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Sample records for coupled phosphoinositide-specific phospholipase

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

    SciTech Connect

    Morrison, W.J.

    1988-01-01

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

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

    PubMed

    Rebecchi, M J; Pentyala, S N

    2000-10-01

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

  3. Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

    PubMed Central

    Kopka, Joachim; Pical, Christophe; Gray, Julie E.; Müller-Röber, Bernd

    1998-01-01

    Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner. PMID:9449844

  4. 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. PMID:19704699

  5. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-09-01

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

  8. A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation.

    PubMed Central

    Payne, W E; Fitzgerald-Hayes, M

    1993-01-01

    We identified a putative Saccharomyces cerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells showed a 10-fold increase in aberrant chromosome segregation compared with the wild type. Molecular analysis revealed that PLC1 encodes a predicted protein of 101 kDa with approximately 50 and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from humans, bovines, rats, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature-sensitive and chromosome missegregation phenotypes exhibited by plc1-1 cells were partially suppressed by exogenous calcium. Images PMID:8391635

  9. Phosphoinositide-specific phospholipase C-delta 1: effect of monolayer surface pressure and electrostatic surface potentials on activity.

    PubMed

    Rebecchi, M; Boguslavsky, V; Boguslavsky, L; McLaughlin, S

    1992-12-29

    We added phospholipase C-delta 1 (PLC-delta) to the aqueous subphase beneath monolayers formed from mixtures of phosphatidylinositol 4,5-bisphosphate (2% PIP2), phosphatidylserine (33% PS), and phosphatidylcholine (65% PC) and then measured the initial rate of hydrolysis of PIP2 after addition of 10 microM free calcium. Increasing the surface pressure of the monolayer, pi, from 20 to 40 mN/m decreased the rate of hydrolysis 200-fold. The rate of hydrolysis depends exponentially on the surface pressure: rate alpha exp(-pi Ap/kT) where k is the Boltzmann constant, T is the temperature, and Ap congruent to 1 nm2. Similar results were obtained with different (1 and 100 microM) free [Ca2+] and with different mole fractions of PIP2. The results are consistent with a model in which PLC-delta binds to PIP2 with high affinity (Ka = 10(6) M-1) in the absence of calcium ions [Rebecchi, M.J., Peterson, A., & McLaughlin, S. (1993) Biochemistry (preceding paper in this issue)], and a portion of PLC-delta of area Ap inserts into the monolayer doing work = pi Ap prior to hydrolysis of PIP2. Removing the monovalent acidic lipid PS from the monolayer decreases the activity of PLC-delta 4-fold, this effect of PS on activity is similar to the effect of monovalent acidic lipids on the binding of PLC-delta to PIP2 in bilayer vesicles. PMID:1334430

  10. Effect of monolayer surface pressure on the activities of phosphoinositide-specific phospholipase C-beta 1, -gamma 1, and -delta 1.

    PubMed

    Boguslavsky, V; Rebecchi, M; Morris, A J; Jhon, D Y; Rhee, S G; McLaughlin, S

    1994-03-15

    Three isoforms of phospholipase C, either PLC-beta 1, PLC-gamma 1, or PLC-delta 1, were added to the aqueous subphase beneath phospholipid monolayers formed at an air-solution interface, and the initial rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate was measured after addition of 10 microM free Ca2+. The monolayers were formed from mixtures of phosphatidylcholine (65% PC), phosphatidylserine (33% PS), and phosphatidylinositol 4,5-biphosphate (2% PIP2). Increasing the surface pressure of the monolayer, pi, from 15 to 25 mN/m decreases the rate of hydrolysis 16-, 13-, and 5-fold for PLC-beta 1, PLC-gamma 1, and PLC-delta 1, respectively. The simplest interpretation of these results is that a portion of each of the enzymes of area Ap must insert into the monolayer, doing work pi Ap, prior to hydrolysis of PIP2; binding studies with simple model compounds of known cross-sectional area are consistent with this interpretation. Removing the monovalent acidic lipid PS from the monolayer decreases the initial rates of hydrolysis of PIP2 about 3-fold for each PLC isoform, which suggests that negative electrostatic surface potentials increase the PLC activity. PMID:8130216

  11. Phosphoinositide-specific Phospholipase C β 1b (PI-PLCβ1b) Interactome: Affinity Purification-Mass Spectrometry Analysis of PI-PLCβ1b with Nuclear Protein*

    PubMed Central

    Piazzi, Manuela; Blalock, William L.; Bavelloni, Alberto; Faenza, Irene; D'Angelo, Antonietta; Maraldi, Nadir M.; Cocco, Lucio

    2013-01-01

    Two isoforms of inositide-dependent phospholipase C β1 (PI-PLCβ1) are generated by alternative splicing (PLCβ1a and PLCβ1b). Both isoforms are present within the nucleus, but in contrast to PLCβ1a, the vast majority of PLCβ1b is nuclear. In mouse erythroid leukemia cells, PI-PLCβ1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLCβ1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLCβ1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLCβ1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule. PMID:23665500

  12. Roles of phosphoinositide-specific phospholipase Cγ1 in brain development.

    PubMed

    Kang, Du-Seock; Yang, Yong Ryoul; Lee, Cheol; Kim, SaetByeol; Ryu, Sung Ho; Suh, Pann-Ghill

    2016-01-01

    Over the past decade, converging evidence suggests that PLCγ1 signaling has key roles in controlling neural development steps. PLCγ1 functions as a signal transducer that converts an extracellular stimulus into intracellular signals by generating second messengers such as DAG and IP3. DAG functions as an activator of either PKC or transient receptor potential cation channels (TRPCs), while IP3 induces the calcium release from intracellular calcium stores. These second messengers regulate the morphological change of neuron, such as neurite outgrowth, migration, axon pathfinding, and synapse formation. These morphological changes depend on finely tuned calcium signaling following receptor tyrosine kinase-mediated PLCγ1 signaling. Thus, deregulation of PLCγ1 signaling causes various abnormalities of neuronal development and it may be associated with diverse neurological disorders. Herein, we discuss the current understanding of the PLCγ1 signaling pathway in neural development and provide recent advances of how PLCγ1 signaling is involved in the formation of neuronal processes for functionally faithful brain development. PMID:26588873

  13. Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes.

    PubMed

    Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Artico, Marco; Cocco, Lucio; Billi, Anna Maria; Fumagalli, Lorenzo; Manzoli, Francesco Antonio

    2007-03-01

    Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level. PMID:17063484

  14. Exploring phospholipase C-coupled Ca(2+) signalling networks using Boolean modelling.

    PubMed

    Bhardwaj, G; Wells, C P; Albert, R; van Rossum, D B; Patterson, R L

    2011-05-01

    In this study, the authors explored the utility of a descriptive and predictive bionetwork model for phospholipase C-coupled calcium signalling pathways, built with non-kinetic experimental information. Boolean models generated from these data yield oscillatory activity patterns for both the endoplasmic reticulum resident inositol-1,4,5-trisphosphate receptor (IP(3)R) and the plasma-membrane resident canonical transient receptor potential channel 3 (TRPC3). These results are specific as randomisation of the Boolean operators ablates oscillatory pattern formation. Furthermore, knock-out simulations of the IP(3)R, TRPC3 and multiple other proteins recapitulate experimentally derived results. The potential of this approach can be observed by its ability to predict previously undescribed cellular phenotypes using in vitro experimental data. Indeed, our cellular analysis of the developmental and calcium-regulatory protein, DANGER1a, confirms the counter-intuitive predictions from our Boolean models in two highly relevant cellular models. Based on these results, the authors theorise that with sufficient legacy knowledge and/or computational biology predictions, Boolean networks can provide a robust method for predictive modelling of any biological system. [Includes supplementary material]. PMID:21639591

  15. Phospholipase Cepsilon regulates ovulation in Caenorhabditis elegans.

    PubMed

    Kariya, Ken-Ichi; Bui, Yen Kim; Gao, Xianlong; Sternberg, Paul W; Kataoka, Tohru

    2004-10-01

    Phospholipase Cepsilon (PLCepsilon) is a novel class of phosphoinositide-specific PLC with unknown physiological functions. Here, we present the first genetic analysis of PLCepsilon in an intact organism, the nematode Caenorhabditis elegans. Ovulation in C. elegans is dependent on an inositol 1,4,5-trisphosphate (IP(3)) signaling pathway activated by the receptor tyrosine kinase LET-23. We generated deletion mutants of the gene, plc-1, encoding C. elegans PLCepsilon. We observed a novel ovulation phenotype whereby oocytes are trapped in the spermatheca due to delayed dilation of the spermatheca-uterine valve. The expression of plc-1 in the adult spermatheca is consistent with its involvement in regulation of ovulation. On the other hand, we failed to observe genetic interaction of plc-1 with let-23-mediated IP(3) signaling pathway genes, suggesting a complex mechanism for control of ovulation. PMID:15355798

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

    PubMed

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

    2016-09-01

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

  17. Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors.

    PubMed

    Kunduri, Govind; Yuan, Changqing; Parthibane, Velayoudame; Nyswaner, Katherine M; Kanwar, Ritu; Nagashima, Kunio; Britt, Steven G; Mehta, Nickita; Kotu, Varshika; Porterfield, Mindy; Tiemeyer, Michael; Dolph, Patrick J; Acharya, Usha; Acharya, Jairaj K

    2014-07-01

    The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking. PMID:25002678

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

    PubMed

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

    2015-01-01

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

  19. 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.

  20. 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

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

    PubMed

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

    2016-06-01

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

  2. 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. PMID:27612188

  3. The turkey erythrocyte beta-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein alpha subunits.

    PubMed Central

    James, S R; Vaziri, C; Walker, T R; Milligan, G; Downes, C P

    1994-01-01

    By contrast with mammalian beta-adrenergic receptors, the avian isoform elicits two distinct effector responses, activation of adenylate cyclase and polyphosphoinositide-specific phospholipase C (PLC) leading to the accumulation of both cyclic adenosine monophosphate (cyclic AMP) and inositol phosphates. We have investigated the mechanisms of beta-adrenergic receptor signalling in turkey erythrocytes. Stimulation of adenylate cyclase by the beta-adrenergic-receptor agonist isoprenaline exhibits a 30-fold lower EC50 than that for PLC activation, which may indicate a marked receptor reserve for the former effector. Similar Ki values were obtained for the inhibition of both responses by four beta-adrenergic antagonists, arguing that a single receptor population is responsible for both effects. Antibodies raised against G-protein peptide sequences were used to show that the identity of the G-protein mediating the PLC response was an avian homologue of G11, the level of expression of which was very similar to that of the stimulatory G-protein of adenylate cyclase, Gs. Thus a single population of beta-adrenergic receptors apparently interacts with distinct G-proteins to activate different effectors. The stoichiometries of the receptor-G-protein-effector interactions are therefore similar for both second-messenger responses and the data are discussed in terms of the different efficacies observed for each response. Images Figure 4 PMID:7998968

  4. Plant phospholipases D and C and their diverse functions in stress responses.

    PubMed

    Hong, Yueyun; Zhao, Jian; Guo, Liang; Kim, Sang-Chul; Deng, Xianjun; Wang, Geliang; Zhang, Gaoyang; Li, Maoyin; Wang, Xuemin

    2016-04-01

    Phospholipases D (PLD) and C (PLC) hydrolyze the phosphodiesteric linkages of the head group of membrane phospholipids. PLDs and PLCs in plants occur in different forms: the calcium-dependent phospholipid binding domain-containing PLDs (C2-PLDs), the plekstrin homology and phox homology domain-containing PLDs (PX/PH-PLDs), phosphoinositide-specific PLC (PI-PLC), and non-specific PLC (NPC). They differ in structures, substrate selectivities, cofactor requirements, and/or reaction conditions. These enzymes and their reaction products, such as phosphatidic acid (PA), diacylglycerol (DAG), and inositol polyphosphates, play important, multifaceted roles in plant response to abiotic and biotic stresses. Here, we review biochemical properties, cellular effects, and physiological functions of PLDs and PLCs, particularly in the context of their roles in stress response along with advances made on the role of PA and DAG in cell signaling in plants. The mechanism of actions, including those common and distinguishable among different PLDs and PLCs, will also be discussed. PMID:26783886

  5. Mammalian phospholipase C.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2013-01-01

    Phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). DAG and IP(3) each control diverse cellular processes and are also substrates for synthesis of other important signaling molecules. PLC is thus central to many important interlocking regulatory networks. Mammals express six families of PLCs, each with both unique and overlapping controls over expression and subcellular distribution. Each PLC also responds acutely to its own spectrum of activators that includes heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca(2+), and phospholipids. Mammalian PLCs are autoinhibited by a region in the catalytic TIM barrel domain that is the target of much of their acute regulation. In combination, the PLCs act as a signaling nexus that integrates numerous signaling inputs, critically governs PIP(2) levels, and regulates production of important second messengers to determine cell behavior over the millisecond to hour timescale. PMID:23140367

