Anne M.; Antosh, Amy L.; Brown, William J.

    2009-01-01

    Previous studies have suggested a role for cytosolic Ca2+-independent phospholipase A2 (PLA2) activity in the formation of endosome membrane tubules that participate in the export of transferrin (Tf) and transferrin receptors (TfR) from sorting endosomes (SEs) and the endocytic recycling compartment (ERC). Here we show that the PLA2 requirement is a general feature of endocytic trafficking. The reversible cytoplasmic PLA2-antagonist ONO-RS-082 (ONO) produced a concentration-dependent, differential block in the endocytic recycling of both low-density lipoprotein receptor (LDLR) and TfRs, and in the degradative pathways of LDL and epidermal growth factor (EGF). These results are consistent with the model that a cytoplasmic PLA2 plays a general role in the export of cargo from multiple endocytic compartments by mediating the formation of membrane tubules. PMID:19695219

  13. Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment.

    PubMed

    Balietti, Marta; Giuli, Cinzia; Fattoretti, Patrizia; Fabbietti, Paolo; Postacchini, Demetrio; Conti, Fiorenzo

    2016-01-01

    We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A.

  14. [Respiration of wheat roots during inhibition of phospholipase A2 by 4-bromphenacylbromide].

    PubMed

    Valitova, Iu N; Gordon, L Kh; Ogorodnikova, T I; Lygin, A V; Ruban, N F

    2001-01-01

    Dependence of oxygen consumption by wheat root cells on the activity of phospholipase A2 (PLA2) was studied. The treatment of excised roots with 4-bromophenacile bromide (BPB), a specific inhibitor of PLA2, caused a decrease in the content of free fatty acids (FFA) and in oxygen consumption of root cells. The latter was prevented by exogenous application of a mixture of FFA. A similar inhibitory effect was caused by BPB after the activation of root respiration by 2,4-dinitrophenol (DNP). These data suggest that FFA may be involved in the regulation of respiration through the formation of succinate. This is supported by the fact of reduction of DNP-induced stimulation of oxygen consumption by malonate, known to be an inhibitor of succinate dehydrogenase, and by stimulation of respiration by exogenous application of succinate.

  15. Impaired brain development and reduced cognitive function in phospholipase D-deficient mice.

    PubMed

    Burkhardt, Ute; Stegner, David; Hattingen, Elke; Beyer, Sandra; Nieswandt, Bernhard; Klein, Jochen

    2014-06-20

    The phospholipases D (PLD1 and 2) are signaling enzymes that catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid, a lipid second messenger involved in cell proliferation, and choline, a precursor of acetylcholine (ACh). In the present study, we investigated development and cognitive function in mice that were deficient for PLD1, or PLD2, or both. We found that PLD-deficient mice had reduced brain growth at 14-27 days post partum when compared to wild-type mice. In adult PLD-deficient mice, cognitive function was impaired in social and object recognition tasks. Using brain microdialysis, we found that wild-type mice responded with a 4-fold increase of hippocampal ACh release upon behavioral stimulation in the open field, while PLD-deficient mice released significantly less ACh. These results may be relevant for cognitive dysfunctions observed in fetal alcohol syndrome and in Alzheimer' disease.

  16. Characterization of Serum Phospholipase A2 Activity in Three Diverse Species of West African Crocodiles

    PubMed Central

    Merchant, Mark; Juneau, Kate; Gemillion, Jared; Falconi, Rodolfo; Doucet, Aaron; Shirley, Matthew H.

    2011-01-01

    Secretory phospholipase A2, an enzyme that exhibits substantial immunological activity, was measured in the serum of three species of diverse West African crocodiles. Incubation of different volumes of crocodile serum with bacteria labeled with a fluorescent fatty acid in the sn-2 position of membrane lipids resulted in a volume-dependent liberation of fluorescent probe. Serum from the Nile crocodile (Crocodylus niloticus) exhibited slightly higher activity than that of the slender-snouted crocodile (Mecistops cataphractus) and the African dwarf crocodile (Osteolaemus tetraspis). Product formation was inhibited by BPB, a specific PLA2 inhibitor, confirming that the activity was a direct result of the presence of serum PLA2. Kinetic analysis showed that C. niloticus serum produced product more rapidly than M. cataphractus or O. tetraspis. Serum from all three species exhibited temperature-dependent PLA2 activities but with slightly different thermal profiles. All three crocodilian species showed high levels of activity against eight different species of bacteria. PMID:22110960

  17. Discovery of desketoraloxifene analogues as inhibitors of mammalian, Pseudomonas aeruginosa, and NAPE phospholipase D enzymes.

    PubMed

    Scott, Sarah A; Spencer, Cierra T; O'Reilly, Matthew C; Brown, Kyle A; Lavieri, Robert R; Cho, Chul-Hee; Jung, Dai-Il; Larock, Richard C; Brown, H Alex; Lindsley, Craig W

    2015-02-20

    Phospholipase D (PLD) hydrolyses cellular lipids to produce the important lipid second messenger phosphatidic acid. A PLD enzyme expressed by Pseudomonas aeruginosa (PldA) has been shown to be important in bacterial infection, and NAPE-PLD has emerged as being key in the synthesis of endocannabinoids. In order to better understand the biology and therapeutic potential of these less explored PLD enzymes, small molecule tools are required. Selective estrogen receptor modulators (SERMs) have been previously shown to inhibit mammalian PLD (PLD1 and PLD2). By targeted screening of a library of SERM analogues, additional parallel synthesis, and evaluation in multiple PLD assays, we discovered a novel desketoraloxifene-based scaffold that inhibited not only the two mammalian PLDs but also structurally divergent PldA and NAPE-PLD. This finding represents an important first step toward the development of small molecules possessing universal inhibition of divergent PLD enzymes to advance the field.

  18. Enhancing seed quality and viability by suppressing phospholipase D in Arabidopsis.

    PubMed

    Devaiah, Shivakumar P; Pan, Xiangqing; Hong, Yueyun; Roth, Mary; Welti, Ruth; Wang, Xuemin

    2007-06-01

    Seed aging decreases the quality of seed and grain and results in agricultural and economic losses. Alterations that impair cellular structures and metabolism are implicated in seed deterioration, but the molecular and biochemical bases for seed aging are not well understood. Ablation of the gene for a membrane lipid-hydrolyzing phospholipase D (PLDalpha1) in Arabidopsis enhanced seed germination and oil stability after storage or exposure of seeds to adverse conditions. The PLDalpha1-deficient seeds exhibited a smaller loss of unsaturated fatty acids and lower accumulation of lipid peroxides than did wild-type seeds. However, PLDalpha1-knockdown seeds were more tolerant of aging than were PLDalpha1-knockout seeds. The results demonstrate the PLDalpha1 plays an important role in seed deterioration and aging in Arabidopsis. A high level of PLDalpha1 is detrimental to seed quality, and attenuation of PLDalpha1 expression has the potential to improve oil stability, seed quality and seed longevity.

  19. Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting.

    PubMed

    Jackson, Angelyca A; Daniels, Emily F; Hammond, John H; Willger, Sven D; Hogan, Deborah A

    2014-10-01

    Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1%) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Δanr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions.

  20. Identification of a secretory phospholipase A2 from Papaver somniferum L. that transforms membrane phospholipids.

    PubMed

    Jablonická, Veronika; Mansfeld, Johanna; Heilmann, Ingo; Obložinský, Marek; Heilmann, Mareike

    2016-09-01

    The full-length sequence of a new secretory phospholipase A2 was identified in opium poppy seedlings (Papaver somniferum L.). The cDNA of poppy phospholipase A2, denoted as pspla2, encodes a protein of 159 amino acids with a 31 amino acid long signal peptide at the N-terminus. PsPLA2 contains a PLA2 signature domain (PA2c), including the Ca(2+)-binding loop (YGKYCGxxxxGC) and the catalytic site motif (DACCxxHDxC) with the conserved catalytic histidine and the calcium-coordinating aspartate residues. The aspartate of the His/Asp dyad playing an important role in animal sPLA2 catalysis is substituted by a serine residue. Furthermore, the PsPLA2 sequence contains 12 conserved cysteine residues to form 6 structural disulfide bonds. The calculated molecular weight of the mature PsPLA2 is 14.0 kDa. Based on the primary structure PsPLA2 belongs to the XIB group of PLA2s. Untagged recombinant PsPLA2 obtained by expression in Escherichia coli, renaturation from inclusion bodies and purification by cation-exchange chromatography was characterized in vitro. The pH optimum for activity of PsPLA2 was found to be pH 7, when using mixed micelles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Triton X-100. PsPLA2 specifically cleaves fatty acids from the sn-2 position of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and shows a pronounced preference for PC over phosphatidyl ethanolamine, -glycerol and -inositol. The active recombinant enzyme was tested in vitro against natural phospholipids isolated from poppy plants and preferably released the unsaturated fatty acids, linoleic acid and linolenic acid, from the naturally occurring mixture of substrate lipids.

  1. Secretory phospholipases A2 induce neurite outgrowth in PC12 cells.

    PubMed Central

    Nakashima, Satoru; Ikeno, Yutaka; Yokoyama, Tatsuya; Kuwana, Masakazu; Bolchi, Angelo; Ottonello, Simone; Kitamoto, Katsuhiko; Arioka, Manabu

    2003-01-01

    sPLA(2)s (secretory phospholipases A(2)) belong to a broad and structurally diverse family of enzymes that hydrolyse the sn -2 ester bond of glycerophospholipids. We previously showed that a secreted fungal 15 kDa protein, named p15, as well as its orthologue from Streptomyces coelicolor (named Scp15) induce neurite outgrowth in PC12 cells at nanomolar concentrations. We report here that both p15 and Scp15 are members of a newly identified group of fungal/bacterial sPLA(2)s. The phospholipid-hydrolysing activity of p15 is absolutely required for neurite outgrowth induction. Mutants with a reduced PLA(2) activity exhibited a comparable reduction in neurite-inducing activity, and the ability to induce neurites closely matched the capacity of various p15 forms to promote fatty acid release from live PC12 cells. A structurally divergent member of the sPLA(2) family, bee venom sPLA(2), also induced neurites in a phospholipase activity-dependent manner, and the same effect was elicited by mouse group V and X sPLA(2)s, but not by group IB and IIA sPLA(2)s. Lysophosphatidylcholine, but not other lysophospholipids, nor arachidonic acid, elicited neurite outgrowth in an L-type Ca(2+) channel activity-dependent manner. In addition, p15-induced neuritogenesis was unaffected by various inhibitors that block arachidonic acid conversion into bioactive eicosanoids. Altogether, these results delineate a novel, Ca(2+)- and lysophosphatidylcholine-dependent neurotrophin-like role of sPLA(2)s in the nervous system. PMID:12967323

  2. 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.

  3. Point of care testing of phospholipase A2 group IIA for serological diagnosis of rheumatoid arthritis

    NASA Astrophysics Data System (ADS)

    Liu, Nathan J.; Chapman, Robert; Lin, Yiyang; Mmesi, Jonas; Bentham, Andrew; Tyreman, Matthew; Abraham, Sonya; Stevens, Molly M.

    2016-02-01

    Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care.Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08423g

  4. Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D.

    PubMed

    Li, Y; Shiels, A J; Maszak, G; Byron, K L

    2001-06-01

    Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.

  5. Influence of (phospho)lipases on properties of mica supported phospholipid layers

    NASA Astrophysics Data System (ADS)

    Jurak, Malgorzata; Chibowski, Emil

    2010-08-01

    The effect of enzymes: lipase from Candida cylindracea (L Cc), phospholipase A 2 from hog pancreas (PLA 2) and phospholipase C from Bacillus cereus (PLC) to modulate wetting properties of solid supported phospholipid bilayers was studied via advancing and receding contact angle measurements of water, formamide and diiodomethane, and calculation of the surface free energy and its components from van Oss et al. (LWAB) and contact angle hysteresis (CAH) approaches. Simultaneously, topography of the studied layers was determined by Atomic Force Microscopy (AFM). The investigated lipid bilayers were transferred on mica plates from subphase of pure water by means of Langmuir-Blodgett and Langmuir-Schaefer techniques. The investigated phospolipid layers were: saturated DPPC (1,2-dipalmitoyl- sn-glycero-3-phosphocholine), unsaturated DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine), and their mixture DPPC/DOPC. The obtained results revealed that the lipid membrane degradation by the enzymes caused increase in its surface free energy due to the amphiphilic hydrolysis products, which may accumulate in the lipid bilayer. In result activity of the enzymes may increase and then break down the bilayer structure takes place. It is likely that after dissolution of the hydrolysis reaction products in the bulk phase, patches of bare mica surface are accessible, which contribute to the apparent surface free energy changes. Comparison of AFM images and the free energy changes of the layers gives better insight into changes of their properties. The observed gradual increase in the layer surface free energy allows controlling of the hydrolysis process to obtain the surfaces of defined properties.

  6. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase

    PubMed Central

    Ruisanchez, Éva; Dancs, Péter; Kerék, Margit; Németh, Tamás; Faragó, Bernadett; Balogh, Andrea; Patil, Renukadevi; Jennings, Brett L.; Liliom, Károly; Malik, Kafait U.; Smrcka, Alan V.; Tigyi, Gabor; Benyó, Zoltán

    2014-01-01

    Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1–3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase–protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.—Ruisanchez, É., Dancs, P., Kerék, M., Németh, T., Faragó, B., Balogh, A., Patil, R., Jennings, B. L., Liliom, K., Malik, K. U., Smrcka, A. V., Tigyi, G., Benyó, Z. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. PMID:24249637

  7. Importance of adenosine triphosphate in phospholipase A2-induced rabbit renal proximal tubule cell injury.

    PubMed Central

    Nguyen, V D; Cieslinski, D A; Humes, H D

    1988-01-01

    The pathogenesis of ischemic renal tubular cell injury involves a complex interaction of different processes, including membrane phospholipid alterations and depletion of high-energy phosphate stores. To assess the role of membrane phospholipid changes due to activation of phospholipases in renal tubule cell injury, suspensions enriched in rabbit renal proximal tubule segments were incubated with exogenous phospholipase A2 (PLA2). Exogenous PLA2 did not produce any significant change in various metabolic parameters reflective of cell injury in control nonhypoxic preparations despite a significant decrease in phosphatidylethanolamine (PE) and moderate increases in lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). In contrast, exogenous PLA2 treatment of hypoxic tubules resulted in a severe degree of cell injury, as demonstrated by marked declines in tubule K+ and ATP contents and significant decreases in tubule uncoupled respiratory rates, and was associated with significant phospholipid alterations, including marked declines in phosphatidylcholine (PC) and PE and significant rises in LPC, LPE, and free fatty acids (FFA). The injurious metabolic effects of exogenous PLA2 on hypoxic tubules were reversed by addition of ATP-MgCl2 to the tubules. The protective effect of ATP-MgCl2 was associated with increases in tubule PC and PE contents and declines in LPC, LPE, and FFA contents. These experiments thus indicate that an increase in exogenous PLA2 activity produces renal proximal tubule cell injury when cell ATP levels decline, at which point phospholipid resynthesis cannot keep pace with phospholipid degradation with resulting depletion of phospholipids and accumulation of lipid by-products. High-energy phosphate store depletion appears to be an important condition for exogenous PLA2 activity to induce renal tubule cell injury. PMID:3417866

  8. Rickettsial phospholipase A2 as a pathogenic mechanism in a model of cell injury by typhus and spotted fever group rickettsiae.

    PubMed

    Walker, D H; Feng, H M; Popov, V L

    2001-12-01

    Phospholipase A2 activity by typhus group rickettsiae causes hemolysis in vitro. Rickettsial phospholipase A2 has been proposed to mediate entry into the host cell, escape from the phagosome, and cause injury to host cells by both typhus and spotted fever group rickettsiae. In a rickettsial contact-associated cytotoxicity model, the interaction of Rickettsia prowazekii or R. conorii with Vero cells caused temperature-dependent release of 51Cr from the cells. Treatment of rickettsiae, but not the cells, with a phospholipase A2 inhibitor (bromophenacyl bromide) or with antibody to king cobra venom inhibited cell injury. Rickettsial treatment with bromophenacyl bromide inhibited the release of free fatty acids from the host cell. Neither the inhibitor nor antivenom impaired rickettsial active transport of L-lysine. Thus, host cell injury was mediated by a rickettsial phospholipase A2-dependent mechanism.

  9. Effect of Darapladib Treatment on Endarterectomy Carotid Plaque Lipoprotein-Associated Phospholipase A2 Activity: A Randomized, Controlled Trial

    PubMed Central

    Johnson, Joel L.; Shi, Yi; Snipes, Rose; Janmohamed, Salim; Rolfe, Timothy E.; Davis, Bill; Postle, Anthony; Macphee, Colin H.

    2014-01-01

    Background The aim of this study was to assess the effects of darapladib, a selective oral investigational lipoprotein-associated phospholipase A2 inhibitor, on both plasma and plaque lipoprotein-associated phospholipase A2 activity. Methods Patients undergoing elective carotid endarterectomy were randomized to darapladib 40 mg (n = 34), 80 mg (n = 34), or placebo (n = 34) for 14 days, followed by carotid endarterectomy 24 hours after the last dose of study medication. Results Darapladib 40 mg and 80 mg reduced plasma lipoprotein-associated phospholipase A2 activity by 52% and 81%, respectively, versus placebo (both P<0.001). Significant reductions in plaque lipoprotein-associated phospholipase A2 activity were also observed compared with placebo (P<0.0001), which equated to a 52% and 80% decrease compared with placebo. No significant differences were observed between groups in plaque lysophosphatidylcholine content or other biomarkers, although a dose-dependent decrease in plaque matrix metalloproteinase-9 mRNA expression was observed with darapladib 80 mg (P = 0.053 vs placebo). In a post-hoc analysis, plaque caspase-3 (P<0.001) and caspase-8 (P<0.05) activity were found to be significantly lower in the darapladib 80-mg group versus placebo. No major safety concerns were identified in the study. Conclusions Short-term treatment (14±4 days) with darapladib produced a robust, dose-dependent reduction in plasma lipoprotein-associated phospholipase A2 activity. More importantly, darapladib demonstrated placebo-corrected reductions in carotid plaque lipoprotein-associated phospholipase A2 activity of similar magnitude. Darapladib was generally well tolerated and no safety concerns were identified. Additional studies of longer duration are needed to explore whether these pharmacodynamic effects are associated with improved clinical outcomes, as might be hypothesized. Trial Registration Information Name of Registry 1: ClinicalTrials.gov Registry Number

  10. Oxytocin signalling in human myometrium.

    PubMed

    Phaneuf, S; Europe-Finner, G N; Carrasco, M P; Hamilton, C H; López Bernal, A

    1995-01-01

    A physiological role for oxytocin in stimulating uterine contractions during labour is well accepted, but has not yet been well defined. Oxytocin activates phospholipase C to produce inositol 1,4,5-trisphosphate, which releases Ca2+ from intracellular stores. There is considerable evidence that G-proteins are involved in this signalling pathway. The objectives of the present study were to determine the mechanisms of action of oxytocin in human myometrium. We have measured the effect of oxytocin on the formation of inositol phosphates (InsPs) in cultured human myometrial cells labelled with [3H] inositol and on changes in intracellular free Ca2+ concentration ([Ca2+i]) in single cells using a dynamic calcium imaging system. Pertussis toxin was used to obtain information on the G-proteins involved. Oxytocin induced InsPs formation and [Ca2+i] mobilisation in a concentration-dependent manner in human myometrial cells. Our data suggest that two distinct types of G-proteins are involved in the oxytocin response: one most probably a member of the Gq family (pertussis toxin-resistant) and another of the Gi family (pertussis toxin-sensitive). Using Western blotting, we have found that the pertussis toxin-resistant G-proteins alpha(q), alpha(11) and alpha(2), and pertussis toxin-sensitive alpha(i1), alpha(i2), and alpha(i3) are expressed in these cells. We have also detected the phospholipase C isoforms beta(1), beta(2) and beta(3) which are regulated by G-proteins, and phospholipase C isoforms gamma(1) and gamma(2), regulated by receptor tyrosine kinase pathways. However, oxytocin does not stimulate tyrosine phosphorylation in myometrial cells. Extracellular Ca2+ does not play a direct role in the activation of phospholipase C by oxytocin. Protein kinase C causes a strong inhibitory feedback on the oxytocin pathway: protein kinase C activators abolish the response to oxytocin while inhibitors potentiate it. Oxytocin responsiveness is upregulated by incubating the cells in

  11. Activity of phospholipase C and release of prostaglandin F2 alpha by endometrial tissue from ovariectomized ewes receiving progesterone and estradiol.

    PubMed

    Raw, R E; Silvia, W J

    1991-03-01

    Progesterone and estradiol interact to regulate secretion of prostaglandin (PG) F2 alpha from the ovine endometrium in response to oxytocin. Two experiments were conducted to determine if these effects were due to changes in activity of phospholipase C or in the second messenger responsive pathways that regulate production of PGF2 alpha. In both experiments, ovariectomized ewes were assigned to one of four treatment groups (control, estradiol, progesterone, progesterone and estradiol). Steroids were administered, in vivo, to mimic the changes that occur during the estrous cycle. On Day 16 of steroid treatment, endometrial tissue was collected and incubated, in vitro, to measure activity of phospholipase C and release of PGF2 alpha. Treatment with progesterone, in vivo, enhanced basal and oxytocin-induced activity of phospholipase C and release of PGF2 alpha, in vitro. Estradiol suppressed oxytocin-induced activity of phospholipase C, both in the presence and absence of progesterone. In contrast to its effects on phospholipase C, estradiol inhibited basal and oxytocin-induced release of PGF2 alpha when administered alone, but not when administered with progesterone. Steroids had similar effects on the release of PGF2 alpha induced by phorbol 12-myristate 13-acetate and A23187. It was concluded that progesterone and estradiol regulate endometrial release of PGF2 alpha by affecting both the activity of phospholipase C and its associated second messenger responsive pathways that may regulate production of PGF2 alpha.