  6. Role of phospholipases in adrenal steroidogenesis.

    PubMed

    Bollag, Wendy B

    2016-04-01

    Phospholipases are lipid-metabolizing enzymes that hydrolyze phospholipids. In some cases, their activity results in remodeling of lipids and/or allows the synthesis of other lipids. In other cases, however, and of interest to the topic of adrenal steroidogenesis, phospholipases produce second messengers that modify the function of a cell. In this review, the enzymatic reactions, products, and effectors of three phospholipases, phospholipase C, phospholipase D, and phospholipase A2, are discussed. Although much data have been obtained concerning the role of phospholipases C and D in regulating adrenal steroid hormone production, there are still many gaps in our knowledge. Furthermore, little is known about the involvement of phospholipase A2, perhaps, in part, because this enzyme comprises a large family of related enzymes that are differentially regulated and with different functions. This review presents the evidence supporting the role of each of these phospholipases in steroidogenesis in the adrenal cortex. PMID:26878860

  7. Involvement of phospholipase D and phosphatidic acid in the light-dependent up-regulation of sorghum leaf phosphoenolpyruvate carboxylase-kinase

    PubMed Central

    Monreal, José Antonio; López-Baena, Francisco Javier; Vidal, Jean; Echevarría, Cristina; García-Mauriño, Sofía

    2010-01-01

    The photosynthetic phosphoenolpyruvate carboxylase (C4-PEPC) is regulated by phosphorylation by a phosphoenolpyruvate carboxylase kinase (PEPC-k). In Digitaria sanguinalis mesophyll protoplasts, this light-mediated transduction cascade principally requires a phosphoinositide-specific phospholipase C (PI-PLC) and a Ca2+-dependent step. The present study investigates the cascade components at the higher integrated level of Sorghum bicolor leaf discs and leaves. PEPC-k up-regulation required light and photosynthetic electron transport. However, the PI-PLC inhibitor U-73122 and inhibitors of calcium release from intracellular stores only partially blocked this process. Analysis of [32P]phosphate-labelled phospholipids showed a light-dependent increase in phospholipase D (PLD) activity. Treatment of leaf discs with n-butanol, which decreases the formation of phosphatidic acid (PA) by PLD, led to the partial inhibition of the C4-PEPC phosphorylation, suggesting the participation of PLD/PA in the signalling cascade. PPCK1 gene expression was strictly light-dependent. Addition of neomycin or n-butanol decreased, and a combination of both inhibitors markedly reduced PPCK1 expression and the concomitant rise in PEPC-k activity. The calcium/calmodulin antagonist W7 blocked the light-dependent up-regulation of PEPC-k, pointing to a Ca2+-dependent protein kinase (CDPK) integrating both second messengers, calcium and PA, which were shown to increase the activity of sorghum CDPK. PMID:20410319

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

    PubMed

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

    1998-04-01

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

  9. Characteristics of muscarinic receptors that selectively couple to inhibition of adenylate cyclase or stimulation of phospholipase C on NG108-15 and 1321N1 cells

    SciTech Connect

    Liang, M.

    1988-01-01

    The purpose of this dissertation was to establish whether different muscarinic receptor proteins selectively couple to different second messenger response system. Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma {times} glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with ({sup 3}H)Propylbenzilylcholine mustard (({sup 3}H)PBCM) and the mobilities of the ({sup 3}H)PBCM-labelled species of both cells were compared by SDS-PAGE. 1321N1 and NG108-15 cells each primarily expressed a single ({sup 3}H)PBCM-labelled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. ({sup 3}H)PBCM labelling was completely inhibited by 1 {mu}M atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the ({sup 3}H)PBCM-labelled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Another approach for determining differences in the muscarinic receptors of 2 cells lines was to study agonist-induced alteration of muscarinic receptor number. Exposure of both cell types to agonists resulted in rapid loss of muscarinic receptors from cell surface without change of total cellular muscarinic receptors followed by subsequently loss of receptors from cells. Muscarinic receptors on both cell lines were regulated by agonist with similar properties.

  10. 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

  11. H2O2-Activated Mitochondrial Phospholipase iPLA2γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein–Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells

    PubMed Central

    Ježek, Jan; Dlasková, Andrea; Zelenka, Jaroslav; Jabůrek, Martin

    2015-01-01

    Abstract Aims: Pancreatic β-cell chronic lipotoxicity evolves from acute free fatty acid (FA)–mediated oxidative stress, unprotected by antioxidant mechanisms. Since mitochondrial uncoupling protein-2 (UCP2) plays antioxidant and insulin-regulating roles in pancreatic β-cells, we tested our hypothesis, that UCP2-mediated uncoupling attenuating mitochondrial superoxide production is initiated by FA release due to a direct H2O2-induced activation of mitochondrial phospholipase iPLA2γ. Results: Pro-oxidant tert-butylhydroperoxide increased respiration, decreased membrane potential and mitochondrial matrix superoxide release rates of control but not UCP2- or iPLA2γ-silenced INS-1E cells. iPLA2γ/UCP2-mediated uncoupling was alternatively activated by an H2O2 burst, resulting from palmitic acid (PA) β-oxidation, and it was prevented by antioxidants or catalase overexpression. Exclusively, nascent FAs that cleaved off phospholipids by iPLA2γ were capable of activating UCP2, indicating that the previously reported direct redox UCP2 activation is actually indirect. Glucose-stimulated insulin release was not affected by UCP2 or iPLA2γ silencing, unless pro-oxidant activation had taken place. PA augmented insulin secretion via G-protein–coupled receptor 40 (GPR40), stimulated by iPLA2γ-cleaved FAs (absent after GPR40 silencing). Innovation and Conclusion: The iPLA2γ/UCP2 synergy provides a feedback antioxidant mechanism preventing oxidative stress by physiological FA intake in pancreatic β-cells, regulating glucose-, FA-, and redox-stimulated insulin secretion. iPLA2γ is regulated by exogenous FA via β-oxidation causing H2O2 signaling, while FAs are cleaved off phospholipids, subsequently acting as amplifying messengers for GPR40. Hence, iPLA2γ acts in eminent physiological redox signaling, the impairment of which results in the lack of antilipotoxic defense and contributes to chronic lipotoxicity. Antioxid. Redox Signal. 23, 958–972. PMID:25925080

  12. Role of lipid packing in the activity of phospholipase C-delta1 as determined by hydrostatic pressure measurements.

    PubMed Central

    Rebecchi, M; Bonhomme, M; Scarlata, S

    1999-01-01

    Previous studies with phospholipid monolayers revealed a large decrease in the activity of phosphoinositide-specific phospholipase C-delta(1) (PLC-delta(1)) which catalyses the hydrolysis of PtdIns(4, 5)P(2) as lateral pressure is applied to the membrane. If stress on the membrane is the sole inhibitor of PLC-delta(1) activity, the enzyme must penetrate the membrane surface to engage its substrate. To test the effect on PLC-delta(1) activity of lipid packing in the absence of a directional stress, we examined the effects of increasing hydrostatic pressure on enzymic activity. We find that, in contrast with monolayer studies, increasing lipid packing by hydrostatic pressure does not affect membrane binding and increases enzymic activity by 90% in going from atmospheric pressure to 10(8) Pa (approx. 1000 atm). The increase in activity could be accounted for mainly by electrostriction of water around the multiply-charged product. Our results show that when there is no net stress on the monolayer, lipid packing does not alter PLC-delta(1) activity, possibly because penetration of the enzyme into the membrane surface is shallow. We suggest that, in biological membranes, the activity of this and possibly other interfacial proteins is independent of headgroup packing. PMID:10417319

  13. Primary phospholipase C and brain disorders.

    PubMed

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

    2016-05-01

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

  14. 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

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

    PubMed

    Parrish, Jason C; Nichols, David E

    2006-11-01

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

  16. Characterization of N-Acyl Phosphatidylethanolamine-Specific Phospholipase-D Isoforms in the Nematode Caenorhabditis elegans

    PubMed Central

    Harrison, Neale; Lone, Museer A.; Kaul, Tiffany K.; Reis Rodrigues, Pedro; Ogungbe, Ifedayo Victor; Gill, Matthew S.

    2014-01-01

    N-acylethanolamines are an important class of lipid signaling molecules found in many species, including the nematode Caenorhabditis elegans (C. elegans) where they are involved in development and adult lifespan. In mammals, the relative activity of the biosynthetic enzyme N-acyl phosphatidylethanolamine-specific phospholipase-D and the hydrolytic enzyme fatty acid amide hydrolase determine N-acylethanolamine levels. C. elegans has two N-acyl phosphatidylethanolamine-specific phospholipase-D orthologs, nape-1 and nape-2, that are likely to have arisen from a gene duplication event. Here, we find that recombinant C. elegans NAPE-1 and NAPE-2 are capable of generating N-acylethanolamines in vitro, confirming their functional conservation. In vivo, they exhibit overlapping expression in the pharynx and the nervous system, but are also expressed discretely in these and other tissues, suggesting divergent roles. Indeed, nape-1 over-expression results in delayed growth and shortened lifespan only at 25°C, while nape-2 over-expression results in significant larval arrest and increased adult lifespan at 15°C. Interestingly, deletion of the N-acylethanolamine degradation enzyme faah-1 exacerbates nape-1 over-expression phenotypes, but suppresses the larval arrest phenotype of nape-2 over-expression, suggesting that faah-1 is coupled to nape-2, but not nape-1, in a negative feedback loop. We also find that over-expression of either nape-1 or nape-2 significantly enhances recovery from the dauer larval stage in the insulin signaling mutant daf-2(e1368), but only nape-1 over-expression reduces daf-2 adult lifespan, consistent with increased levels of the N-acylethanolamine eicosapentaenoyl ethanolamine. These results provide evidence that N-acylethanolamine biosynthetic enzymes in C. elegans have conserved function and suggest a temperature-dependent, functional divergence between the two isoforms. PMID:25423491

  17. A specific phospholipase C activity regulates phosphatidylinositol levels in lung surfactant of patients with acute respiratory distress syndrome.

    PubMed

    Spyridakis, Spyros; Leondaritis, George; Nakos, George; Lekka, Marilena E; Galanopoulou, Dia

    2010-03-01

    Lung surfactant (LS) is a lipid-rich material lining the inside of the lungs. It reduces surface tension at the liquid/air interface and thus, it confers protection of the alveoli from collapsing. The surface-active component of LS is dipalmitoyl-phosphatidylcholine, while anionic phospholipids such as phosphatidylinositol (PtdIns) and primarily phosphatidylglycerol are involved in the stabilization of the LS monolayer. The exact role of PtdIns in this system is not well-understood; however, PtdIns levels change dramatically during the acute respiratory distress syndrome (ARDS) evolution. In this report we present evidence of a phosphoinositide-specific phospholipase C (PI-PLC) activity in bronchoalveolar lavage (BAL) fluid, which may regulate PtdIns levels. Characterization of this extracellular activity showed specificity for PtdIns and phosphatidylinositol 4,5-bisphosphate, sharing the typical substrate concentration-, pH-, and calcium-dependencies with mammalian PI-PLCs. Fractionation of BAL fluid showed that PI-PLC did not co-fractionate with large surfactant aggregates, but it was found mainly in the soluble fraction. Importantly, analysis of BAL samples from control subjects and from patients with ARDS showed that the PI-PLC specific activity was decreased by 4-fold in ARDS samples concurrently with the increase in BAL PtdIns levels. Thus, we have identified for the first time an extracellular PI-PLC enzyme activity that may be acutely involved in the regulation of PtdIns levels in LS. PMID:19491339

  18. Thromboxane-insensitive dog platelets have impaired activation of phospholipase C due to receptor-linked G protein dysfunction.

    PubMed Central

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

    1993-01-01

    Human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G proteins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2- platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, [125I]BOP and [3H]U46619, and antagonist, [3H]SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2- platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2- platelets. Antisera to Gq class alpha subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTP gamma S yielded significantly less PA and IP3 in TXA2- platelets. Immunotransfer blotting revealed comparable quantities of Gq class alpha-subunits in all three platelet types. Thus, TXA2- dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a member of the Gq class. Images PMID:8227362

  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. [Immobilization of phospholipase A2 from Central Asian cobra venom on polyamide sorbents].

    PubMed

    Akhmedzhanov, R A; Salikhova, Z T; Aripov, T F; Rakhimov, M M

    1988-01-01

    The effect of the immobilization technique and the ligand nature on catalytic properties of phospholipase A2 from the cobra venom was studied. Preparations of phospholipase A2 adsorbed on and covalently bound to polyamide sorbents were obtained. The enzyme was coupled to polyamide beads modified with glutaraldehyde. In this case only 9% of the enzyme activity was retained. The enzyme adsorbed on polyamide modified with phosphatidylethanolamine retained up to 20% of the initial activity. The binding selectivity of phospholipase A2 was maximum in case of the sorbent with a binary ligand, e. g. phosphatidylethanolamine+cytotoxin, the sorbent capacity for the bound enzyme increased 2-3 times (460-600 units/g sorbent. The specific activity of the adsorbed phospholipase A2 was 17-40 units/g sorbent in contrast to 8.6 units/g sorbent for the covalently bound enzyme. Immobilization of the enzyme on polyamide sorbents resulted in changes of the pH-optimum, sensitivity to Ca2+ ions and the character of the enzyme-substrate interactions. Heart stability of the adsorbed phospholipase A2 was lower than that of the covalently bound enzyme. However, the adsorbed enzyme can be used, for example, in affinity chromatography due to its higher specific activity, selectivity and reversibility of the sorption. PMID:3244675

  1. Phospholipase A2 and Phospholipase B Activities in Fungi

    PubMed Central

    Köhler, Gerwald A.; Brenot, Audrey; Haas-Stapleton, Eric; Agabian, Nina; Deva, Rupal; Nigam, Santosh

    2007-01-01

    As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A2 (PLA2) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host’s immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA2 activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host. PMID:17081801

  2. Assaying nonspecific phospholipase C activity.

    PubMed

    Pejchar, Přemysl; Scherer, Günther F E; Martinec, Jan

    2013-01-01

    Plant nonspecific phospholipase C (NPC) is a recently described enzyme which plays a role in membrane rearrangement during phosphate starvation. It is also involved in responses of plants to brassinolide, abscisic acid (ABA), elicitors, and salt. The NPC activity is decreased in cells treated with aluminum. In the case of salt stress, the molecular mechanism of NPC action is based on accumulation of diacylglycerol (DAG) by hydrolysis of phospholipids and conversion of DAG, the product of NPC activity, to phosphatidic acid (PA) that participates in ABA signaling pathways. Here we describe a step-by-step protocol, which can be used to determine in situ or in vitro NPC activity. Determination is based on quantification of fluorescently labeled DAG as a product of cleavage of the fluorescently labeled substrate lipid, phosphatidylcholine. High-performance thin-layer chromatography is used for separation of fluorescent DAG. The spot is visualized with a laser scanner and the relative amounts of fluorescent DAG are quantified using imaging software. PMID:23681535

  3. 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. PMID:27307578

  4. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Lecithins, phospholipase A2-hydrolyzed... Substances § 721.4585 Lecithins, phospholipase A2-hydrolyzed. (a) Chemical substances and significant new..., phospholipase A2-hydrolyzed (PMN P-93-333) is subject to reporting under this section for the significant...

  5. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Lecithins, phospholipase A2-hydrolyzed... Substances § 721.4585 Lecithins, phospholipase A2-hydrolyzed. (a) Chemical substances and significant new..., phospholipase A2-hydrolyzed (PMN P-93-333) is subject to reporting under this section for the significant...

  6. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Lecithins, phospholipase A2-hydrolyzed... Substances § 721.4585 Lecithins, phospholipase A2-hydrolyzed. (a) Chemical substances and significant new..., phospholipase A2-hydrolyzed (PMN P-93-333) is subject to reporting under this section for the significant...

  7. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Lecithins, phospholipase A2-hydrolyzed... Substances § 721.4585 Lecithins, phospholipase A2-hydrolyzed. (a) Chemical substances and significant new..., phospholipase A2-hydrolyzed (PMN P-93-333) is subject to reporting under this section for the significant...

  8. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Lecithins, phospholipase A2-hydrolyzed... Substances § 721.4585 Lecithins, phospholipase A2-hydrolyzed. (a) Chemical substances and significant new..., phospholipase A2-hydrolyzed (PMN P-93-333) is subject to reporting under this section for the significant...

  9. COUPLING

    DOEpatents

    Hawke, B.C.

    1963-02-26

    This patent relates to a releasable coupling connecting a control rod to a control rod drive. This remotely operable coupling mechanism can connect two elements which are laterally and angviarly misaligned, and provides a means for sensing the locked condition of the elements. The coupling utilizes a spherical bayonet joint which is locked against rotation by a ball detent lock. (AEC)

  10. 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. PMID:22426737

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

    PubMed

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

    2002-06-15

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

  12. COUPLING

    DOEpatents

    Frisch, E.; Johnson, C.G.

    1962-05-15

    A detachable coupling arrangement is described which provides for varying the length of the handle of a tool used in relatively narrow channels. The arrangement consists of mating the key and keyhole formations in the cooperating handle sections. (AEC)

  13. Studies on the phospholipases of rat intestinal mucosa

    PubMed Central

    Subbaiah, P. V.; Ganguly, J.

    1970-01-01

    1. Subcellular distribution and characteristics of different phospholipases of rat intestinal mucosa were studied. 2. The presence of free fatty acid was necessary for the maximal hydrolysis of lecithin (phosphatidylcholine), but there was no accumulation of lysolecithin (1 or 2-acylglycerophosphorylcholine);lysolecithin accumulated when the reaction was carried out in the presence of sodium deoxycholate and at or above pH8.0. 3. The fatty acid-activated phospholipase B as well as lysolecithinase showed optimum activity at pH6.5, whereas for the phospholipase A it was about pH8.6. 4. The bulk of the phospholipase A was present in the microsomal fraction, whereas the phospholipase B and lysolecithinase activities were distributed between the microsomal and soluble fractions of the mucosal homogenate. 5. Phospholipase A was equally distributed between the brush border and brush-border-free particulate fraction, with the brush border having highest specific activity, whereas the other two activities were distributed between the brush-border-free particulate and soluble fractions. 6. Various treatments showed marked differences between the phospholipase A and phospholipase B activities, but not between phospholipase B and lysolecithinase activities. 7. By using (β[1-14C]-oleoyl) lecithin it was shown that the mucosal phospholipase A was specific for the β-ester linkage of the lecithin molecule. PMID:5484667

  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. Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells.

    PubMed

    Mah, S J; Ades, A M; Mir, R; Siemens, I R; Williamson, J R; Fluharty, S J

    1992-08-01

    The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In the present study, polyclonal antibodies raised against a PLC isoenzyme, PLC-alpha, reacted with a 60-kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-alpha with cell surface AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. Anti-PLC-alpha antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent

  16. 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.

  17. Purification and characterization of phospholipase C of Salmonella gallinarum.

    PubMed

    Singh, B R; Sharma, V D

    1998-12-01

    Phospholipase C was isolated from an outbreak strain of Salmonella gallinarum with ciprofloxacin extraction, dialysis, gel filtration, ion exchange chromatography and chromatofocussing. Purified phospholipase C (mol wt. 65 KDa; isoelectric point, pI 3.5) was resistant to pasteurization, stomach enzyme (pepsin), bacterial protease and lipase but lost its activity on trypsin and chymotrypsin treatment. It was sensitive to pH > or = 8.0. It was haemolytic, embryotoxic, enterohaemorrhagic, lethal to birds, cytotoxic to Vero and MDBK cells, dermonecrotoxic in rabbit and antigenically active protein. Antisera raised against purified phospholipase C neutralized its all biological activities and agglutinated the producer Salmonella strains. Serologically it was proved similar to phospholipase C of Klebsiella pneumoniae and S. weltevreden. Fluorescent antibody technique (FAT) was standardized to detect phospholipase producer strains. PMID:10093508

  18. The Role of Phospholipase D in Regulated Exocytosis.

    PubMed

    Rogasevskaia, Tatiana P; Coorssen, Jens R

    2015-11-27

    There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca(2+) sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se. PMID:26433011

  19. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  20. Reminiscence of phospholipase B in Penicillium notatum

    PubMed Central

    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, Fe3+, 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. PMID:25391318

  1. 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. PMID:25391318

  2. The role of phospholipase D in Glut-4 translocation.

    PubMed

    Huang, Ping; Frohman, Michael A

    2003-01-01

    Insulin-stimulated Glut-4 translocation is regulated through a complex pathway. Increasing attention is being paid to the role undertaken in this process by Phospholipase D, a signal transduction-activated enzyme that generates the lipid second-messenger phosphatidic acid. Phospholipase D facilitates Glut-4 translocation at potentially multiple steps in its outward movement. Current investigation is centered on Phospholipase D promotion of Glut-4-containing membrane vesicle trafficking and vesicle fusion into the plasma membrane, in part through activation of atypical protein kinase C isoforms. PMID:14648804

  3. Induction of cytosolic phospholipase A2 activity by phosphatidic acid and diglycerides in permeabilized human neutrophils: interrelationship between phospholipases D and A2.

    PubMed Central

    Bauldry, S A; Wooten, R E

    1997-01-01

    Relationships between phospholipases are poorly understood, but phosphatidic acid (PA) and diglycerides (DGs), produced by phospholipase D (PLD) and phosphatidate phosphohydrolase actions, might function as second messengers coupling cell stimulation to cellular responses. This study investigates the role of PLD-mediated PA and DG formation in inducing phospholipase A2 (PLA2) activity in intact human neutrophils (PMNs) and in PMNs permeabilized with Staphylococcus aureus alpha-toxin. PMNs were labelled with [3H]arachidonic acid (AA) to assess AA release and metabolism and diacylglycerol formation, or with [3H]1-O-hexadecyl-2-lyso-glycerophosphatidylcholine for the determination of platelet-activating factor (PAF), PA and alkylacylglycerol production. In intact PMNs primed with tumour necrosis factor alpha before stimulation with N-formyl-Met-Leu-Phe, AA release and metabolism and PAF formation increased in parallel with enhanced PA and DG formation, and inhibition of PA and DG production led to a decrease in both AA release and PAF accumulation. In alpha-toxin-permeabilized PMNs, AA release and PAF production result from the specific activation of cytosolic PLA2 (cPLA2). In this system, PA and DG formation were always present when cPLA2 activation occurred; blocking PA and DG production inhibited AA release and PAF accumulation. Adding either PA or DG back to permeabilized cells (with endogenous PA and DG formation blocked) led to a partial restoration of AA release and PAF formation; a combination of PA and DGs reconstituted full cPLA2 activity. These results strongly suggest that products of PLD participate in activating cPLA2 in PMNs. PMID:9065750

  4. Cloning and expression analysis of murine phospholipase D1.

    PubMed Central

    Colley, W C; Altshuller, Y M; Sue-Ling, C K; Copeland, N G; Gilbert, D J; Jenkins, N A; Branch, K D; Tsirka, S E; Bollag, R J; Bollag, W B; Frohman, M A

    1997-01-01

    Activation of phosphatidylcholine-specific phospholipase D(PLD) occurs as part of the complex signal-transduction cascade initiated by agonist stimulation of tyrosine kinase and G-protein-coupled receptors. A variety of mammalian PLD activities have been described, and cDNAs for two PLDs recently reported (human PLD1 and murine PLD2). We describe here the cloning and chromosomal localization of murine PLD1. Northern-blot hybridization and RNase protection analyses were used to examine the expression of murine PLD1 and PLD2 ina variety of cell lines and tissues. PLD1 and PLD2 were expressed in all RNA samples examined, although the absolute expression of each isoform varied, as well as the ratio of PLD1 to PLD2. Moreover, in situ hybridization of adult brain and murine embryo sections revealed high levels of expression of individual PLDs in some cell types and no detectable expression in others. Thus the two PLDs probably carry out distinct roles in restricted subsets of cells rather than ubiquitous roles in all cells. PMID:9307024

  5. Probing phospholipase a(2) with fluorescent phospholipid substrates.

    PubMed

    Wichmann, Oliver; Gelb, Michael H; Schultz, Carsten

    2007-09-01

    The Foerster resonance energy transfer-based sensor, PENN, measures intracellular phospholipase A(2) (PLA(2)) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn-2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic-acid-mimicking fatty acids were prepared by copper-mediated coupling reactions. Probes with a single double bond in the 5-position exhibited favorable substrate properties for secretory PLA(2)s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA(2), while the O-methyl-derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S-acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane-permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted. PMID:17661302

  6. Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena▿‡

    PubMed Central

    Leondaritis, George; Sarri, Theoni; Dafnis, Ioannis; Efstathiou, Antonia; Galanopoulou, Dia

    2011-01-01

    Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], produce the Ca2+-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca2+. One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca2+, modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P2 levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P3-Ca2+ regulatory axis in ciliates. PMID:21169416

  7. Ubiquitin Activates Patatin-Like Phospholipases from Multiple Bacterial Species

    PubMed Central

    Anderson, David M.; Sato, Hiromi; Dirck, Aaron T.; Feix, Jimmy B.