  12. Purification, sequencing, and phylogenetic analyses of novel Lys-49 phospholipases A(2) from the venoms of rattlesnakes and other pit vipers.

    PubMed

    Tsai, I H; Chen, Y H; Wang, Y M; Tu, M C; Tu, A T

    2001-10-15

    Basic phospholipase A(2) homologs with Lys49 substitution at the essential Ca(2+)-binding site are present in the venom of pit vipers under many genera. However, they have not been found in rattlesnake venoms before. We have now screened for this protein in the venom of rattlesnakes and other less studied pit vipers. By gel filtration chromatography and RP-HPLC, Lys49-phospholipase-like proteins were purified from the venoms of two rattlers, Crotalus atrox and Crotalus m. molossus, and five nonrattlers, Porthidium nummifer, Porthidium godmani, Bothriechis schlegelii, Trimeresurus puniceus, and Trimeresurus albolabris. Their N-terminal amino acid sequences were shown to be characteristic for this phospholipase subfamily. The purified basic proteins from rattlesnakes caused myonecrosis and edema in experimental animals. We have also cloned the cDNAs and solved the complete sequences of four novel Lys49-phospholipases from the venom glands of C. atrox, P. godmani, B. schlegelii, and Deinagkistrodon acutus (hundred-pace). Phylogenetic analyses based on the amino acid sequences of 28 Lys49-phospholipases separate the pitviper of the New World from those of the Old World, and the arboreal Asiatic species from the terrestrial Asiatic species. The implications of the phylogeny tree to the systematics of pit vipers, and structure-function relationship of the Lys49-phospholipases are discussed.

  13. Cytosolic phospholipase A2 regulates alcohol-mediated astrocyte inflammatory responses in HIV-associated neurocognitive disorders

    PubMed Central

    Pandey, R; Ghorpade, A

    2015-01-01

    Alcohol (EtOH) abuse and HIV-1 infection remain leading public health problems not only in the United States but also across the world. Alcohol abusers have a significantly greater risk of HIV-1 infection than non-drinkers globally. In the United States, prevalence of EtOH abuse is over two-fold higher in HIV-1-positive individuals than that of the general population. Although alcohol abusers show neurodegeneration, exacerbated neuroinflammation and oxidative damage, the mechanism(s) by which EtOH regulates astrocyte inflammatory responses in HIV-associated neurocognitive disorders is unknown. Thus, we explored signaling pathway(s) involved in EtOH-mediated activation of human astrocytes with HIV-1 and subsequent alterations in their inflammatory functions. Alcohol exposure altered the morphology of astrocytes, proinflammatory responses and induced cytotoxicity in a dose-dependent manner. Time-dependent changes were also evaluated. EtOH and HIV-1 cotreatment decreased cell viability and proliferation, while increasing apoptosis and mitochondrial depolarization. EtOH and HIV-1 together increased the levels of proinflammatory molecules, interleukin-1β, tumor necrosis factor-α, CXCL8, tissue inhibitor of metalloproteinases-1 and more importantly, arachidonic acid, a known downstream target of cytosolic phospholipase A2 (cPLA2). Consistent with this observation, phospho-cPLA2 levels were augmented in HIV-1 and EtOH cotreatment as compared with HIV-1 or EtOH alone. Cyclooxygenase 2 was upregulated as measured by real-time PCR and western blot, whereas cotreatment of HIV-1 and EtOH decreased cytochrome P450-2E1 levels as compared with EtOH alone. Furthermore, we confirmed that blocking cPLA2 with arachidonyl tri floro methyl ketone, a cPLA2-specific inhibitor, effectively prevented cPLA2 phosphorylation and downstream outcomes. Thus, the present findings suggest that cPLA2 has a critical role in alcohol and HIV-induced astrocyte inflammation. In the future, cPLA2

  14. Inhibition of phosphatidylcholine-specific phospholipase C downregulates HER2 overexpression on plasma membrane of breast cancer cells

    PubMed Central

    2010-01-01

    Introduction Overexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop. We recently showed that inactivation of phosphatidylcholine-specific phospholipase C (PC-PLC) may exert a pivotal role in selectively modulating the expression on the membrane of specific receptors or proteins relevant to cell function. In the present study, we investigated the capability of PC-PLC inhibition to target the molecular mechanisms controlling HER2 overexpression on the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation. Methods Localization on the membrane and interaction of PC-PLC with HER2, EGFR, and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, by using confocal laser scanning microscopy, flow cytometry, cell-surface biotinylation, isolation of lipid rafts, and immunoprecipitation experiments. The effects of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) on HER2 expression on the membrane and on the levels of overall HER2, HER2-HER3, and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, after either transient or continuous receptor engagement with anti-HER2 monoclonal antibodies, including trastuzumab. Changes of HER2 expression and cell proliferation were examined in SKBr3, BT-474, and MDA-MB-453 cells continuously exposed to D609 alone or combined with trastuzumab. Results PC-PLC selectively accumulates on the plasma membrane of HER2-overexpressing cells, where it colocalizes and associates with

  15. Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages* ♦

    PubMed Central

    Sokolowska, Milena; Chen, Li-Yuan; Eberlein, Michael; Martinez-Anton, Asuncion; Liu, Yueqin; Alsaaty, Sara; Qi, Hai-Yan; Logun, Carolea; Horton, Maureen; Shelhamer, James H.

    2014-01-01

    Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4−/− and Myd88−/− mice, but not in Cd44−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2high and COX1/ALOX15/ALOX5/LTA4Hlow gene and PGE2/PGD2/15-HETEhigh and LXA4low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism. PMID:24366870

  16. Secreted phospholipase A2 inhibitors are also potent blockers of binding to the M-type receptor.

    PubMed

    Boilard, Eric; Rouault, Morgane; Surrel, Fanny; Le Calvez, Catherine; Bezzine, Sofiane; Singer, Alan; Gelb, Michael H; Lambeau, Gérard

    2006-11-07

    Mammalian secreted phospholipases A(2) (sPLA(2)s) constitute a family of structurally related enzymes that are likely to play numerous biological roles because of their phospholipid hydrolyzing activity and binding to soluble and membrane-bound proteins, including the M-type receptor. Over the past decade, a number of competitive inhibitors have been developed against the inflammatory-type human group IIA (hGIIA) sPLA(2) with the aim of specifically blocking its catalytic activity and pathophysiological functions. The fact that many of these inhibitors, including the indole analogue Me-Indoxam, inhibit several other sPLA(2)s that bind to the M-type receptor prompted us to investigate the impact of Me-Indoxam and other inhibitors on the sPLA(2)-receptor interaction. By using a Ca(2+) loop mutant derived from a venom sPLA(2) which is insensitive to hGIIA inhibitors but still binds to the M-type receptor, we demonstrate that Me-Indoxam dramatically decreases the affinity of various sPLA(2)s for the receptor, yet an sPLA(2)-Me-Indoxam-receptor complex can form at very high sPLA(2) concentrations. Me-Indoxam inhibits the binding of iodinated mouse sPLA(2)s to the mouse M-type receptor expressed on live cells but also enhances binding of sPLA(2) to phospholipids. Because Me-Indoxam and other competitive inhibitors protrude out of the sPLA(2) catalytic groove, it is likely that the inhibitors interfere with the sPLA(2)-receptor interaction by steric hindrance and to different extents that depend on the type of sPLA(2) and inhibitor. Our finding suggests that the various anti-inflammatory therapeutic effects of sPLA(2) inhibitors may be due not only to inhibition of enzymatic activity but also to modulation of binding of sPLA(2) to the M-type receptor or other as yet unknown protein targets.

  17. A novel Time-resolved Fluoroimmunoassay for the quantitative detection of Antibodies against the Phospholipase A2 Receptor

    PubMed Central

    Huang, Biao; Wang, Liang; Zhang, Yi; Zhang, Jue; Zhang, Qiuhua; Xiao, Hualong; Zhou, Bin; Sun, Zhuxing; Cao, Ya-nan; Chen, Yu; Hu, Zhigang; Sheng, Huiming

    2017-01-01

    A highly sensitive time-resolved fluoroimmunoassay (TRFIA) was developed to quantify serum antibodies against the phospholipase A2 receptor (anti-PLA2R-IgG) for differential diagnosis of membranous nephropathy. Recombinant PLA2R (rPLA2R) was coated onto 96-well plates as a capture. A goat-anti-human IgG tracer was prepared with europium-chelate for detection. After bound/free separation by washing, the fluorescence counts of bound tracer were measured for quantifying serum anti-PLA2R-IgG concentration. A purified anti-PLA2R-IgG calibrator was first prepared for ensuring that consistent quantitative results could be obtained. The assay detection limit was 0.03 mg/L with linear measurement range of 0.03–340 mg/L. The intra- and inter-assay coefficients of variation (CVs) were 3.8% and 6.2%, respectively. The average serum anti-PLA2R-IgG concentration in 45 healthy volunteers, 31 IgA nephropathy, 9 lupus nephropathy, and 52 idiopathic membranous nephropathy patients was 0.53 ± 0.18 mg/L, 0.70 ± 0.41 mg/L, 1.08 ± 0.65 mg/L, and 9.00 ± 11.82 mg/L, respectively. The cut-off point for an abnormal anti-PLA2R-IgG concentration was defined as >0.89 mg/L. The positive rates in serum from patients with IgA nephropathy, lupus nephropathy, and idiopathic membranous nephropathy were 29.0%, 44.4%, and 88.5%, respectively. The availability of this quantitation method will facilitate the use of serum anti-PLA2R-IgG for diagnosing idiopathic membranous nephropathy.

  18. Endothelin stimulates phospholipase C, Na+/H+ exchange, c-fos expression, and mitogenesis in rat mesangial cells.

    PubMed Central

    Simonson, M S; Wann, S; Mené, P; Dubyak, G R; Kester, M; Nakazato, Y; Sedor, J R; Dunn, M J

    1989-01-01

    A recently described peptide hormone, endothelin, is a potent vasoconstrictor, but it is unclear whether endothelin has other biological actions. These experiments extend the range of biological actions of endothelin to stimulation of mitogenesis. Endothelin at low concentrations (0.1-10 nM) induced mitogenesis by quiescent rat glomerular mesangial cells in culture. Mitogenesis induced by endothelin was accompanied by activation of phospholipase C with increased inositol phosphate turnover and increments of intracellular [Ca2+]. Endothelin also activated Na+/H+ exchange, causing cytosolic alkalinization, and enhanced transcription of the c-fos protooncogene, additional biochemical signals closely linked to proliferation. In addition to being a vasoconstrictor, endothelin thus also functions as a mitogen, presumably through activation of phospholipase C. Images PMID:2536405

  19. Membrane translocation of protein kinase Ctheta during T lymphocyte activation requires phospholipase C-gamma-generated diacylglycerol.