    2014-01-01

    Phospholipase A2 enzymes are ubiquitously distributed throughout the prokaryotic and eukaryotic kingdoms and are utilized in a wide array of cellular processes and physiological and immunological responses. Several patatin-like phospholipase homologs of ExoU from Pseudomonas aeruginosa were selected on the premise that ubiquitin activation of this class of bacterial enzymes was a conserved process. We found that ubiquitin activated all phospholipases tested in both in vitro and in vivo assays via a conserved serine-aspartate catalytic dyad. Ubiquitin chains versus monomeric ubiquitin were superior in inducing catalysis, and ubiquitin-like proteins failed to activate phospholipase activity. Toxicity studies in a prokaryotic dual-expression system grouped the enzymes into high- and low-toxicity classes. Toxicity measured in eukaryotic cells also suggested a two-tiered classification but was not predictive of the severity of cellular damage, suggesting that each enzyme may correspond to unique properties perhaps based on its specific biological function. Additional studies on lipid binding preference suggest that some enzymes in this family may be differentially sensitive to phosphatidyl-4,5-bisphosphate in terms of catalytic activation enhancement and binding affinity. Further analysis of the function and amino acid sequences of this enzyme family may lead to a useful approach to formulating a unifying model of how these phospholipases behave after delivery into the cytoplasmic compartment. PMID:25404699

  8. Phospholipase A2 as a mechanosensor.

    PubMed Central

    Lehtonen, J Y; Kinnunen, P K

    1995-01-01

    Osmotic swelling of large unilamellar vesicles (LUVs) causes membrane stretching and thus reduces the lateral packing of lipids. This is demonstrated to modulate strongly the catalytic activity of phospholipase A2 (PLA2) toward a fluorescent phospholipid, 1-palmitoyl-2-[(6-pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) residing in LUVs composed of different unsaturated and saturated phosphatidylcholines. The magnitude of the osmotic pressure gradient delta omega required for maximal PLA2 activity as well as the extent of activation depend on the degree of saturation of the membrane phospholipid acyl chains. More specifically, delta omega needed for maximal hydrolytic activity increases in the sequence DOPC < SOPC < DMPC in accordance with the increment in the intensity of chain-chain van der Waals interactions. Previous studies on the hydrolysis of substrate monolayers by C. adamanteus and N. naja PLA2 revealed maximal hydrolytic rates for these two enzymes to be achieved at lipid packing densities corresponding to surface pressures of 12 and 18 mN m-1, respectively. In keeping with the above the magnitudes of delta omega producing maximal activity of Crotalus adamanteus and Naja naja toward PPDPC/DMPC LUVs were 40 and 20 mOsm/kg, respectively. Our findings suggest a novel possibility of regulating the activity of PLA2 and perhaps also other lipid packing density-dependent enzymes in vivo by osmotic forces applied on cellular membranes. Importantly, our results reveal serendipitously that the responsiveness of membranes to osmotic stress is modulated by the acyl chain composition of the lipids. PMID:7612831

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  11. Structural deterioration in produce: Phospholipase D, membrane deterioration and senescence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Following cloning of the first phospholipase D (PLD) gene from castor bean, there has been good progress in determining physiological roles of members of the plant PLD gene family, now known to comprise six classes: alpha, beta, gamma, delta, epsilon and zeta. Most notably, phosphatidic acid derived...

  12. Phospholipase A2 activity during cold acclimation of wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phospholipase A2 (EC 3.1.1.4; PLA2) activity in wheat (Triticum aestivum L.) crown tissue from plants undergoing cold acclimation and/or chilling stress was investigated in a moderately cold tolerant winter wheat, a spring wheat, and a poorly cold tolerant winter wheat. Activity levels were inv...

  13. Diagnosis of snake envenomation using a simple phospholipase A2 assay

    PubMed Central

    Maduwage, Kalana; O'Leary, Margaret A.; Isbister, Geoffrey K.

    2014-01-01

    Diagnosis of snake envenomation is challenging but critical for deciding on antivenom use. Phospholipase A2 enzymes occur commonly in snake venoms and we hypothesized that phospholipase activity detected in human blood post-bite may be indicative of envenomation. Using a simple assay, potentially a bedside test, we detected high phospholipase activity in sera of patients with viper and elapid envenomation compared to minimal activity in non-envenomed patients. PMID:24777205

  14. Purification of phospholipase D from citrus callus tissue.

    PubMed

    Witt, W; Yelenosky, G; Mayer, R T

    1987-11-15

    Phospholipase D in extracts of soluble proteins from callus cultures derived from cotyledons of Citrus sinensis (L.) Osbeck is activated by Ca2+ and anionic detergents and has a pH optimum of 6.5. The enzyme was purified 703-fold over the crude protein extract with a yield of 15% by ammonium sulfate precipitation, ion exchange chromatography, gel filtration, hydrophobic interaction chromatography, and preparative acrylamide gel electrophoresis. Preparative electrophoresis was carried out using conventional slab gel equipment and electroelution of the sliced gel. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified phospholipase revealed two bands of the same staining intensity running at 94.2K and 90.5K. PMID:3688883

  15. Odorant-sensitive phospholipase C in insect antennae.

    PubMed

    Boekhoff, I; Strotmann, J; Raming, K; Tareilus, E; Breer, H

    1990-01-01

    Exogenous tritiated phosphatidylinositol bisphosphate added to antennal preparations from locust and cockroach was hydrolysed releasing inositol trisphosphate. High activity of phospholipase C was detected in the soluble as well as in the membrane fraction. At low free calcium concentrations hydrolysis of the labelled lipid was stimulated by odorants and pheromones in a GTP-dependent manner. Consequently the level of inositol trisphosphate in antennal preparations increased upon odorant stimulation. PMID:2176800

  16. Purification and characterization of a phospholipase by Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum.

    PubMed

    Hsu, Po-Yuan; Lee, Kuo-Kau; Hu, Chih-Chuang; Liu, Ping-Chung

    2014-09-01

    Toxicity of the extracellular products (ECPs) and the lethal attributes of phospholipase secreted by pathogenic Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum was studied. An extracellular lethal toxin in the ECPs was partially purified by using Fast Protein Liquid Chromatography system. A protein band (27 kDa) exhibited phospholipase activity on Native-PAGE (by 0.3% egg yolk agar-overlay), was excised and eluted. The pI value of the purified phospholipase was determined as 3.65 and was determined as a phospholipase C by using the Amplex™ Red phosphatidylcholine -Specific phospholipase C Assay kit. The phospholipase showed maximum activity at temperature around 4-40 °C and maximal activity at pH between 8 and 9. The enzyme was inhibited by ethylenediamine-tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS); but was activated by Ca(2+) and Mg(2+) and inactivated by Zn(2+) and Cu(2+) . Both the ECPs and phospholipase were hemolytic against erythrocytes of cobia and lethal to the fish with LD50 values of 3.25 and 0.91 µg protein g(-1) fish, respectively. In toxicity neutralization test, the rabbit antisera against the phospholipase could neutralize the toxicity of ECPs, indicating that the phospholipase is a major extracellular toxin produced by the bacterium. PMID:23787821

  17. [Do phospholipases, key enzymes in sperm physiology, represent therapeutic challenges?].

    PubMed

    Arnoult, Christophe; Escoffier, Jessica; Munch, Léa; Pierre, Virginie; Hennebicq, Sylviane; Lambeau, Gérard; Ray, Pierre

    2012-05-01

    The spermatozoon is one of the most differentiated cells in mammals and its production requires an extremely complex machinery. Subtle but critical molecular changes take place during capacitation, which comprises the last series of maturation steps that naturally occur between the cauda epididymidis where spermatozoa are stored and their ultimate destination inside the oocyte. Phospholipases, by hydrolyzing various phospholipids, have been found to be critical in sperm processes such as 1) the control of flagellum beats, 2) capacitation - the molecular transformations preparing the sperm for fertilization, 3) acrosome reaction and 4) oocyte activation by eliciting calcium oscillations. The emerging important role of phospholipases is also emphasized by the fact that alterations of sperm lipids can lead to infertility. Phospholipases may represent valuable targets to develop anti- and pro-fertility drugs. Results obtained in mice are encouraging, since treatment of sperm with recombinant sPLA(2) of group X, known to be involved in capacitation, improves fertilization in vitro, while co-injection of PLCζ RNA with infertile sperm restores oocyte activation. PMID:22643005

  18. Phospholipase C in Beijing strains of Mycobacterium tuberculosis

    PubMed Central

    Mirsamadi, ES; Farnia, P; Jahani Sherafat, S; Esfahani, M; Faramarzi, N

    2010-01-01

    Background and Objectives Phospholipase of Mycobacterium tuberculosis plays an important role in pathogenesis through breaking up phospholipids and production of diacylglycerol. In this study, we examined the Beijing strains of Mycobacterium tuberculosis isolated from Iranian patients for the genes encoding this enzyme. Materials and Methods DNA extraction was performed using CTAB (cetyltrimethylammonium bromide) from positive culture specimens in tuberculosis patients. PCR was then used to amplify the plcA, plcB, plcC genes of Beijing strain, and non-Beijing strains were identified by spoligotyping. Results Of 200 specimens, 19 (9.5%) were Beijing strain and 181 (90.5%) were non-Beijing strains. The results of PCR for Beijing strains were as follows: 16 strains (84.2%) were positive for plcA, 17 (89.4%) were positive for plcB and 17 (89.4%) were positive for plcC genes. The standard strain (H37RV) was used as control. Conclusion The majority of Beijing strains have phospholipase C genes which can contribute to their pathogenesis but we need complementary studies to confirm the role of phospholipase C in pathogenecity of Mycobacterium tuberculosis. PMID:22347572

  19. Restoration of Responsiveness of Phospholipase Cγ2-Deficient Platelets by Enforced Expression of Phospholipase Cγ1

    PubMed Central

    Zheng, Yongwei; Adams, Tamara; Zhi, Huiying; Yu, Mei; Wen, Renren; Newman, Peter J.; Wang, Demin; Newman, Debra K.

    2015-01-01

    Receptor-mediated platelet activation requires phospholipase C (PLC) activity to elevate intracellular calcium and induce actin cytoskeleton reorganization. PLCs are classified into structurally distinct β, γ, δ, ε, ζ, and η isoforms. There are two PLCγ isoforms (PLCγ1, PLCγ2), which are critical for activation by tyrosine kinase-dependent receptors. Platelets express both PLCγ1 and PLCγ2. Although PLCγ2 has been shown to play a dominant role in platelet activation, the extent to which PLCγ1 contributes has not been evaluated. To ascertain the relative contributions of PLCγ1 and PLCγ2 to platelet activation, we generated conditionally PLCγ1-deficient, wild-type (WT), PLCγ2-deficient, and PLCγ1/PLCγ2 double-deficient mice and measured the ability of platelets to respond to different agonists. We found that PLCγ2 deficiency abrogated αIIbβ3-dependent platelet spreading, GPVI-dependent platelet aggregation, and thrombus formation on collagen-coated surfaces under shear conditions, which is dependent on both GPVI and αIIbβ3. Addition of exogenous ADP overcame defective spreading of PLCγ2-deficient platelets on immobilized fibrinogen, suggesting that PLCγ2 is required for granule secretion in response to αIIbβ3 ligation. Consistently, αIIbβ3-mediated release of granule contents was impaired in the absence of PLCγ2. In contrast, PLCγ1-deficient platelets spread and released granule contents normally on fibrinogen, exhibited normal levels of GPVI-dependent aggregation, and formed thrombi normally on collagen-coated surfaces. Interestingly, enforced expression of PLCγ1 fully restored GPVI-dependent aggregation and αIIbβ3-dependent spreading of PLCγ2-deficient platelets. We conclude that platelet activation through GPVI and αIIbβ3 utilizes PLCγ2 because PLCγ1 levels are insufficient to support responsiveness, but that PLCγ1 can restore responsiveness if expressed at levels normally achieved by PLCγ2. PMID:25793864

  20. Phospholipase activity of Mycobacterium leprae harvested from experimentally infected armadillo tissue.

    PubMed Central

    Wheeler, P R; Ratledge, C

    1991-01-01

    Three types of phospholipase activity--phospholipase A1, A2, and lysophospholipase--were detected in Mycobacterium leprae harvested from armadillo tissue at about 25% of the specific activity found in a slowly growing mycobacterium, Mycobacterium microti, which was grown in medium to optimize its phospholipase activity. The highest activity found was lysophospholipase, which released fatty acid from 2-lyso-phosphatidylcholine. Phospholipase activity was detected by using phosphatidylcholine and phosphatidylethanolamine. Differences in relative activities with these three types of substrate distinguished phospholipase activity in M. leprae extracts from armadillo liver extracts. Furthermore, retention of activity in M. leprae after NaOH treatment showed that the activity associated with M. leprae was not host derived. The specific activity of phospholipase was 20 times higher in extracts of M. leprae than in intact M. leprae organisms. Diazotization, a treatment which abolishes activities of surface enzymes exposed to the environment by the formation of covalent azide bonds with exposed amino groups, did not affect M. leprae's phospholipase activity, with one exception: release of arachidonic acid from phosphatidylcholine, which was partially inhibited. Phenolic glycolipid I, the major excreted amphipathic lipid of M. leprae, inhibited phospholipase activity, including release of arachidonic acid, for both M. leprae- and armadillo-derived activity. PMID:1855994

  1. The distribution of phospholipase D in developing and mature plants

    PubMed Central

    Quarles, R. H.; Dawson, R. M. C.