    PubMed

    Díaz-Flores, Ernesto; Siliceo, María; Martínez-A, Carlos; Mérida, Isabel

    2003-08-01

    Protein kinase C (PKC) is the only PKC isoform recruited to the immunological synapse after T cell receptor stimulation, suggesting that its activation mechanism differs from that of the other isoforms. Previous studies have suggested that this selective PKC recruitment may operate via a Vav-regulated, cytoskeletal-dependent mechanism, independent of the classical phospholipase C/diacylglycerol pathway. Here, we demonstrate that, together with tyrosine phosphorylation of PKC in the regulatory domain, binding of phospholipase C-dependent diacylglycerol is required for PKC recruitment to the T cell synapse. In addition, we demonstrate that diacylglycerol kinase alpha-dependent diacylglycerol phosphorylation provides the negative signal required for PKC inactivation, ensuring fine control of the T cell activation response.

  20. Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae.

    PubMed

    Petrovic, Uros; Sribar, Jernej; Matis, Maja; Anderluh, Gregor; Peter-Katalinić, Jasna; Krizaj, Igor; Gubensek, Franc

    2005-10-15

    Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to g and e isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.

  1. Developmental Regulation of the (1,3)-beta-Glucan (Callose) Synthase from Tomato : Possible Role of Endogenous Phospholipases.

    PubMed

    Ma, S; Gross, K C; Wasserman, B P

    1991-06-01

    Activity levels of UDP-glucose: (1,3)-beta-glucan (callose) synthase in microsomal membranes of pericarp tissue from tomato fruit (Lycoperisicon esculentum Mill, cv Rutgers) were determined during development and ripening. Addition of the phospholipase inhibitors O-phosphorylcholine and glycerol-1-phosphate to homogenization buffers was necessary to preserve enzyme activity during homogenization and membrane isolation. Enzyme activity declined 90% from the immature green to the red ripe stage. The polypeptide composition of the membranes did not change significantly during ripening. The enzyme from immature fruit was inactivated by exogenously added phospholipases A(2), C, and D. These results suggest that the decline in callose synthase activity during ontogeny may be a secondary effect of endogenous lipase action.

  2. Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system

    PubMed Central

    He, Yan; Li, Linghua; Hu, Fengyu; Chen, Wanshan; Lei, Huali; Chen, Xiejie; Cai, Weiping; Tang, Xiaoping

    2016-01-01

    Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS–polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation. PMID:27876784

  3. Comparison Between Biofilm Production, Phospholipase and Haemolytic Activity of Different Species of Candida Isolated from Dental Caries Lesions in Children

    PubMed Central

    Shenoy, Neetha

    2016-01-01

    Introduction C.albicans is the most commonly isolated fungal pathogen in the oral cavity, but isolation of non-albicans Candida is increasing in recent years. We wish to demonstrate the virulence factors of Candida spp. isolated from the dental caries lesion of the children as presence of virulence factors determines the pathogenic potential of any microorganism. Aim To compare biofilm production, phospholipase and haemolytic activity of C.albicans with that of non-albicans species of Candida isolated from dental caries lesions of children to evaluate the role of non- albicans species of Candida in formation of dental caries. Materials and Methods Oral swabs were collected from caries lesion of 100 school children of age 5-10 years with dental caries. Candida isolates were tested for biofilm production, phospholipase and haemolytic activity. Statistical analysis was done by Chi-Square test and Mann-Whitney U test wherever applicable using SPSS version 11.5. Results Out of the 100 children with dental caries 37 were positive for Candida by smear or culture and 31 by culture. C.albicans was the most prevalent isolate followed by C.krusei, C.tropicalis and C.albicans. Out of 21 C.albicans isolates, 10 (47.6%) showed phospholipase activity and 18 (85.71%) produced biofilm. Of the 10 non-albicans strains, 5 (50%) showed phospholipase activity and 6 (60%) produced biofilm. All isolates of Candida produced haemolysin (100%). Conclusion There was no statistically relevant difference between the virulence factor production by C.albicans and non-albicans species of Candida. In other words, our study shows that both C.albicans and non-albicans species of Candida isolated from caries lesions of the children, produce these virulence factors. So we can say that non-albicans species of Candida also are involved in caries formation. PMID:27190803

  4. Roles of various phospholipases A2 in providing lysophospholipid acceptors for fatty acid phospholipid incorporation and remodelling.

    PubMed Central

    Balsinde, Jesús

    2002-01-01

    In the present study the lysophospholipid sources for arachidonic (AA) and eicosapentaenoic acid (EPA) incorporation into and redistribution within the phospholipids of phorbol-ester-differentiated U937 cells was investigated. Initially, AA incorporated primarily into choline glycerophospholipids (PC), whereas EPA incorporated mainly into ethanolamine glycerophospholipids (PE). Bromoenol lactone (BEL), an inhibitor of the Group VI Ca2+-independent phospholipase A2 (iPLA2), diminished both lysophosphatidylcholine levels and the incorporation of AA into phospholipids. However BEL had little effect on EPA incorporation. In concanavalin A-activated cells, EPA, but not AA, incorporation was also affected by methyl arachidonyl fluorophosphonate (MAFP), suggesting an additional role for the group IV cytosolic phospholipase A2. In the activated cells AA and EPA did not compete with each other for incorporation, indicating that the pathways for AA and EPA incorporation are partially different. The AA and EPA initially incorporated into PC slowly moved to PE in a process that took several hours. The transfer of AA and EPA from PC to PE was not inhibited by BEL, MAFP or LY311727 [3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulphonic acid], raising the possibility that an as-yet-undetermined phospholipase A2 may be involved in fatty acid phospholipid remodelling. A strong candidate to be involved in these reactions is a novel Ca2+-independent phospholipase A2 that, unlike all known iPLA2s, is resistant to inhibition by BEL and also to MAFP and LY311727. The enzyme activity cleaves both PC and PE and is thus able to provide the lysoPC and lysoPE acceptors required for the fatty acid acylation reactions. PMID:12049633

  5. 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.

  6. Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles.

    PubMed

    Chiou, Yi-Ling; Chen, Ying-Jung; Lin, Shinne-Ren; Chang, Long-Sen

    2011-11-01

    CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions.

  7. Phospholipase D1 Couples CD4+ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication

    PubMed Central

    Taylor, Harry E.; Simmons, Glenn E.; Mathews, Thomas P.; Khatua, Atanu K.; Popik, Waldemar; Lindsley, Craig W.; D’Aquila, Richard T.; Brown, H. Alex

    2015-01-01

    Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis. PMID:26020637

  8. Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

    PubMed

    Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H; Boilard, Eric

    2015-01-01

    Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

  9. Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

    PubMed Central

    Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A.; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H.; Boilard, Eric

    2015-01-01

    Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes. PMID:25587983

  10. A Single Nucleotide Polymorphism in the Phospholipase D1 Gene is Associated with Risk of Non-Small Cell Lung Cancer

    PubMed Central

    Ahn, Myung-Ju; Park, Shin-Young; Kim, Won Kyu; Cho, Ju Hwan; Chang, Brian Junho; Kim, Dong Jo; Ahn, Jin Seok; Park, Keunchil; Han, Joong-Soo

    2012-01-01

    Phospholipase D (PLD) has an important role in various biological functions including vesicular transport, endocytosis, exocytosis, cell migration, and mitosis. These cellular biological processes are deregulated in the development of various human tumors. In order to explore the relationship between the PLD1 gene and risk of non-small cell lung cancer (NSCLC), single nucleotide polymorphisms (SNP) in the PLD1 exon region were surveyed in 211 NSCLC patients and 205 normal controls. In this study, we identified six SNPs at exon 23 in the PLD1 gene. Among the six SNPs, the most notable was a heterozygous A to C transition at nucleotide 2698 (A2698C, p<0.001). In addition, the genotype frequencies of A2744C (AC+CC) and A2756C (AC+CC) were associated with gender (female, A2744C and A2756C: p=0.071) in NSCLC patients. Interestingly, although the SNP A2698C did not cause change in amino acid, correlation between odd ratio of NSCLC patients and the SNP A2698C was observed to be statistically significant. PMID:23675264

  11. Requirement of Phospholipase C and Protein Kinase C in Cholecystokinin-mediated Facilitation of NMDA Channel Function and Anxiety-like Behavior

    PubMed Central

    Xiao, Zhaoyang; Jaiswal, Manoj; Deng, Pan-Yue; Matsui, Toshimitsu; Shin, Hee-Sup; Porter, James E.; Lei, Saobo

    2011-01-01

    Whereas cholecystokinin (CCK) has long been known to exert anxiogenic effects in both animal anxiety models and humans, the underlying cellular and molecular mechanisms are ill-defined. CCK interacts with CCK-1 and CCK-2 receptors resulting in up-regulation of phospholipase C (PLC) and protein kinase C (PKC). However, the roles of PLC and PKC in CCK-mediated anxiogenic effects have not been determined. We have shown previously that CCK facilitates glutamate release in the hippocampus especially at the synapses formed by the perforant path and dentate gyrus granule cells via activations of PLC and PKC. Here we further demonstrated that CCK enhanced NMDA receptor function in dentate gyrus granule cells via activation of PLC and PKC pathway. At the single-channel level, CCK increased NMDA single-channel open probability and mean open time, reduced the mean close time and had no effects on the conductance of NMDA channels. Because elevation of glutamatergic functions results in anxiety, we explored the roles of PLC and PKC in CCK-induced anxiogenic actions using the Vogel Conflict Test (VCT). Our results from both pharmacological approach and knockout mice demonstrated that microinjection of CCK into the dentate gyrus concentration-dependently increased anxiety-like behavior via activation of PLC and PKC. Our results provide a novel unidentified signaling mechanism whereby CCK increases anxiety. PMID:22072552

  12. Lipoprotein-associated phospholipase A2 as a predictive biomarker of sub-clinical inflammation in cardiovascular diseases

    PubMed Central

    Cojocaru, Manole; Cojocaru, Inimioara Mihaela; Silosi, Isabela

    2010-01-01

    ABSTRACT Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a predictor biomarker for incident atherosclerotic disease. Lp-PLA2 has been identified in atherosclerotic plaques, however, its role in atherosclerosis is still under investigation. Lp-PLA2 belongs to the superfamily of phospholipase A2 enzymes. It is produced by macrophages that appears to play a role in the atherosclerotic vessel wall. Emerging data seem to suggest that Lp-PLA2 may be proatherogenic, which is an effect thought to be mediated by lypophosphatidylcholine and oxidized nonesterified fatty acids, two mediators generated by Lp-PLA2. Phospholipase A2 plays an essential role in metabolism of membrane phospholipids, it is related to inflammatory reactions, secretion of amyloid precursor protein. Several studies have documented the strong association of Lp-PLA2 with coronary heart disease and stroke in the general population. Lp-PLA2 may be a stronger predictor of recurrent stroke risk. Inflammatory markers have been associated with ischemic stroke risk. Their relationship to prognosis after stroke is unsettled. The present review article focuses particularly on the characteristics of the Lp(a)-associated Lp-PLA2 and discusses the possible role of this enzyme in view of the new data. PMID:21977119

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

    PubMed

    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.

  14. Interaction of a trehalose lipid biosurfactant produced by Rhodococcus erythropolis 51T7 with a secretory phospholipase A2.