    1969-01-01

    1. The distribution of phospholipase D (phosphatidylcholine phosphatido-hydrolase, EC 3.1.4.4) was examined in the tissues of a number of plants and seeds. 2. The highest activities were found in various swollen storage tissues of certain plants: cabbage, central stalk; cauliflower, flower; celery, swollen leaf stalk; Kohl rabi, swollen stem; carrot, root; pea and marrow, seed. 3. Appreciable activity was retained in pea seeds for at least 1 year after drying. After germination and growth in the dark the total activity present in the cotyledons and also in the whole seedling decreased. 4. In the growing pea seedling (7 days old), about 3% of the total activity was in the plumule, 9% in the root and the remainder in the cotyledons. However, the activity in the root on a dry-weight basis was higher than that in the cotyledons. In both the root and the plumule the activity on a wet- or a dry-weight basis was highest in the growing tip. 5. The activity per dry weight in the roots and aerial parts of pea plants declined to low values as growth continued, but roots struck from cuttings of mature plants showed the same high activity as found in roots from young seedlings with cotyledons attached. 6. The total phospholipids present in the cotyledons of pea seeds were depleted on germination and growth. Of the individual phospholipids, phosphatidylcholine and phosphatidylethanolamine showed the same loss in 11 days as the whole phospholipid fraction, whereas phosphatidylinositol was decreased to a greater extent and cardiolipin and phosphatidylserine were not decreased. There was no increase of phosphatidic acid, as might have been expected if the phospholipids had disappeared through phospholipase D hydrolysis. 7. It is concluded that phospholipase D in plant storage tissues and seeds may be related to the rapid growth involved in their formation rather than being necessary for the utilization of their food reserve substances. PMID:4309675

  2. A role for Phospholipase D in Drosophila embryonic cellularization

    PubMed Central

    LaLonde, Mary; Janssens, Hilde; Yun, Suyong; Crosby, Juan; Redina, Olga; Olive, Virginie; Altshuller, Yelena M; Choi, Seok-Yong; Du, Guangwei; Gergen, J Peter; Frohman, Michael A

    2006-01-01

    Background Cellularization of the Drosophila embryo is an unusually synchronous form of cytokinesis in which polarized membrane extension proceeds in part through incorporation of new membrane via fusion of apically-translocated Golgi-derived vesicles. Results We describe here involvement of the signaling enzyme Phospholipase D (Pld) in regulation of this developmental step. Functional analysis using gene targeting revealed that cellularization is hindered by the loss of Pld, resulting frequently in early embryonic developmental arrest. Mechanistically, chronic Pld deficiency causes abnormal Golgi structure and secretory vesicle trafficking. Conclusion Our results suggest that Pld functions to promote trafficking of Golgi-derived fusion-competent vesicles during cellularization. PMID:17156430

  3. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of active Gα protein. PMID:27124090

  4. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A/sub 2/

    SciTech Connect

    Burch, R.M.; Axelrod, J.

    1987-09-01

    In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E/sub 2/ (PGE/sub 2/) synthesis. The EC/sub 50/ values for stimulation of PGE/sub 2/ synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-(..gamma..-thio)triphosphate stimulated PGE/sub 2/ synthesis and InsP formation, and guanosine-5'-(..beta..-thio)diphosphate inhibited both PGE/sub 2/ synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE/sub 2/ synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE/sub 2/ synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE/sub 2/ synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE/sub 2/ synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with (/sup 3/H) choline, the phospholipase A/sub 2/ products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A/sub 2/ and that phospholipase A/sub 2/ is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.

  5. Interactions of phospholipase D and cytochrome P450 protein stability

    SciTech Connect

    Zangar, Richard C.; Fan, Yang-Yi; Chapkin, Robert S.

    2004-08-01

    Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.

  6. Effects of Phospholipase A2 Inhibitors on Bilayer Lipid Membranes.

    PubMed

    Dubinin, Mikhail V; Astashev, Maxim E; Penkov, Nikita V; Gudkov, Sergey V; Dyachenko, Igor A; Samartsev, Victor N; Belosludtsev, Konstantin N

    2016-06-01

    The work examines the effect of inhibitors of cytosolic Ca(2+)-dependent and Ca(2+)-independent phospholipases A2 on bilayer lipid membranes. It was established that trifluoroperazine (TFP) and, to a lesser extent, arachidonyl trifluoromethyl ketone (AACOCF3) and palmitoyl trifluoromethyl ketone (PACOCF3) were able to permeabilize artificial lipid membranes (BLM and liposomes). It was shown that AACOCF3 lowered the temperature of phase transition of DMPC liposomes, inducing disordering of the hydrophobic region of lipid bilayer. TFP disordered membranes both in the hydrophobic region and in the region of hydrophilic heads, this being accompanied by changes in the membrane permeability: appearance of a channel-like BLM activity and leakage of sulforhodamine B from liposomes. In contrast to AACOCF3 and TFP, PACOCF3 increased membrane orderliness in the hydrophobic region (heightened the temperature of phase transition of DMPC liposomes) and in the region of lipid heads. The effectiveness of AACOCF3 and PACOCF3 as inductors of BLM and liposome permeabilization was considerably lower comparatively to TFP. As revealed by dynamic light scattering, incorporation of TFP, AACOCF3 and PACOCF3 into the membrane of liposomes resulted in the increase of the average size of particles in the suspension, presumably due to their aggregation or fusion. The paper discusses possible mechanisms of the influence of phospholipase A2 inhibitors on bilayer lipid membranes. PMID:26762382

  7. Biochemical and monolayer characterization of Tunisian snake venom phospholipases.

    PubMed

    Baîram, Douja; Aissa, Imen; Louati, Hanen; Othman, Houcemeddine; Abdelkafi-Koubaa, Zaineb; Krayem, Najeh; El Ayeb, Mohamed; Srairi-Abid, Najet; Marrakchi, Naziha; Gargouri, Youssef

    2016-08-01

    The present study investigated the kinetic and interfacial properties of two secreted phospholipases isolated from Tunisian vipers'venoms: Cerastes cerastes (CC-PLA2) and Macrovipera lebetina transmediterranea (MVL-PLA2). Results show that these enzymes have great different abilities to bind and hydrolyse phospholipids. Using egg-yolk emulsions as substrate at pH 8, we found that MVL-PLA2 has a specific activity of 1473U/mg at 37°C in presence of 1mM CaCl2. Furthermore the interfacial kinetic and binding data indicate that MVL-PLA2 has a preference to the zwitterionic phosphatidylcholine monolayers (PC). Conversely, CC-PLA2 was found to be able to hydrolyse preferentially negatively charged head group phospholipids (PG and PS) and exhibits a specific activity 9 times more important (13333U/mg at 60°C in presence of 3mM CaCl2). Molecular models of both CC-PLA2 and MVL-PLA2 3D structures have been built and their electrostatic potentials surfaces have been calculated. A marked anisotropy of the overall electrostatic charge distribution leads to a significantly difference in the dipole moment intensity between the two enzymes explaining the great differences in catalytic and binding properties, which seems to be governed by the electrostatic and hydrophobic forces operative at the surface of the two phospholipases. PMID:27164498

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

    SciTech Connect

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

    1989-07-01

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

  9. Phospholipase A2 activity in Epstein-Barr virus-transformed lymphoblast cells from schizophrenic patients.

    PubMed

    Bennett, E R; Yedgar, S; Lerer, B; Ebstein, R P

    1991-06-01

    We examined the activity of phospholipase A2 in Epstein-Barr virus-transformed lymphoblast cell lines established from ten schizophrenic patients and ten controls. A novel method for determination of enzyme activity in whole cells was employed, by measuring the hydrolysis of a fluorescent analogue of phosphatidylcholine. No significant difference in phospholipase A2 activity was found between the groups. These results suggest that the previously reported changes in phospholipase A2 activity in plasma and in fresh peripheral cells are indicative of environmental influences and not of "trait" characteristics intrinsic to schizophrenia. PMID:1651772

  10. 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. PMID:15019494

  11. Gβ1γ2 activates phospholipase A2-dependent Golgi membrane tubule formation

    PubMed Central

    Bechler, Marie E.; Brown, William J.

    2014-01-01

    Heterotrimeric G proteins transduce the ligand binding of transmembrane G protein coupled receptors into a variety of intracellular signaling pathways. Recently, heterotrimeric Gβγ subunit signaling at the Golgi complex has been shown to regulate the formation of vesicular transport carriers that deliver cargo from the Golgi to the plasma membrane. In addition to vesicles, membrane tubules have also been shown to mediate export from the Golgi complex, which requires the activity of cytoplasmic phospholipase A2 (PLA2) enzyme activity. Through the use of an in vitro reconstitution assay with isolated Golgi complexes, we provide evidence that Gβ1γ2 signaling also stimulates Golgi membrane tubule formation. In addition, we show that an inhibitor of Gβγ activation of PLA2 enzymes inhibits in vitro Golgi membrane tubule formation. Additionally, purified Gβγ protein stimulates membrane tubules in the presence of low (sub-threshold) cytosol concentrations. Importantly, this Gβγ stimulation of Golgi membrane tubule formation was inhibited by treatment with the PLA2 antagonist ONO-RS-082. These studies indicate that Gβ1γ2 signaling activates PLA2 enzymes required for Golgi membrane tubule formation, thus establishing a new layer of regulation for this process. PMID:25019068

  12. Expression and Function of Group IIE Phospholipase A2 in Mouse Skin.

    PubMed

    Yamamoto, Kei; Miki, Yoshimi; Sato, Hiroyasu; Nishito, Yasumasa; Gelb, Michael H; Taketomi, Yoshitaka; Murakami, Makoto

    2016-07-22

    Recent studies using knock-out mice for various secreted phospholipase A2 (sPLA2) isoforms have revealed their non-redundant roles in diverse biological events. In the skin, group IIF sPLA2 (sPLA2-IIF), an "epidermal sPLA2" expressed in the suprabasal keratinocytes, plays a fundamental role in epidermal-hyperplasic diseases such as psoriasis and skin cancer. In this study, we found that group IIE sPLA2 (sPLA2-IIE) was expressed abundantly in hair follicles and to a lesser extent in basal epidermal keratinocytes in mouse skin. Mice lacking sPLA2-IIE exhibited skin abnormalities distinct from those in mice lacking sPLA2-IIF, with perturbation of hair follicle ultrastructure, modest changes in the steady-state expression of a subset of skin genes, and no changes in the features of psoriasis or contact dermatitis. Lipidomics analysis revealed that sPLA2-IIE and -IIF were coupled with distinct lipid pathways in the skin. Overall, two skin sPLA2s, hair follicular sPLA2-IIE and epidermal sPLA2-IIF, play non-redundant roles in distinct compartments of mouse skin, underscoring the functional diversity of multiple sPLA2s in the coordinated regulation of skin homeostasis and diseases. PMID:27226633

  13. Phospholipase D2-dependent inhibition of the nuclear hormone receptor PPARgamma by cyclic phosphatidic acid.

    PubMed

    Tsukahara, Tamotsu; Tsukahara, Ryoko; Fujiwara, Yuko; Yue, Junming; Cheng, Yunhui; Guo, Huazhang; Bolen, Alyssa; Zhang, Chunxiang; Balazs, Louisa; Re, Fabio; Du, Guangwei; Frohman, Michael A; Baker, Daniel L; Parrill, Abby L; Uchiyama, Ayako; Kobayashi, Tetsuyuki; Murakami-Murofushi, Kimiko; Tigyi, Gabor

    2010-08-13

    Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists. PMID:20705243

  14. Glucose and carbachol activate phospholipase C in digitonin-permeabilized islets

    SciTech Connect

    Wolf, B.A.; Florholmen, J.; Turk, J.; McDaniel, M.L.