    PubMed

    Zaragoza, Ana; Teruel, José A; Aranda, Francisco J; Ortiz, Antonio

    2013-10-15

    Trehalose-containing glycolipid biosurfactants form an emerging group of interesting compounds, which alter the structure and properties of phospholipid membranes, and interact with enzymatic and non-enzymatic proteins. Phospholipases A2 constitute a class of enzymes that hydrolyze the sn-2 ester of glycerophospholipids, and are classified into secreted phospholipases A2 (sPLA2) and intracellular phospholipases A2. In this work, pancreatic sPLA2 was chosen as a model enzyme to study the effect of the trehalose lipid biosurfactant on enzymes acting on interfaces. By using this enzyme, it is possible to study the modulation of enzyme activity, either by direct interaction of the biosurfactant with the protein, or as a result of the incorporation of the glycolipid on the phospholipid target membrane. It is shown that the succinoyl trehalose lipid isolated from Rhodococcus erythropolis 51T7 interacts with porcine pancreatic sPLA2 and inhibits its catalytic activity. Two modes of inhibition are observed, which are clearly differentiated by its timescale. First, a slow inhibition of sPLA2 activity upon preincubation of the enzyme with trehalose lipid in the absence of substrate is described. Second, incorporation of trehalose lipid into the phospholipid target membrane gives rise to a fast enzyme inhibition. These results are discussed in the light of previous data on sPLA2 inhibitors and extend the list of interesting biological activities reported for this R. erythropolis trehalose lipid biosurfactant.

  15. Real-time Visualization of Phospholipid Degradation by Outer Membrane Phospholipase A using High-Speed Atomic Force Microscopy.

    PubMed

    Rangl, Martina; Rima, Luca; Klement, Jessica; Miyagi, Atsushi; Keller, Sandro; Scheuring, Simon

    2017-03-07

    Phospholipases are abundant in various types of cells and compartments, where they play key roles in physiological processes as diverse as digestion, cell proliferation, and neural activation. In Gram-negative bacteria, outer membrane phospholipase A (OmpLA) is involved in outer-membrane lipid homeostasis and bacterial virulence. Although the enzymatic activity of OmpLA can be probed with an assay relying on an artificial monoacyl thioester substrate, only little is known about its activity on diacyl phospholipids. Here, we used high-speed atomic force microscopy (HS-AFM) to directly image enzymatic phospholipid degradation by OmpLA in real time. In the absence of Ca(2+), reconstituted OmpLA diffused within a phospholipid bilayer without revealing any signs of phospholipase activity. Upon addition of Ca(2+), OmpLA was activated and degraded the membrane with a turnover of ~2 phospholipid molecules per second, per OmpLA dimer until most of the membrane phospholipids were hydrolyzed and the protein became tightly packed.

  16. Lipoprotein-associated phospholipase A2 as a predictive biomarker of sub-clinical inflammation in cardiovascular diseases.

    PubMed

    Cojocaru, Manole; Cojocaru, Inimioara Mihaela; Silosi, Isabela

    2010-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a predictor biomarker for incident atherosclerotic disease. Lp-PLA2 has been identified in atherosclerotic plaques, however, its role in atherosclerosis is still under investigation. Lp-PLA2 belongs to the superfamily of phospholipase A2 enzymes. It is produced by macrophages that appears to play a role in the atherosclerotic vessel wall. Emerging data seem to suggest that Lp-PLA2 may be proatherogenic, which is an effect thought to be mediated by lypophosphatidylcholine and oxidized nonesterified fatty acids, two mediators generated by Lp-PLA2. Phospholipase A2 plays an essential role in metabolism of membrane phospholipids, it is related to inflammatory reactions, secretion of amyloid precursor protein. Several studies have documented the strong association of Lp-PLA2 with coronary heart disease and stroke in the general population. Lp-PLA2 may be a stronger predictor of recurrent stroke risk. Inflammatory markers have been associated with ischemic stroke risk. Their relationship to prognosis after stroke is unsettled. The present review article focuses particularly on the characteristics of the Lp(a)-associated Lp-PLA2 and discusses the possible role of this enzyme in view of the new data.

  17. Phospholipase A2 as a point of care alternative to serum amylase and pancreatic lipase

    NASA Astrophysics Data System (ADS)

    Liu, Nathan J.; Chapman, Robert; Lin, Yiyang; Bentham, Andrew; Tyreman, Matthew; Philips, Natalie; Khan, Shahid A.; Stevens, Molly M.

    2016-06-01

    Acute pancreatitis is a relatively common and potentially fatal condition, but the presenting symptoms are non-specific and diagnosis relies largely on the measurement of amylase activity by the hospital clinical laboratory. In this work we develop a point of care test for pancreatitis measuring concentration of secretory phospholipase A2 group IB (sPLA2-IB). Novel antibodies for sPLA2-IB were raised and used to design an ELISA and a lateral flow device (LFD) for the point of care measurement of sPLA2-IB concentration, which was compared to pancreatic amylase activity, lipase activity, and sPLA2-IB activity in 153 serum samples. 98 of these samples were obtained from the pathology unit of a major hospital and classified retrospectively according to presence or absence of pancreatitis, and the remaining 55 were obtained from commercial sources to serve as high lipase (n = 20), CA19-9 positive (n = 15), and healthy (n = 20) controls. sPLA2-IB concentration correlated well with the serum activity of both amylase and lipase, and performed at least as well as either markers in the differentiation of pancreatitis from controls.Acute pancreatitis is a relatively common and potentially fatal condition, but the presenting symptoms are non-specific and diagnosis relies largely on the measurement of amylase activity by the hospital clinical laboratory. In this work we develop a point of care test for pancreatitis measuring concentration of secretory phospholipase A2 group IB (sPLA2-IB). Novel antibodies for sPLA2-IB were raised and used to design an ELISA and a lateral flow device (LFD) for the point of care measurement of sPLA2-IB concentration, which was compared to pancreatic amylase activity, lipase activity, and sPLA2-IB activity in 153 serum samples. 98 of these samples were obtained from the pathology unit of a major hospital and classified retrospectively according to presence or absence of pancreatitis, and the remaining 55 were obtained from commercial sources to

  18. SKELETAL MUSCLE GROUP VIA PHOSPHOLIPASE A2 (iPLA2β): EXPRESSION AND ROLE IN FATTY ACID OXIDATION†

    PubMed Central

    Carper, Michael J.; Zhang, Sheng; Turk, John; Ramanadham, Sasanka

    2009-01-01

    Among the phospholipases A2 (PLA2s) are the Group VI Ca2+-independent PLA2s (iPLA2s) and expression of multiple transcripts of iPLA2 in skeletal muscle has been reported. In the present study, phospholipase activity and sequential ATP and calmodulin affinity column chromatography analyses reveal that skeletal muscle iPLA2 exhibits properties characteristic of the iPLA2β isoform. The phospholipase activity of iPLA2β has been demonstrated to participate in signal transduction, cell proliferation, and apoptosis. We also report here that skeletal muscle from iPLA2β-null mice, relative to wild type muscle, exhibits a reduced capacity to oxidize palmitate but not palmitoyl-CoA or acetyl-CoA in the absence of changes in fatty acid transporters CD36 and CPT1 or β-hydroxyacyl-CoA dehydrogenase activity. Recently, purified iPLA2β was demonstrated to manifest a thioesterase activity which catalyzes hydrolysis of fatty acyl-CoAs. The liberated CoA-SH facilitates fatty acid transport into the mitochondria. In this regard, we find that fractions eluted from the ATP column and containing iPLA2β phospholipase activity also contained acyl-CoA thioesterase activity that was inhibited by the bromoenol lactone (BEL) suicide inhibitor of iPLA2β. We further find that acyl-CoA thioesterase activity in skeletal muscle preparations from iPLA2β-null mice is significantly reduced, relative to WT activity. These findings suggest that the absence of acyl-CoA thioesterase activity of iPLA2β can lead to reduced fatty acyl-CoA generation and impair fatty acid oxidation in iPLA2β-null mice. Our findings therefore reveal a novel function of iPLA2β, related not to its phospholipase activity but to its thioesterase activity, which contributes to optimal fatty acid oxidation in skeletal muscle. PMID:18937505

  19. Disruption of TrkB-mediated phospholipase Cgamma signaling inhibits limbic epileptogenesis.

    PubMed

    He, Xiao Ping; Pan, Enhui; Sciarretta, Carla; Minichiello, Liliana; McNamara, James O

    2010-05-05

    The BDNF receptor, TrkB, is critical to limbic epileptogenesis, but the responsible downstream signaling pathways are unknown. We hypothesized that TrkB-dependent activation of phospholipase Cgamma1 (PLCgamma1) signaling is the key pathway and tested this in trkB(PLC/PLC) mice carrying a mutation (Y816F) that uncouples TrkB from PLCgamma1. Biochemical measures revealed activation of both TrkB and PLCgamma1 in hippocampi in the pilocarpine and kindling models in wild-type mice. PLCgamma1 activation was decreased in hippocampi isolated from trkB(PLC/PLC) compared with control mice. Epileptogenesis assessed by development of kindling was inhibited in trkB(PLC/PLC) compared with control mice. Long-term potentiation of the mossy fiber-CA3 pyramid synapse was impaired in slices of trkB(PLC/PLC) mice. We conclude that TrkB-dependent activation of PLCgamma1 signaling is an important molecular mechanism of limbic epileptogenesis. Elucidating signaling pathways activated by a cell membrane receptor in animal models of CNS disorders promises to reveal novel targets for specific and effective therapeutic intervention.

  20. 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

  1. Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins.

    PubMed

    Shuaibu, M N; Kanbara, H; Yanagi, T; Ameh, D A; Bonire, J J; Nok, A J

    2001-11-01

    Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.

  2. 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).

  3. Secretory phospholipases A2 induce cytokine release from blood and synovial fluid monocytes.

    PubMed

    Triggiani, Massimo; Granata, Francescopaolo; Oriente, Alfonso; Gentile, Marco; Petraroli, Angelica; Balestrieri, Barbara; Marone, Gianni

    2002-01-01

    Secretory phospholipases A2 (sPLA2) are released in the blood of patients with various inflammatory diseases and exert proinflammatory activities by releasing arachidonic acid (AA), the precursor of eicosanoids. We examined the ability of four sPLA2 to activate blood and synovial fluid monocytes in vitro. Monocytes were purified from blood of healthy donors or from synovial fluid of patients with rheumatoid arthritis by negative immunoselection and by adherence to plastic dishes, respectively. The cells were incubated with group IA, IB, IIA and III sPLA2 and the release of TNF-alpha, IL-6 and IL-12 was determined by ELISA. Group IA, IB and IIA sPLA2 induced a concentration-dependent release of TNF-alpha and IL-6 from blood monocytes. These sPLA2 activated IL-12 production only in monocytes preincubated with IFN-gamma. Group IA and IIA sPLA2 also induced TNF-alpha and IL-6 release from synovial fluid monocytes. TNF-alpha and IL-6 release paralleled an increase in their mRNA expression and was independent from the capacity of sPLA2 to mobilize AA. These results indicate that sPLA2 stimulate cytokine release from blood and synovial fluid monocytes by a mechanism at least partially unrelated to their enzymatic activity. This effect may concur with the generation of AA in the proinflammatory activity of sPLA2 released during inflammatory diseases.

  4. Secretory phospholipase A2 in the pathogenesis of acute dengue infection

    PubMed Central

    Jeewandara, Chandima; Gomes, Laksiri; Udari, Sukhitha; Paranavitane, S.A.; Shyamali, N.L.A.; Ogg, Graham S.

    2016-01-01

    Abstract Introduction Platelet activating factor (PAF) is an important mediator of vascular leak in acute dengue. Phospholipase A2s (PLA2) are inflammatory lipid enzymes that generate and regulate PAF and other mediators associated with mast cells. We sought to investigate if mast cell activation and increases in secretory sPLA2s are associated with an increase in PAF and occurrence of dengue haemorrhagic fever (DHF). Methods The changes in the levels of mast cell tryptase, PAF and the activity of sPLA2 were determined throughout the course of illness in 13 adult patients with DHF, and 30 patients with dengue fever (DF). Results We found that sPLA2 activity was significantly higher in patients with DHF when compared to those with DF, during the first 120 h of clinical illness. sPLA2 activity was significantly associated with PAF levels, which were also significantly higher in patients with DHF. Although levels of mast cell tryptase were higher in patients with DHF, the difference was not significant, and the levels were not above the reference ranges. sPLA2 activity significantly correlated with the degree of viraemia in patients with DHF but not in those with DF. Conclusion sPLA2 appears to play an important role in the pathogenesis of dengue. Since its activity is significantly increased during the early phase of infection in patients with DHF, this suggests that understanding the underlying mechanisms may provide opportunities for early intervention. PMID:28250920

  5. Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1.

    PubMed

    Ranjan Moharana, Tushar; Byreddy, Avinesh R; Puri, Munish; Barrow, Colin; Rao, Nalam Madhusudhana

    2016-01-01

    Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.

  6. Hydrolysis of mixed monomolecular films of tricaprylin/dilauroylphosphatidylcholine by lipase and phospholipase A₂.

    PubMed

    Mircheva, K; Ivanova, Tz; Panaiotov, I; Verger, R

    2011-08-01

    The purpose of this article was to describe the kinetics of the enzymatic action of one or more enzymes on mixture of substrates organized in 2D structures in order to mimic some situations existing in biological or industrial systems. Hydrolysis of the mixed monomolecular films of tricaprylin/dilauroylphosphatidylcholine (TC8/DiC12PC) by Thermomyces lanuginosus lipase (TLL) and phospholipase A₂ (PLA₂) was studied by measuring the decrease of the surface area and change of the surface potential at barostatic conditions. The decrease of the surface area detects the transition of the substrate into reaction products and their solubilization while the change of the surface potential detects the contribution of dipole moment of the molecules remaining at the interface during the hydrolysis. The kinetic models, describing the interfacial hydrolysis allowed us to estimate the values of the global kinetic constants for TC8 and DiC12PC hydrolysis, respectively. The role of interaction between all participants of the catalytic act in that complex catalytic system is shown. The catalytic activity of TLL and PLA₂ is affected by the molecular environment in TC8/DiC12PC mixed monolayers.

  7. Bee Venom Phospholipase A2: Yesterday’s Enemy Becomes Today’s Friend

    PubMed Central

    Lee, Gihyun; Bae, Hyunsu

    2016-01-01

    Bee venom therapy has been used to treat immune-related diseases such as arthritis for a long time. Recently, it has revealed that group III secretory phospholipase A2 from bee venom (bee venom group III sPLA2) has in vitro and in vivo immunomodulatory effects. A growing number of reports have demonstrated the therapeutic effects of bee venom group III sPLA2. Notably, new experimental data have shown protective immune responses of bee venom group III sPLA2 against a wide range of diseases including asthma, Parkinson’s disease, and drug-induced organ inflammation. It is critical to evaluate the beneficial and adverse effects of bee venom group III sPLA2 because this enzyme is known to be the major allergen of bee venom that can cause anaphylactic shock. For many decades, efforts have been made to avoid its adverse effects. At high concentrations, exposure to bee venom group III sPLA2 can result in damage to cellular membranes and necrotic cell death. In this review, we summarized the current knowledge about the therapeutic effects of bee venom group III sPLA2 on several immunological diseases and described the detailed mechanisms of bee venom group III sPLA2 in regulating various immune responses and physiopathological changes. PMID:26907347

  8. Phospholipase Dε enhances Braasca napus growth and seed production in response to nitrogen availability.

    PubMed

    Lu, Shaoping; Yao, Shuaibing; Wang, Geliang; Guo, Liang; Zhou, Yongming; Hong, Yueyun; Wang, Xuemin

    2016-03-01

    Phospholipase D (PLD), which hydrolyses phospholipids to produce phosphatidic acid, has been implicated in plant response to macronutrient availability in Arabidopsis. This study investigated the effect of increased PLDε expression on nitrogen utilization in Brassica napus to explore the application of PLDε manipulation to crop improvement. In addition, changes in membrane lipid species in response to nitrogen availability were determined in the oil seed crop. Multiple PLDε over expression (PLDε-OE) lines displayed enhanced biomass accumulation under nitrogen-deficient and nitrogen-replete conditions. PLDε-OE plants in the field produced more seeds than wild-type plants but have no impact on seed oil content. Compared with wild-type plants, PLDε-OE plants were enhanced in nitrate transporter expression, uptake and reduction, whereas the activity of nitrite reductase was higher under nitrogen-depleted, but not at nitrogen-replete conditions. The level of nitrogen altered membrane glycerolipid metabolism, with greater impacts on young than mature leaves. The data indicate increased expression of PLDε has the potential to improve crop plant growth and production under nitrogen-depleted and nitrogen-replete conditions.

  9. Lithium activates brain phospholipase A2 and improves memory in rats: implications for Alzheimer's disease.

    PubMed

    Mury, Fábio B; da Silva, Weber C; Barbosa, Nádia R; Mendes, Camila T; Bonini, Juliana S; Sarkis, Jorge Eduardo Souza; Cammarota, Martin; Izquierdo, Ivan; Gattaz, Wagner F; Dias-Neto, Emmanuel

    2016-10-01

    Phospholipase A2 (Pla2) is required for memory retrieval, and its inhibition in the hippocampus has been reported to impair memory acquisition in rats. Moreover, cognitive decline and memory deficits showed to be reduced in animal models after lithium treatment, prompting us to evaluate possible links between Pla2, lithium and memory. Here, we evaluated the possible modulation of Pla2 activity by a long-term treatment of rats with low doses of lithium and its impact in memory. Wistar rats were trained for the inhibitory avoidance task, treated with lithium for 100 days and tested for perdurability of long-term memory. Hippocampal samples were used for quantifying the expression of 19 brain-expressed Pla2 genes and for evaluating the enzymatic activity of Pla2 using group-specific radio-enzymatic assays. Our data pointed to a significant perdurability of long-term memory, which correlated with increased transcriptional and enzymatic activities of certain members of the Pla2 family (iPla2 and sPla2) after the chronic lithium treatment. Our data suggest new possible targets of lithium, add more information on its pharmacological activity and reinforce the possible use of low doses of lithium for the treatment of neurodegenerative conditions such as the Alzheimer's disease.

  10. Salicylic-acid elicited phospholipase D responses in Capsicum chinense cell cultures.

    PubMed

    Rodas-Junco, B A; Muñoz-Sánchez, J A; Vázquez-Flota, F; Hernández-Sotomayor, S M T

    2015-05-01

    The plant response to different stress types can occur through stimulus recognition and the subsequent signal transduction through second messengers that send information to the regulation of metabolism and the expression of defense genes. The phospholipidic signaling pathway forms part of the plant response to several phytoregulators, such as salicylic acid (SA), which has been widely used to stimulate secondary metabolite production in cell cultures. In this work, we studied the effects of SA treatment on [(32)-P]Pi phospholipid turnover and phospholipase D (PLD) activity using cultured Capsicum chinense cells. In cultured cells, the PIP2 turnover showed changes after SA treatment, while the most abundant phospholipids (PLs), such as phosphatidylcholine (PC), did not show changes during the temporal course. SA treatment significantly increased phosphatidic acid (PA) turnover over time compared to control cells. The PA accumulation in cells treated with 1-butanol showed a decrease in messengers; at the same time, there was a 1.5-fold increase in phosphatidylbutanol. These results suggest that the participation of the PLD pathway is a source of PA production, and the activation of this mechanism may be important in the cell responses to SA treatment.

  11. Monitoring Phospholipase A2 Activity with Gd-encapsulated Phospholipid Liposomes

    PubMed Central

    Cheng, Zhiliang; Tsourkas, Andrew

    2014-01-01

    To date, numerous analytical methods have been developed to monitor phospholipase A2 (PLA2) activity. However, many of these methods require the use of unnatural PLA2 substrates that may alter enzyme kinetics, and probes that cannot be extended to applications in more complex environments. It would be desirable to develop a versatile assay that monitors PLA2 activity based on interactions with natural phospholipids in complex biological samples. Here, we developed an activatable T1 magnetic resonance (MR) imaging contrast agent to monitor PLA2 activity. Specifically, the clinically approved gadolinium (Gd)-based MR contrast agent, gadoteridol, was encapsulated within nanometer-sized phospholipid liposomes. The encapsulated Gd exhibited a low T1-weighted signal, due to low membrane permeability. However, when the phospholipids within the liposomal membrane were hydrolyzed by PLA2, encapsulated Gd was released into bulk solution, resulting in a measureable change in the T1-relaxation time. These activatable MR contrast agents can potentially be used as nanosensors for monitoring of PLA2 activity in biological samples with minimal sample preparation. PMID:25376186

  12. Phospholipase D2 mediates survival signaling through direct regulation of Akt in glioblastoma cells.

    PubMed

    Bruntz, Ronald C; Taylor, Harry E; Lindsley, Craig W; Brown, H Alex

    2014-01-10

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipaseD(PLD) is a novel regulator of Akt inGBM.Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase.