    1987-05-01

    Stimulation of intact islets with D-glucose, the major insulin secretagogue, or with carbachol, a muscarinic agonist, results in the accumulation of inositoltrisphosphate (IP/sub 3/) suggesting that activation of phospholipase C (PLC) has a major role in stimulus-secretion coupling. Carbachol activation of PLC is an example of receptor-mediated activation in islets, whereas, the mechanism of glucose activation of PLC is controversial since a glucose receptor has not been identified. They have measured PLC activity in digitonin-permeabilized islets. Islets were labeled with /sup 3/H-inositol, permeabilized and IP/sub 3/ accumulation measured by HPLC. Carbachol, in the presence of ATP, GTP and 1 ..mu..M free Ca/sup 2 +/ released two-fold more Ins 1,3,4-P/sub 3/ than control in a time-dependent manner. Glucose, under the same conditions also significantly released more Ins 1,3,4-P/sub 3/ than control. This effect was not due to metabolism of glucose nor to an effect on the IP/sub 3/-phosphomonoesterase. Preliminary Ca/sup 2 +/-dependency studies indicate that PLC is not activated by Ca/sup 2 +/ in the submicromolar range. In conclusion, these studies show that Ca/sup 2 +/ does not activate PLC, and furthermore, that D-glucose may be recognized directly by PLC.

  15. Inactivation of the phospholipase B gene PLB5 in wild-type Candida albicans reduces cell-associated phospholipase A2 activity and attenuates virulence

    PubMed Central

    Theiss, Stephanie; Ishdorj, Ganchimeg; Brenot, Audrey; Kretschmar, Marianne; Lan, Chung-Yu; Nichterlein, Thomas; Hacker, Jörg; Nigam, Santosh; Agabian, Nina; Köhler, Gerwald A.

    2008-01-01

    Phospholipases are critical for modification and redistribution of lipid substrates, membrane remodeling and microbial virulence. Among the many different classes of phospholipases, fungal phospholipase B (Plb) proteins show the broadest range of substrate specificity and hydrolytic activity, hydrolyzing acyl ester bonds in phospholipids and lysophospholipids and further catalyzing lysophospholipase-transacylase reactions. The genome of the opportunistic fungal pathogen Candida albicans encodes a PLB multigene family with five putative members; we present the first characterization of this group of potential virulence determinants. CaPLB5, the third member of this multigene family characterized herein is a putative secretory protein with a predicted GPI-anchor attachment site. Real-time RT-PCR gene expression analysis of CaPLB5 and the additional CaPLB gene family members revealed that filamentous growth and physiologically relevant environmental conditions are associated with increased phospholipase B gene activity. The phenotypes expressed by null mutant and revertant strains of CaPLB5 indicate that this lipid hydrolase plays an important role for cell-associated phospholipase A2 activity and in vivo organ colonization. PMID:16759910

  16. Loss of phospholipase D2 impairs VEGF-induced angiogenesis

    PubMed Central

    Lee, Chang Sup; Ghim, Jaewang; Song, Parkyong; Suh, Pann-Ghill; Ryu, Sung Ho

    2016-01-01

    Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and critical for normal embryonic development and repair of pathophysiological conditions in adults. Although phospholipase D (PLD) activity has been implicated in angiogenic processes, its role in VEGF signaling during angiogenesis in mammals is unclear. Here, we found that silencing of PLD2 by siRNA blocked VEGF-mediated signaling in immortalized human umbilical vein endothelial cells (iHUVECs). Also, VEGF-induced endothelial cell survival, proliferation, migration, and tube formation were inhibited by PLD2 silencing. Furthermore, while Pld2-knockout mice exhibited normal development, loss of PLD2 inhibited VEGF-mediated ex vivo angiogenesis. These findings suggest that PLD2 functions as a key mediator in the VEGF-mediated angiogenic functions of endothelial cells. [BMB Reports 2016; 49(3): 191-196] PMID:26818087

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

    PubMed

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

    2004-03-26

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

  18. Purification and kinetics of extracellular phospholipase A of Salmonella newport.

    PubMed

    Neena, S; Asnani, P J; Bhandari, S; Vohra, R

    1992-01-01

    Attempts were made to purify and study the kinetics of extracellular phospholipase A of Salmonella newport (6,8, eb; 1,2). The enzyme was purified by salt precipitation followed by gel filtration, using different grades of Sephadex. The enzymically active purified preparation was found to be a protein, having molar mass ranging between 43 and 67 kDa. The enzyme had a pH optimum at 7.5, giving 18.2 micrograms of lysophosphatidylcholine per mg protein. Its activity was enhanced by all metal ions except potassium, by solvents and surfactants except sodium dodecyl sulfate. It hydrolyzed the membrane phospholipids of red blood cells and was inhibitory to the growth of other microorganisms. PMID:1505883

  19. Modulation of phospholipase A2 activity in human fibroblasts.

    PubMed Central

    Solito, E.; Parente, L.

    1989-01-01

    1. Human embryonic skin fibroblasts (HSF) incubated overnight with either human recombinant interleukin-1 alpha (rIL-1 alpha) or interleukin-1 beta (rIL-1 beta) released large amounts of prostaglandin E2 (PGE2). 2. rIL-1 beta, bradykinin (Bk) and arachidonic acid (AA) significantly stimulated PGE2 release from HSF incubated overnight in the presence of either interleukin. 3. Hydrocortisone inhibited the PGE2 release induced by rIL-1 beta and Bk, but not by AA. 4. The steroid inhibitory effect was reversed by actinomycin D as well as by an anti-lipocortin monoclonal antibody. 5. The results suggest that in HSF, rIL-1 beta is able to stimulate both cyclo-oxygenase and phospholipase A2 (PLA2) activity. 6. The stimulation of PLA2 activity by rIL-1 beta is inhibited by hydrocortisone, probably via induction of lipocortin-like proteins. PMID:2785834

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

    PubMed

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

    2016-01-01

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

  1. 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. PMID:23192346

  2. 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

  3. 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

  4. Biochemical signal transmitted by Fc gamma receptors: phospholipase A2 activity of Fc gamma 2b receptor of murine macrophage cell line P388D1.

    PubMed Central

    Suzuki, T; Saito-Taki, T; Sadasivan, R; Nitta, T

    1982-01-01

    The detergent lysate of the P388D1 macrophage cell line was subjected to affinity chromatography on two different media, Sepharose coupled to heat-aggregated human IgG (IgG-Sepharose) and Sepharose coupled to the phosphatidylcholine analog rac-1-(9-carboxyl)nonyl-2-hexadecylglycero-3-phosphocholine (PC-Sepharose). Both IgG- and phosphatidylcholine-binding proteins were further purified by Sephadex G-100 gel filtration and isoelectric focusing in the presence of 6 M urea. The isolated IgG-binding proteins specifically bound to IgG2a, but not to IgG2b, whereas the isolated phosphatidylcholine-binding proteins specifically bound to IgG2b but not to IgG2a. Phosphatidylcholine-binding proteins possessed a typical phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4), which was maximal (10 mumol/min per mg of protein) at pH 9.5, depended on Ca2+, and was specific for cleavage of fatty acid from the C-2 position of the glycerol backbone of phosphatidylcholine. The noted enzymatic activity was augmented 4-fold by preincubating phosphatidylcholine-binding proteins with heat-aggregated murine IgG2b but not with IgG2a. IgG-binding proteins, on the other hand, are devoid of any detectable phospholipase A2 activity. Thus, the functional significance of Fc gamma 2b receptor of P388D1 macrophage cell line would be the generation of phospholipase A2 activity at the cell surface upon specific binding to Fc gamma 2b fragment. PMID:6804944

  5. 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

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

    PubMed

    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

  7. Modulation of membrane phospholipids, the cytosolic calcium influx and cell proliferation following treatment of B16-F10 cells with recombinant phospholipase-D from Loxosceles intermedia (brown spider) venom.

    PubMed

    Wille, Ana Carolina Martins; Chaves-Moreira, Daniele; Trevisan-Silva, Dilza; Magnoni, Mariana Gabriel; Boia-Ferreira, Marianna; Gremski, Luiza Helena; Gremski, Waldemiro; Chaim, Olga Meiri; Senff-Ribeiro, Andrea; Veiga, Silvio Sanches

    2013-06-01

    The mechanism through which brown spiders (Loxosceles genus) cause dermonecrosis, dysregulated inflammatory responses, hemolysis and platelet aggregation, which are effects reported following spider bites, is currently attributed to the presence of phospholipase-D in the venom. In the present investigation, through two-dimensional immunoblotting, we observed immunological cross-reactivity for at least 25 spots in crude Loxosceles intermedia venom, indicating high expression levels for different isoforms of phospholipase-D. Using a recombinant phospholipase-D from the venom gland of L. intermedia (LiRecDT1) in phospholipid-degrading kinetic experiments, we determined that this phospholipase-D mainly hydrolyzes synthetic sphingomyelin in a time-dependent manner, generating ceramide 1-phosphate plus choline, as well as lysophosphatidylcholine, generating lysophosphatidic acid plus choline, but exhibits little activity against phosphatidylcholine. Through immunofluorescence assays with antibodies against LiRecDT1 and using a recombinant GFP-LiRecDT1 fusion protein, we observed direct binding of LiRecDT1 to the membrane of B16-F10 cells. We determined that LiRecDT1 hydrolyzes phospholipids in detergent extracts and from ghosts of B16-F10 cells, generating choline, indicating that the enzyme can access and modulate and has activity against membrane phospholipids. Additionally, using Fluo-4, a calcium-sensitive fluorophore, it was shown that treatment of cells with phospholipase-D induced an increase in the calcium concentration in the cytoplasm, but without altering viability or causing damage to cells. Finally, based on the known endogenous activity of phospholipase-D as an inducer of cell proliferation and the fact that LiRecDT1 binds to the cell surface, hydrolyzing phospholipids to generate bioactive lipids, we employed LiRecDT1 as an exogenous source of phospholipase-D in B16-F10 cells. Treatment of the cells was effective in increasing their proliferation in a

  8. 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. PMID:26691755

  9. Phospholipases and arachidonic acid contribute independently to sensory transduction and associative neuronal facilitation in Hermissenda type B photoreceptors.

    PubMed

    Talk, A C; Muzzio, I A; Matzel, L D

    1997-03-21

    During contiguous pairings of light and rotation, B photoreceptors in the Hermissenda eye undergo an increase in excitability that contributes to a modification of several light-elicited behaviors. This excitability increase requires a light-induced rise in intracellular Ca2+ in the photoreceptor concomitant with transmitter binding to G protein-coupled receptors as a result of presynaptic vestibular hair cell stimulation. Phospholipases and arachidonic acid (ArA) are here reported to be involved in independent signal transduction pathways that underlie both receptor function and activity-dependent facilitation of the B photoreceptor. 4-Bromophenacyl bromide (BPB), an inhibitor of phospholipases A2 (PLA2) and C (PLC), blocked the generation of light-induced depolarizing generator potentials, but had no affect on the inhibitory postsynaptic potential (IPSP) in the B cell that results from hair cell stimulation. Quinacrine, which predominantly blocks the activity of PLA2 in neurons, had no affect on either the light response or the IPSP, but did block increases in excitability (i.e. increased input resistance and elicited spike rate) of the B cell that results from pairings of light and presynaptic vestibular stimulation (i.e., in vitro associative conditioning). Neither nordihydroquararetic acid (NDGA), which inhibits metabolism of ArA by cyclooxygenase, nor indomethacin, which inhibits lipoxygenase metabolism of ArA, affected the light response or IPSP, but both blocked the increases in excitability in the B cell that accompanied in vitro conditioning. In combination with earlier results, these data suggest that ArA activates PKC in a synergistic fashion with Ca2+ and diacylglycerol in the B cell, and suggest that PLA2-induced ArA release, though not necessary for transduction of light or the hair cell-induced IPSP in the B cell, is a critical component of the convergence of signals that precipitates associative facilitation in this system. PMID:9099806

  10. Amygdala-Hippocampal Phospholipase D (PLD) Signaling As Novel Mechanism of Cocaine-Environment Maladaptive Conditioned Responses

    PubMed Central

    2016-01-01

    Background: Drug-environment associative memory mechanisms and the resulting conditioned behaviors are key contributors in relapse to cocaine dependence. Recently, we reported rat amygdala phospholipase D as a key convergent downstream signaling partner in the expression of cocaine-conditioned behaviors mediated by glutamatergic and dopaminergic pathways. In the present study, 1 of the 2 known upstream serotonergic targets of phospholipase D, the serotonin (5-hydroxytryptamine) 2C receptor, was investigated for its role in recruiting phospholipase D signaling in cocaine-conditioned behaviors altered in the rat amygdala and dorsal hippocampus. Methods: Using Western-blot analysis, amygdala phospholipase D phosphorylation and total expression of phospholipase D/5-hydroxytryptamine 2C receptor were observed in early (Day-1) and late (Day-14) withdrawal (cocaine-free) states among male Sprague-Dawley rats subjected to 7-day cocaine-conditioned hyperactivity training. Functional studies were conducted using Chinese Hamster Ovary cells with stably transfected human unedited isoform of 5-hydroxytryptamine 2C receptor. Results: Phosphorylation of phospholipase D isoforms was altered in the Day-1 group of cocaine-conditioned animals, while increased amygdala and decreased dorsal hippocampus phospholipase D/5-hydroxytryptamine 2C receptor protein expression were observed in the Day-14 cocaine-conditioned rats. Functional cellular studies established that increased p phospholipase D is a mechanistic response to 5-HT2CR activation and provided the first evidence of a biased agonism by specific 5-hydroxytryptamine 2C receptor agonist, WAY163909 in phospholipase D phosphorylation 2, but not phospholipase D phosphorylation 1 activation. Conclusions: Phospholipase D signaling, activated by dopaminergic, glutamatergic, and serotonergic signaling, can be a common downstream element recruited in associative memory mechanisms altered by cocaine, where increased expression in amygdala

  11. 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. PMID:25529980

  12. Phospholipase Dε and Phosphatidic Acid Enhance Arabidopsis Growth

    PubMed Central

    Hong, Yueyun; Devaiah, Shivakumar P.; Bahn, SungChul; Thamasandra, Bharath N.; Li, Maoyin; Welti, Ruth; Wang, Xuemin

    2014-01-01

    Summary The activation of phospholipase D (PLD) produces phosphatidic acid (PA), a new lipid messenger implicated in cell growth and proliferation, but direct evidence for PLD and PA promotion of growth at an organismal level is lacking. Here we characterized a new PLD, PLDε, and show that PLDε plays a role in promoting Arabidopsis growth. PLDε is mainly associated with the plasma membrane and is the most permissive of all PLDs tested in activity requirements. Knockout (KO) of PLDε decreases, whereas overexpression (OE) of PLDε enhances root growth and biomass accumulation. The level of PA was higher in OE, but lower in KO than in wild-type plants, and suppression of PLD-mediated PA formation by alcohol alleviated the growth-promoting effect of PLDε. OE and KO of PLDε had the opposite effect on lateral root elongation in response to nitrogen (N). Increased expression of PLDε also promoted root hair elongation and primary root growth at severe N deprivation. The results suggest that PLDε and PA promote organismal growth and play a role in N response. The lipid signaling process may play a role in translating the membrane sensing of nutrient status to increasing plant growth and biomass production. PMID:19143999

  13. G Protein Activation Stimulates Phospholipase D Signaling in Plants.

    PubMed Central

    Munnik, T.; Arisz, S. A.; De Vrije, T.; Musgrave, A.

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals. PMID:12242371

  14. 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. PMID:27129182

  15. Molecular characteristics of horse phospholipase C zeta (PLCζ).

    PubMed

    Sato, Kana; Wakai, Takuya; Seita, Yasunari; Takizawa, Akiko; Fissore, Rafael A; Ito, Junya; Kashiwazaki, Naomi

    2013-04-01

    A sperm-specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca(2+) ]i ) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT-PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long-lasting [Ca(2+) ]i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca(2+) ]i oscillation-inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse. PMID:23590511

  16. Going into labor and beyond: phospholipase A2 in pregnancy.

    PubMed

    Besenboeck, Carolin; Cvitic, Silvija; Lang, Uwe; Desoye, Gernot; Wadsack, Christian

    2016-06-01

    The phospholipase A2 (PLA2) family is a very diverse group of enzymes, all serving in the cleavage of phospholipids, thereby releasing high amounts of arachidonic acid (AA) and lysophospholipids. AA serves as a substrate for prostaglandin production, which is of special importance in pregnancy for the onset of parturition. Novel research demonstrates that PLA2 action affects the immune response of the mother toward the child and is therefore probably implied in the tolerance of the fetus and prevention of miscarriage. This review presents data on the biochemical and enzymatic properties of PLA2 during gestation with a special emphasis on its role for the placental function and development of the fetus. We also critically discuss the possible pathophysiological significance of PLA2 alterations and its possible functional consequences. These alterations are often associated with pregnancy pathologies such as preeclampsia and villitis or pregnancy complications such as obesity and diabetes in the mother as well as preterm onset of labor. PMID:26908920

  17. Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders*

    PubMed Central

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

    2015-01-01

    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. 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. PMID:25752604

  18. Membrane and inhibitor interactions of intracellular phospholipases A2.

    PubMed

    Mouchlis, Varnavas D; Dennis, Edward A

    2016-05-01

    Studying phospholipases A2 (PLA2s) is a challenging task since they act on membrane-like aggregated substrates and not on monomeric phospholipids. Multidisciplinary approaches that include hydrogen/deuterium exchange mass spectrometry (DXMS) and computational techniques have been employed with great success in order to address important questions about the mode of interactions of PLA2 enzymes with membranes, phospholipid substrates and inhibitors. Understanding the interactions of PLA2s is crucial since these enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid (AA) and other polyunsaturated fatty acids (PUFA). The liberation of AA by PLA2 enzymes sets off a cascade of molecular events that involves downstream regulators such as cyclooxygenase (COX) and lipoxygenase (LOX) metabolites leading to inflammation. Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) work by inhibiting COX, while Zileuton inhibits LOX and both rely on PLA2 enzymes to provide them with AA. That means PLA2 enzymes can potentially also be targeted to diminish inflammation at an earlier point in the process. In this review we describe extensive efforts reported in the past to define the interactions of PLA2 enzymes with membranes, substrate phospholipids and inhibitors using DXMS, molecular docking, and molecular dynamics (MD) simulations. PMID:26774606

  19. 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

  20. 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. PMID:22450361

  1. Are events after endotoxemia related to circulating phospholipase A2?

    PubMed Central

    Santos, A A; Browning, J L; Scheltinga, M R; Lynch, E A; Brown, E F; Lawton, P; Chambers, E; Dougas, I; Benjamin, C D; Dinarello, C A

    1994-01-01

    OBJECTIVE: The authors sought to determine whether the signs and symptoms of endotoxemia were related to the endotoxin-stimulated increase in circulating phospholipase A2 (PLA2) activity. BACKGROUND: Because hypotension and pulmonary injury have been associated with elevated PLA2 activity in septic shock and PLA2 levels are reduced with the administration of glucocorticoids, the PLA2 response to endotoxin was investigated in volunteers pretreated with and without hydrocortisone. METHODS: Carefully screened human subjects were studied under four conditions: (1) saline, (2) hydrocortisone, (3) endotoxin, and (4) hydrocortisone administration before endotoxin exposure. Pulse rate, blood pressure, temperature, and symptoms of endotoxemia were serially measured. Plasma for tumor necrosis factor concentrations and PLA2 activity was obtained. RESULTS: After lipopolysaccharide, pulse rate and tumor necrosis factor concentrations rose at 1 to 2 hours; temperature increased maximally at 4 hours. PLA2 activity reached peak levels at 24 hours. With hydrocortisone pretreatment, a 50% reduction in the concentrations of tumor necrosis factor and PLA2 occurred. Significant correlations between other variables and PLA2 activity were not observed. The enzyme identified by monoclonal antibody was the secreted nonpancreatic PLA2 (SNP-PLA2). CONCLUSIONS: The results of this study suggest that elevations in circulating SNP-PLA2 activity and systemic events associated with intravenous endotoxin administration are unrelated. PMID:8129489

  2. Bee Venom Phospholipase A2: Yesterday's Enemy Becomes Today's Friend.

    PubMed

    Lee, Gihyun; Bae, Hyunsu

    2016-02-01

    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

  3. Stability of soybean oil degumming using immobilized phospholipase A(2).

    PubMed

    Yu, Dianyu; Ma, Ying; Jiang, Lianzhou; Walid, Elfalleh; He, Shenghua; He, Yanming; Xiaoyu, Zhou; Zhang, Jianing; Hu, Lizhi

    2014-01-01

    The aim of this study was evaluation of stability of immobilized phospholipase A2 (PLA2) for soybean oil degumming. Also, the effect of reaction time on residual phosphorus levels was investigated according to the optimum pH and temperature. The free PLA2 and three immobilized PLA2 demonstrated significant differences in optimum operation conditions. pH, temperature and reaction time increased upon immobilization for three different immobilized PLA2 (PLA2-CA, PLA2-CAC and PLA2-CAG). Immobilized PLA2 showed enhanced thermal stability and retained more than 74% of relative activity after 1 h of incubation at 60°C, while the free PLA2 retained only 33%. The three immobilized PLA2 retained 30% to 60% of initial activities after 7 recycles. In particular, PLA2-CAC has more significant profiles in pH, temperature, reaction time and showed the highest remaining activity, thermal stability, reusability. Therefore, PLA2-CAC is a suitable immobilized enzyme for soybean oil degumming process. PMID:24371193

  4. Cell wounding activates phospholipase D in primary mouse keratinocytes

    PubMed Central

    Arun, Senthil N.; Xie, Ding; Howard, Amber C.; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L.; Bollag, Wendy B.

    2013-01-01

    Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D3, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing. PMID:23288946

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

    PubMed

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

    2015-12-01

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

  6. 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

  7. Cell wounding activates phospholipase D in primary mouse keratinocytes.

    PubMed

    Arun, Senthil N; Xie, Ding; Howard, Amber C; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L; Bollag, Wendy B

    2013-03-01

    Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D₃, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing. PMID:23288946

  8. Activation of human inflammatory cells by secreted phospholipases A2.

    PubMed

    Triggiani, Massimo; Granata, Francescopaolo; Frattini, Annunziata; Marone, Gianni

    2006-11-01

    Secreted phospholipases A(2) (sPLA(2)s) are enzymes detected in serum and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Different isoforms of sPLA(2)s are expressed and released by human inflammatory cells, such as neutrophils, eosinophils, T cells, monocytes, macrophages and mast cells. sPLA(2)s generate arachidonic acid and lysophospholipids thus contributing to the production of bioactive lipid mediators in inflammatory cells. However, sPLA(2)s also activate human inflammatory cells by mechanisms unrelated to their enzymatic activity. Several human and non-human sPLA(2)s induce degranulation of mast cells, neutrophils and eosinophils and activate exocytosis in macrophages. In addition some, but not all, sPLA(2) isoforms promote cytokine and chemokine production from macrophages, neutrophils, eosinophils, monocytes and endothelial cells. These effects are primarily mediated by binding of sPLA(2)s to specific membrane targets (heparan sulfate proteoglycans, M-type, N-type or mannose receptors) expressed on effector cells. Thus, sPLA(2)s may play an important role in the initiation and amplification of inflammatory reactions by at least two mechanisms: production of lipid mediators and direct activation of inflammatory cells. Selective inhibitors of sPLA(2)-enzymatic activity and specific antagonists of sPLA(2) receptors are current being tested for pharmacological treatment of inflammatory and autoimmune diseases. PMID:16952481

  9. Differential regulation of renal phospholipase C isoforms by catecholamines.

    PubMed

    Yu, P Y; Asico, L D; Eisner, G M; Jose, P A

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam or pramipexole) or antinatriuresis (NE) occurred, the kidneys were removed for analysis of PLC isoform protein expression activity. Western blot analysis revealed that in renal cortical membranes, fenoldopam and pramipexole increased expression of PLC beta 1 and decreased expression of PLC gamma 1; PLC delta was unchanged. In the cytosol, pramipexole and fenoldopam increased expression of both PLC beta 1 and PLC gamma 1. No effects were noted in the medulla. A preferential D1 antagonist, SKF 83742, which by itself had no effect, blocked the effects of pramipexole, thus confirming the involvement of the D1 receptor. In contrast, NE also increased PLC beta 1 but did not affect PLC gamma 1 protein expression in membranes. The changes in PLC isoform expression were accompanied by similar changes in PLC isoform activity. These studies demonstrate for the first time differential regulation of PLC isoforms by catecholamines. PMID:7814630

  10. 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

  11. 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.