  13. Phospholipase D1 is required for angiogenesis of intersegmental blood vessels in zebrafish

    PubMed Central

    Zeng, Xin-Xin I.; Zheng, Xiangjian; Xiang, Yun; Cho, Hyekyung P.; Jessen, Jason R.; Zhong, Tao P.; Solnica-Krezel, Lilianna; Brown, H. Alex

    2009-01-01

    Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid and choline. Studies in cultured cells and Drosophila melanogaster have implicated PLD in the regulation of many cellular functions, including intracellular vesicle trafficking, cell proliferation and differentiation. However, the function of PLD in vertebrate development has not been explored. Here we report cloning and characterization of a zebrafish PLD1 (pld1) homolog. Like mammalian PLDs, zebrafish Pld1 contains two conservative HKD motifs. Maternally contributed pld1 transcripts are uniformly distributed in early embryo. Localized expression of pld1 is observed in the notochord during early segmentation, in the somites during later segmentation and in the liver at the larval stages. Studies in intact and cell-free preparations demonstrate evolutionary conservation of regulation. Inhibition of Pld1 expression using antisense morpholino oligonucleotides (MO) interfering with the translation or splicing of pld1 impaired intersegmental vessel (ISV) development. Incubating embryos with 1-butanol, which diverts production of phosphatidic acid to a phosphatidylalcohol, caused similar ISV defects. To determine where pld1 is required for ISV development we performed transplantation experiments. Analyses of the mosaic pld1 deficient embryos showed partial suppression of ISV defects in the segments containing transplanted wild-type somitic and notochord cells, or notochord cells alone. These results provide the first evidence that function of Pld1 in the developing notochord is essential for vascular development in vertebrates. PMID:19389349

  14. Phospholipase D2 Mediates Survival Signaling through Direct Regulation of Akt in Glioblastoma Cells*♦

    PubMed Central

    Bruntz, Ronald C.; Taylor, Harry E.; Lindsley, Craig W.; Brown, H. Alex

    2014-01-01

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipase D (PLD) is a novel regulator of Akt in GBM. Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase. PMID:24257753

  15. Evidence that phospholipase D mediates ADP ribosylation factor- dependent formation of Golgi coated vesicles

    PubMed Central

    1996-01-01

    Formation of coatomer-coated vesicles from Golgi-enriched membranes requires the activation of a small GTP-binding protein, ADP ribosylation factor (ARF). ARF is also an efficacious activator of phospholipase D (PLD), an activity that is relatively abundant on Golgi- enriched membranes. It has been proposed that ARF, which is recruited onto membranes from cytosolic pools, acts directly to promote coatomer binding and is in a 3:1 stoichiometry with coatomer on coated vesicles. We present evidence that cytosolic ARF is not necessary for initiating coat assembly on Golgi membranes from cell lines with high constitutive PLD activity. Conditions are also described under which ARF is at most a minor component relative to coatomer in coated vesicles from all cell lines tested, including Chinese hamster ovary cells. Formation of coated vesicles was sensitive to ethanol at concentrations that inhibit the production of phosphatidic acid (PA) by PLD. When PA was produced in Golgi membranes by an exogenous bacterial PLD, rather than with ARF and endogenous PLD, coatomer bound to Golgi membranes. Purified coatomer also bound selectively to artificial lipid vesicles that contained PA and phosphatidylinositol (4,5)-bisphosphate (PIP2). We propose that activation of PLD and the subsequent production of PA are key early events for the formation of coatomer-coated vesicles. PMID:8707816

  16. Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness

    PubMed Central

    Scott, Sarah A; Selvy, Paige E; Buck, Jason R; Cho, Hyekyung P; Criswell, Tracy L; Thomas, Ashley L; Armstrong, Michelle D; Arteaga, Carlos L; Lindsley, Craig W; Brown, H Alex

    2013-01-01

    Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. The lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. We used a diversity-oriented synthetic approach and developed a library of PLD inhibitors with considerable pharmacological characterization. Here we report the rigorous evaluation of that library, which contains highly potent inhibitors, including the first isoform-selective PLD inhibitors. Specific members of this series inhibit isoforms with > 100-fold selectivity both in vitro and in cells. A subset of inhibitors was shown to block invasiveness in metastatic breast cancer models. These findings demonstrate the power of diversity-oriented synthesis combined with biochemical assays and mass spectrometric lipid profiling of cellular responses to develop the first isoform-selective PLD inhibitors—a new class of antimetastatic agents. PMID:19136975

  17. Group IVA phospholipase A2 participates in the progression of hepatic fibrosis.

    PubMed

    Ishihara, Keiichi; Miyazaki, Akira; Nabe, Takeshi; Fushimi, Hideaki; Iriyama, Nao; Kanai, Shiho; Sato, Takashi; Uozumi, Naonori; Shimizu, Takao; Akiba, Satoshi

    2012-10-01

    Group IVA phospholipase A2 (IVA-PLA2) is an enzyme that intiates the arachidonic acid pathway and plays an important role in inflammation. We demonstrate that IVA-PLA2 deficiency suppresses lipid deposition in the liver, which was induced by administration of a high-fat and -cholesterol diet (HFCD) for 16 wk in mice. Herein, we performed 2-dimensional gel-based comparative proteomics to further define the suppressive effect of IVA-PLA2 deficiency on fatty liver formation. In comparisons among 4 groups, wild-type (WT)/normal diet (ND), IVA-PLA2-deficient knockout (KO)/ND, WT/HFCD, and KO/HFCD, 4 proteins, 3 of which are associated with hepatic fibrosis, were identified as molecules, of which altered expression by HFCD was suppressed in KO mice compared to WT mice. Therefore, we assessed the effect of IVA-PLA2 deficiency on hepatic fibrosis induced by HFCD or carbon tetrachloride (CCl4) in mouse models. Biochemical and histological analyses revealed that IVA-PLA2 deficiency markedly reduced overall collagen accumulation in the liver of HFCD- and CCl4-derived mouse models. We found that IVA-PLA2 deficiency prevented activation of hepatic stellate cells and infiltration of F4/80-positive macrophages without affecting other immunocytes such as CD8+ lymphocytes and natural killer cells. In summary, IVA-PLA2 deficiency attenuates not only lipid deposition in the liver but also hepatic fibrosis formation.

  18. Quantum-dynamical picture of a multistep enzymatic process: reaction catalyzed by phospholipase A(2).

    PubMed Central

    Bała, P; Grochowski, P; Nowiński, K; Lesyng, B; McCammon, J A

    2000-01-01

    A quantum-classical molecular dynamics model (QCMD), applying explicit integration of the time-dependent Schrödinger equation (QD) and Newtonian equations of motion (MD), is presented. The model is capable of describing quantum dynamical processes in complex biomolecular systems. It has been applied in simulations of a multistep catalytic process carried out by phospholipase A(2) in its active site. The process includes quantum-dynamical proton transfer from a water molecule to histidine localized in the active site, followed by a nucleophilic attack of the resulting OH(-) group on a carbonyl carbon atom of a phospholipid substrate, leading to cleavage of an adjacent ester bond. The process has been simulated using a parallel version of the QCMD code. The potential energy function for the active site is computed using an approximate valence bond (AVB) method. The dynamics of the key proton is described either by QD or classical MD. The coupling between the quantum proton and the classical atoms is accomplished via Hellmann-Feynman forces, as well as the time dependence of the potential energy function in the Schrödinger equation (QCMD/AVB model). Analysis of the simulation results with an Advanced Visualization System revealed a correlated rather than a stepwise picture of the enzymatic process. It is shown that an sp(2)--> sp(3) configurational change at the substrate carbonyl carbon is mostly responsible for triggering the activation process. PMID:10968989

  19. Involvement of Oxidative Pathways in Cytokine-induced Secretory Phospholipase A2-IIA in Astrocytes

    PubMed Central

    Jensen, Michael D.; Sheng, Wenwen; Simonyi, Agnes; Johnson, Gary S.; Sun, Albert Y.; Sun, Grace Y.

    2009-01-01

    Recent studies have suggested the involvement of secretory phospholipase A2-IIA (sPLA2-IIA) in neuroinflammatory diseases. Although sPLA2-IIA is transcriptionally induced through the NF-κB pathway by pro-inflammatory cytokines, whether this induction pathway is affected by other intracellular signaling pathways has not been investigated in detail. In this study, we demonstrated the induction of sPLA2-IIA mRNA and protein expression in astrocytes by cytokines and detected the protein in the culture medium after stimulation. We further investigated the effects of oxidative pathways and botanical antioxidants on the induction pathway and observed that IL-1β-induced sPLA2-IIA mRNA expression in astrocytes is dependent on ERK1/2 and PI-3 kinase, but not p38 MAPK. In addition to apocynin, a known NADPH oxidase inhibitor, botanical antioxidants, such as resveratrol and epigallocatechin gallate, also inhibited IL-1β-induced sPLA2-IIA mRNA expression. These compounds also suppressed IL-1β-induced ERK1/2 activation and translocation of the NADPH oxidase subunit p67 phox from cytosol to membrane fraction. Taken together, these results support the involvement of reactive oxygen species from NADPH oxidase in cytokine induction of sPLA2-IIA in astrocytes and promote the use of botanical antioxidants as protective agents for inhibition of inflammatory responses in these cells. PMID:19375465

  20. Comparative proteomics and subtyping of venom phospholipases A2 and disintegrins of Protobothrops pit vipers.

    PubMed

    Tsai, Inn-Ho; Chen, Yi-Hsuan; Wang, Ying-Ming

    2004-10-01

    To explore the venom diversity and systematics of pit vipers under the genus Protobothrops, the venom phospholipases A2 (PLA2s) of P. mangshanensis, P. elegans and P. tokarensis were purified and characterized for the first time. The results were compared with the corresponding venom data of other co-generic species including P. mucrosquamatus, P. flavoviridis and P. jerdonii. Based on sequence features at the N-terminal regions, we identified five PLA2 subtypes, i.e., the Asp49-PLA2s with N6, E6 or R6 substitution and the Lys49-PLA2. However, not all subtypes were expressed in each of the species. Venom N6-PLA2s from P. mangshanensis and P. tokarensis venom were weakly neurotoxic toward chick biventer cervicis tissue preparations. The venoms of P. tokarensis and P. flavoviridis contained identical PLA2 isoforms. In most Protobothrop disintegrins, sequences flanking the RGD-motif are conserved. Phylogenetic analyses based on amino acid sequences of both families of the acidic PLA2s and the disintegrins clarify that these species could belong to a monophyletic group.

  1. Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight

    PubMed Central

    Nieswandt, Bernhard; Stegner, David; Sumara, Grzegorz

    2016-01-01

    Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1-/- and Pld2-/- mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes. PMID:27299737

  2. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid.