  12. A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

    PubMed Central

    Soragni, Elisabetta; Bolchi, Angelo; Balestrini, Raffaella; Gambaretto, Claudio; Percudani, Riccardo; Bonfante, Paola; Ottonello, Simone

    2001-01-01

    Important morphogenetic transitions in fungi are triggered by starvation-induced changes in the expression of structural surface proteins. Here, we report that nutrient deprivation causes a strong and reversible up-regulation of TbSP1, a surface-associated, Ca2+-dependent phospholipase from the mycorrhizal fungus Tuber borchii. TbSP1 is the first phospholipase A2 to be described in fungi and identifies a novel class of phospholipid-hydrolyzing enzymes. The TbSP1 phospholipase, which is synthesized initially as a pre-protein, is processed efficiently and secreted during the mycelial phase. The mature protein, however, also localizes to the inner cell wall layer, close to the plasma membrane, in both free-living and symbiosis-engaged hyphae. It thus appears that a dual localization phospholipase A2 is involved in the adaptation of a symbiotic fungus to conditions of persistent nutritional limitation. Moreover, the fact that TbSP1-related sequences are present in Streptomyces and Neurospora, and not in wholly sequenced non-filamentous microorganisms, points to a general role for TbSP1 phospholipases A2 in the organization of multicellular filamentous structures in bacteria and fungi. PMID:11566873

  13. Active site mapping of Loxosceles phospholipases D: Biochemical and biological features.

    PubMed

    Vuitika, L; Chaves-Moreira, D; Caruso, I; Lima, M A; Matsubara, F H; Murakami, M T; Takahashi, H K; Toledo, M S; Coronado, M A; Nader, H B; Senff-Ribeiro, A; Chaim, O M; Arni, R K; Veiga, S S

    2016-09-01

    Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D. PMID:27233517

  14. Nuclear phospholipase C-β1 and diacylglycerol LIPASE-α in brain cortical neurons.

    PubMed

    García del Caño, Gontzal; Montaña, Mario; Aretxabala, Xabier; González-Burguera, Imanol; López de Jesús, Maider; Barrondo, Sergio; Sallés, Joan

    2014-01-01

    Phosphoinositide (PtdIns) signaling involves the generation of lipid second messengers in response to stimuli in a receptor-mediated manner at the plasma membrane. In neuronal cells of adult brain, the standard model proposes that activation of metabotropic receptors coupled to Phospholipase C-β1 (PLC-β1) is linked to endocannabinoid signaling through the production of diacylglycerol (DAG), which could be systematically metabolized by 1,2-diacylglycerol Lipases (DAGL) to produce an increase of 2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid in the brain. However, the existence of a nuclear PtdIns metabolism independent from that occurring elsewhere in the cell is now widely accepted, suggesting that the nucleus constitutes both a functional and a distinct compartment for PtdIns metabolism. In this review, we shall highlight the main achievements in the field of neuronal nuclear inositol lipid metabolism with particular attention to progress made linked to the 2-AG biosynthesis. Our aim has been to identify potential sites of 2-AG synthesis other than the neuronal cytoplasmic compartment by determining the subcellular localization of PLC-β1 and DAGL-α, which is much more abundant than DAGL-β in brain. Our data show that PLC-β1 and DAGL-α are detected in discrete brain regions, with a marked predominance of pyramidal morphologies of positive cortical cells, consistent with their role in the biosynthesis and release of 2-AG by pyramidal neurons to control their synaptic inputs. However, as novelty, we showed here an integrated description of the localization of PLC-β1 and DAGL-α in the neuronal nuclear compartment. We discuss our comparative analysis of the expression patterns of PLC-β1 and DAGL-α, providing some insight into the potential autocrine role of 2-AG production in the neuronal nuclear compartment that probably subserve additional roles to the recognized activation of the CB1 cannabinoid receptor. PMID:24076015

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    2012-01-01

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

  18. Effects of endotoxin and dexamethasone on group I and II phospholipase A2 in rat ileum and stomach.

    PubMed Central

    Lilja, I; Dimberg, J; Sjödahl, R; Tagesson, C; Gustafson-Svärd, C

    1994-01-01

    Phospholipase A2 (EC 3.1.1.4) is a key enzyme in inflammation and is thought to play an important part in inflammatory diseases of the gastrointestinal tract. To investigate the nature and regulation of phospholipase A2 activity in the gastrointestinal mucosa, the distribution of messenger ribonucleic acid (mRNA) for group II phospholipase A2 in various parts of the rat gastrointestinal tract was studied, as well as the influence of endotoxin or dexamethasone, or both, on the group I and II phospholipase A2 mRNA expression and activity in the rat glandular stomach and distal ileum. The results show that (a) group II phospholipase A2 is present along the whole gastrointestinal tract, but in particularly large amounts in the distal ileum, (b) endotoxin increases group II, but not group I, phospholipase A2 mRNA expression in the glandular stomach and distal ileum, and (c) dexamethasone reduces the endotoxin induced increases in group II phospholipase mRNA expression and activity in the gastrointestinal mucosa. These findings suggest that phospholipase A2 of type II is a mediator of endotoxin effects in the gastrointestinal mucosa and that its expression at the mRNA level can be inhibited by corticosteroids. Images Figure 1 PMID:8307447

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

    SciTech Connect

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

    1990-12-14

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

  20. 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. PMID:27059515

  1. 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. PMID:12351830

  2. Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development.

    PubMed

    Stith, Bradley J

    2015-05-15

    This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

  3. Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

    PubMed Central

    Stith, Bradley J.

    2015-01-01

    This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

  4. Phospholipase A2 regulates eicosanoid class switching during inflammasome activation.

    PubMed

    Norris, Paul C; Gosselin, David; Reichart, Donna; Glass, Christopher K; Dennis, Edward A

    2014-09-01

    Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA4 receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X7, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2-derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X7 results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A2, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways. PMID:25139986

  5. 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. PMID:26056930

  6. Extracellular Paracoccidioides brasiliensis phospholipase B involvement in alveolar macrophage interaction

    PubMed Central

    2010-01-01

    Background Phospholipase B (PLB) has been reported to be one of the virulence factors for human pathogenic fungi and has also been described as necessary for the early events in infection. Based on these data, we investigated the role of PLB in virulence and modulation of the alveolar pulmonary immune response during infection using an in-vitro model of host-pathogen interaction, i.e. Paracoccidioides brasiliensis yeast cells infecting alveolar macrophage (MH-S) cells. Results The effect of PLB was analyzed using the specific inhibitor alexidine dihydrochloride (0.25 μM), and pulmonary surfactant (100 μg mL-1), during 6 hours of co-cultivation of P. brasiliensis and MH-S cells. Alexidine dihydrochloride inhibited PLB activity by 66% and significantly decreased the adhesion and internalization of yeast cells by MH-S cells. Genes involved in phagocytosis (trl2, cd14) and the inflammatory response (nfkb, tnf-α, il-1β) were down-regulated in the presence of this PLB inhibitor. In contrast, PLB activity and internalization of yeast cells significantly increased in the presence of pulmonary surfactant; under this condition, genes such as clec2 and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the levels of interleukin-10 (IL-10) and increased the levels of IL-12 and tumor necrosis factor-α (TNF-α). In addition, gene expression analysis of plb1, sod3 and icl1 suggests that P. brasiliensis gene re-programming is effective in facilitating adaptation to this inhospitable environment, which mimics the lung-environment interaction. Conclusion P. brasiliensis PLB activity is involved in the process of adhesion and internalization of yeast cells at the MH-S cell surface and may enhance virulence and subsequent down-regulation of macrophage activation. PMID:20843362

  7. Structural and functional changes in rabbit ileum by purified extracellular phospholipase A of Salmonella newport.

    PubMed

    Neena; Asnani, P J

    1991-01-01

    As phospholipases of Salmonella species may play a role in the pathogenesis of gastrointestinal tract diseases. Salmonella newport, the causative agent of infantile diarrhoea was examined for the production of phospholipase. The enzyme was purified by gel filtration chromatography and was found to be a protein of molar mass ranging from 43 to 67 kDa. The purified enzyme alone or in combination with organisms, produced both structural and functional changes in rabbit ileum, contributing towards pathogenesis of diarrhoea due to this organism. PMID:1841873

  8. Secretory Phospholipase A2-IIA and Cardiovascular Disease

    PubMed Central

    Holmes, Michael V.; Simon, Tabassome; Exeter, Holly J.; Folkersen, Lasse; Asselbergs, Folkert W.; Guardiola, Montse; Cooper, Jackie A.; Palmen, Jutta; Hubacek, Jaroslav A.; Carruthers, Kathryn F.; Horne, Benjamin D.; Brunisholz, Kimberly D.; Mega, Jessica L.; van Iperen, Erik P.A.; Li, Mingyao; Leusink, Maarten; Trompet, Stella; Verschuren, Jeffrey J.W.; Hovingh, G. Kees; Dehghan, Abbas; Nelson, Christopher P.; Kotti, Salma; Danchin, Nicolas; Scholz, Markus; Haase, Christiane L.; Rothenbacher, Dietrich; Swerdlow, Daniel I.; Kuchenbaecker, Karoline B.; Staines-Urias, Eleonora; Goel, Anuj; van 't Hooft, Ferdinand; Gertow, Karl; de Faire, Ulf; Panayiotou, Andrie G.; Tremoli, Elena; Baldassarre, Damiano; Veglia, Fabrizio; Holdt, Lesca M.; Beutner, Frank; Gansevoort, Ron T.; Navis, Gerjan J.; Mateo Leach, Irene; Breitling, Lutz P.; Brenner, Hermann; Thiery, Joachim; Dallmeier, Dhayana; Franco-Cereceda, Anders; Boer, Jolanda M.A.; Stephens, Jeffrey W.; Hofker, Marten H.; Tedgui, Alain; Hofman, Albert; Uitterlinden, André G.; Adamkova, Vera; Pitha, Jan; Onland-Moret, N. Charlotte; Cramer, Maarten J.; Nathoe, Hendrik M.; Spiering, Wilko; Klungel, Olaf H.; Kumari, Meena; Whincup, Peter H.; Morrow, David A.; Braund, Peter S.; Hall, Alistair S.; Olsson, Anders G.; Doevendans, Pieter A.; Trip, Mieke D.; Tobin, Martin D.; Hamsten, Anders; Watkins, Hugh; Koenig, Wolfgang; Nicolaides, Andrew N.; Teupser, Daniel; Day, Ian N.M.; Carlquist, John F.; Gaunt, Tom R.; Ford, Ian; Sattar, Naveed; Tsimikas, Sotirios; Schwartz, Gregory G.; Lawlor, Debbie A.; Morris, Richard W.; Sandhu, Manjinder S.; Poledne, Rudolf; Maitland-van der Zee, Anke H.; Khaw, Kay-Tee; Keating, Brendan J.; van der Harst, Pim; Price, Jackie F.; Mehta, Shamir R.; Yusuf, Salim; Witteman, Jaqueline C.M.; Franco, Oscar H.; Jukema, J. Wouter; de Knijff, Peter; Tybjaerg-Hansen, Anne; Rader, Daniel J.; Farrall, Martin; Samani, Nilesh J.; Kivimaki, Mika; Fox, Keith A.A.; Humphries, Steve E.; Anderson, Jeffrey L.; Boekholdt, S. Matthijs; Palmer, Tom M.; Eriksson, Per; Paré, Guillaume; Hingorani, Aroon D.; Sabatine, Marc S.; Mallat, Ziad; Casas, Juan P.; Talmud, Philippa J.

    2013-01-01

    Objectives This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease. Background Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is causal. A recent phase III clinical trial of an sPLA2 inhibitor (varespladib) was stopped prematurely for lack of efficacy. Methods We conducted a Mendelian randomization meta-analysis of 19 general population studies (8,021 incident, 7,513 prevalent major vascular events [MVE] in 74,683 individuals) and 10 acute coronary syndrome (ACS) cohorts (2,520 recurrent MVE in 18,355 individuals) using rs11573156, a variant in PLA2G2A encoding the sPLA2-IIA isoenzyme, as an instrumental variable. Results PLA2G2A rs11573156 C allele associated with lower circulating sPLA2-IIA mass (38% to 44%) and sPLA2 enzyme activity (3% to 23%) per C allele. The odds ratio (OR) for MVE per rs11573156 C allele was 1.02 (95% confidence interval [CI]: 0.98 to 1.06) in general populations and 0.96 (95% CI: 0.90 to 1.03) in ACS cohorts. In the general population studies, the OR derived from the genetic instrumental variable analysis for MVE for a 1-log unit lower sPLA2-IIA mass was 1.04 (95% CI: 0.96 to 1.13), and differed from the non-genetic observational estimate (OR: 0.69; 95% CI: 0.61 to 0.79). In the ACS cohorts, both the genetic instrumental variable and observational ORs showed a null association with MVE. Instrumental variable analysis failed to show associations between sPLA2 enzyme activity and MVE. Conclusions Reducing sPLA2-IIA mass is unlikely to be a useful therapeutic goal for preventing cardiovascular events. PMID:23916927

  9. Phospholipase A2 activating protein and idiopathic inflammatory bowel disease.

    PubMed Central

    Peterson, J W; Dickey, W D; Saini, S S; Gourley, W; Klimpel, G R; Chopra, A K

    1996-01-01

    BACKGROUND: Crohn's disease and ulcerative colitis are idiopathic inflammatory bowel diseases (IBD) involving synthesis of eicosanoids from arachidonic acid (AA), which is released from membrane phospholipids by phospholipase A2 (PLA2). A potentially important regulator of the production of these mediators is a protein activator of PLA2, referred to as PLA2 activating protein (PLAP). AIMS: The purpose of this investigation was