    PubMed Central

    Lin, L L; Lin, A Y; Knopf, J L

    1992-01-01

    Cytosolic phospholipase A2 (cPLA2) binds to natural membrane vesicles in a Ca(2+)-dependent fashion, resulting in the selective release of arachidonic acid, thus implicating cPLA2 in the hormonally regulated production of eicosanoids. Here we report that the treatment of Chinese hamster ovary (CHO) cells overexpressing cPLA2 with ATP or thrombin resulted in an increased release of arachidonic acid as compared with parental CHO cells, demonstrating the hormonal coupling of cPLA2. In contrast, CHO cells overexpressing a secreted form of mammalian PLA2 (sPLA2-II) failed to show any increased hormonal responsiveness. Interestingly, we have noted that the activation of cPLA2 with a wide variety of agents stimulates the phosphorylation of cPLA2 on serine residues. Pretreatment of cells with staurosporin blocked the ATP-mediated phosphorylation of cPLA2 and strongly inhibited the activation of the enzyme. Increased cPLA2 activity was also observed in lysates prepared from ATP-treated cells and was sensitive to phosphatase treatment. These results suggest that in addition to Ca2+, the phosphorylation of cPLA2 plays an important role in the agonist-induced activation of cPLA2. Images PMID:1631101

  3. Phospholipase D Is Involved in Myogenic Differentiation through Remodeling of Actin Cytoskeleton

    PubMed Central

    Komati, Hiba; Naro, Fabio; Mebarek, Saida; De Arcangelis, Vania; Adamo, Sergio; Lagarde, Michel; Prigent, Annie-France; Némoz, Georges

    2005-01-01

    We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation. PMID:15616193

  4. Denervation Induces Cytosolic Phospholipase A2-mediated Fatty Acid Hydroperoxide Generation by Muscle Mitochondria*

    PubMed Central

    Bhattacharya, Arunabh; Muller, Florian L.; Liu, Yuhong; Sabia, Marian; Liang, Hanyu; Song, Wook; Jang, Youngmok C.; Ran, Qitao; Van Remmen, Holly

    2009-01-01

    Previously, we demonstrated that mitochondria from denervated muscle exhibited dramatically higher Amplex Red dependent fluorescence (thought to be highly specific for hydrogen peroxide) compared with control muscle mitochondria. We now demonstrate that catalase only partially inhibits the Amplex Red signal in mitochondria from denervated muscle. In contrast, ebselen (a glutathione peroxidase mimetic and inhibitor of fatty acid hydroperoxides) significantly inhibits the Amplex Red signal. This suggests that the majority of the Amplex Red signal in mitochondria from denervated muscle is not derived from hydrogen peroxide. Because Amplex Red cannot react with substrates in the lipid environment, we hypothesize that lipid hydroperoxides formed within the mitochondrial lipid bilayer are released as fatty acid hydroperoxides and react with the Amplex Red probe. We also suggest that the release of fatty acid hydroperoxides from denervated muscle mitochondria may be an important determinant of muscle atrophy. In support of this, muscle atrophy and the Amplex Red signal are inhibited in caloric restricted mice and in transgenic mice that overexpress the lipid hydroperoxide-detoxifying enzyme glutathione peroxidase 4. Finally, we propose that cytosolic phospholipase A2 may be a potential source of these hydroperoxides. PMID:19001413

  5. Cbl competitively inhibits epidermal growth factor-induced activation of phospholipase C-gamma1.

    PubMed

    Choi, Jang Hyun; Bae, Sun Sik; Park, Jong Bae; Ha, Sang Hoon; Song, Hebok; Kim, Jae-Ho; Cocco, Lucio; Ryu, Sung Ho; Suh, Pann-Ghill

    2003-04-30

    Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation through its two Src homology (SH) 2 domains and its single SH3 domain, which interact with signaling molecules in response to various growth factors and hormones. However, the role of the SH domains in the growth factor-induced regulation of PLC-gamma1 is unclear. By peptide-mass fingerprinting analysis we have identified Cbl as a binding protein for the SH3 domain of PLC-gamma1 from rat pheochromatocyte PC12 cells. Association of Cbl with PLC-gamma1 was induced by epidermal growth factor (EGF) but not by nerve growth factor (NGF). Upon EGF stimulation, both Cbl and PLC-gamma1 were recruited to the activated EGF receptor through their SH2 domains. Mutation of the SH2 domains of either Cbl or PLC-gamma1 abrogated the EGF-induced interaction of PLC-gamma1 with Cbl, indicating that SH2-mediated translocation is essential for the association of PLC-gamma1 and Cbl. Overexpression of Cbl attenuated EGF-induced tyrosine phosphorylation and the subsequent activation of PLC-gamma1 by interfering competitively with the interaction between PLC-gamma1 and EGFR. Taken together, these results provide the first indications that Cbl may be a negative regulator of intracellular signaling following EGF-induced PLC-gamma1 activation.

  6. Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation

    PubMed Central

    Inaba, Takehiko; Kishimoto, Takuma; Murate, Motohide; Tajima, Takuya; Sakai, Shota; Abe, Mitsuhiro; Makino, Asami; Tomishige, Nario; Ishitsuka, Reiko; Ikeda, Yasuo; Takeoka, Shinji; Kobayashi, Toshihide

    2016-01-01

    Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cβ1 (PLCβ1) as a new candidate. PLCβ1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCβ1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCβ1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCβ1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCβ1: plasma membrane remodeling, and in particular, caveolae formation. PMID:27342861

  7. The Role of Phospholipase D in Modulating the MTOR Signaling Pathway in Polycystic Kidney Disease

    PubMed Central

    Liu, Yang; Käch, Andres; Ziegler, Urs; Ong, Albert C. M.; Wallace, Darren P.; Arcaro, Alexandre; Serra, Andreas L.

    2013-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in polycystic kidney disease (PKD). Emerging evidence suggests that phospholipase D (PLD) and its product phosphatidic acid (PA) regulate mTOR activity. In this study, we assessed in vitro the regulatory function of PLD and PA on the mTOR signaling pathway in PKD. We found that the basal level of PLD activity was elevated in PKD cells. Targeting PLD by small molecule inhibitors reduced cell proliferation and blocked mTOR signaling, whereas exogenous PA stimulated mTOR signaling and abolished the inhibitory effect of PLD on PKD cell proliferation. We also show that blocking PLD activity enhanced the sensitivity of PKD cells to rapamycin and that combining PLD inhibitors and rapamycin synergistically inhibited PKD cell proliferation. Furthermore, we demonstrate that targeting mTOR did not induce autophagy, whereas targeting PLD induced autophagosome formation. Taken together, our findings suggest that deregulated mTOR pathway activation is mediated partly by increased PLD signaling in PKD cells. Targeting PLD isoforms with pharmacological inhibitors may represent a new therapeutic strategy in PKD. PMID:24009738

  8. Phospholipase D-mediated autophagic regulation is a potential target for cancer therapy

    PubMed Central

    Jang, Y H; Choi, K Y; Min, D S

    2014-01-01

    Autophagy is a catabolic process in which cell components are degraded to maintain cellular homeostasis by nutrient limitations. Defects of autophagy are involved in numerous diseases, including cancer. Here, we demonstrate a new role of phospholipase D (PLD) as a regulator of autophagy. PLD inhibition enhances autophagic flux via ATG1 (ULK1), ATG5 and ATG7, which are essential autophagy gene products critical for autophagosome formation. Moreover, PLD suppresses autophagy by differentially modulating phosphorylation of ULK1 mediated by mTOR and adenosine monophosphate-activated protein kinase (AMPK), and by suppressing the interaction of Beclin 1 with vacuolar-sorting protein 34 (Vps34), indicating that PLD coordinates major players of the autophagic pathway, AMPK-mTOR-ULK1 and Vps34/Beclin 1. Ultimately, PLD inhibition significantly sensitized in vitro and in vivo cancer regression via genetic and pharmacological inhibition of autophagy, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PLD inhibition. Collectively, we show a novel role for PLD in the molecular machinery regulating autophagy. PMID:24317201

  9. Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

    PubMed

    Staudt, Emanuel; Ramasamy, Pathmanaban; Plattner, Helmut; Simon, Martin

    2016-12-01

    Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP3. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface.

  10. Amyloid-type fiber formation in control of enzyme action: interfacial activation of phospholipase A2.

    PubMed

    Code, Christian; Domanov, Yegor; Jutila, Arimatti; Kinnunen, Paavo K J

    2008-07-01

    The lag-burst behavior in the action of phospholipase A(2) (PLA(2)) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T(m) of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA(2), evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA(2), involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form "mature" fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.

  11. Amyloid-Type Fiber Formation in Control of Enzyme Action: Interfacial Activation of Phospholipase A2

    PubMed Central

    Code, Christian; Domanov, Yegor; Jutila, Arimatti; Kinnunen, Paavo K. J.

    2008-01-01

    The lag-burst behavior in the action of phospholipase A2 (PLA2) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature Tm of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA2, evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA2, involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form “mature” fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis. PMID:18339749

  12. Differential regulation of Saccharomyces cerevisiae phospholipase D in sporulation and Sec14-independent secretion.

    PubMed Central

    Rudge, Simon A; Zhou, Chun; Engebrecht, JoAnne

    2002-01-01

    Saccharomyces cerevisiae Spo14, a phosphatidylcholine-specific, phosphatidylinositol (4,5) bisphosphate-activated phospholipase D (PLD), is essential for meiosis and spore formation. Spo14 is also required for secretion in the absence of the phosphatidylinositol/phosphatidylcholine transfer protein Sec14 (i.e., Sec14-independent secretion). In sporulating cells Spo14 is phosphorylated and relocalized within the cell. In contrast, Spo14 does not relocalize and is not phosphorylated in Sec14-independent secretion. Analysis of a partially phosphatidylinositol (4,5) bisphosphate-activated Spo14 mutant, spo14(R894G), revealed that Spo14 function in Sec14-independent secretion, unlike the situation in meiosis, requires fully stimulated PLD activity. Consistent with the differential regulation of Spo14 function during sporulation and secretion, we isolated a mutant allele, spo14-S251P, the product of which is improperly phosphorylated and fails to relocalize and rescue the sporulation phenotype of homozygous spo14 diploids, but supports Sec14-independent secretion. Furthermore, we show that the N-terminal domain of Spo14 is both phosphorylated and sufficient for prospore membrane localization during sporulation. These data indicate that Spo14 phosphorylation and relocalization are essential for the process of sporulation, but dispensable for Sec14-independent secretion. Finally, we demonstrate that Spo14 phosphorylation and relocalization are initiated by nitrogen and glucose limitation and occur independently of the process of meiosis. PMID:11973292

  13. Phospholipase D is involved in myogenic differentiation through remodeling of actin cytoskeleton.

    PubMed

    Komati, Hiba; Naro, Fabio; Mebarek, Saida; De Arcangelis, Vania; Adamo, Sergio; Lagarde, Michel; Prigent, Annie-France; Némoz, Georges

    2005-03-01

    We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation.

  14. Phospholipase D1 deficiency in mice causes nonalcoholic fatty liver disease via an autophagy defect

    PubMed Central

    Hur, Jang Ho; Park, Shi-Young; Dall’Armi, Claudia; Lee, Jae Sung; Di Paolo, Gilbert; Lee, Hui-Young; Yoon, Mee-Sup; Min, Do Sik; Choi, Cheol Soo

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of triglycerides (TG) as lipid droplets in the liver. Although lipid-metabolizing enzymes are considered important in NAFLD, the involvement of phospholipase D1 (PLD1) has not yet been studied. Here, we show that the genetic ablation of PLD1 in mice induces NAFLD due to an autophagy defect. PLD1 expression was decreased in high-fat diet-induced NAFLD. Subsequently, PLD1 deficiency led to an increase in hepatic TGs and liver weight. Autophagic flux was blocked in Pld1−/− hepatocytes, with decreased β-oxidation rate, reduced oxidation-related gene expression, and swollen mitochondria. The dynamics of autophagy was restored by treatment with the PLD product, phosphatidic acid (PA) or adenoviral PLD1 expression in Pld1−/− hepatocytes, confirming that lysosomal PA produced by PLD1 regulates autophagy. Notably, PLD1 expression in Pld1−/− liver significantly reduced hepatic lipid accumulation, compared with Pld1−/− liver. Thus, PLD1 plays an important role in hepatic steatosis via the regulation of autophagy. PMID:27976696

  15. Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration.

    PubMed

    Teng, Shuzhi; Stegner, David; Chen, Qin; Hongu, Tsunaki; Hasegawa, Hiroshi; Chen, Li; Kanaho, Yasunori; Nieswandt, Bernhard; Frohman, Michael A; Huang, Ping

    2015-02-01

    Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase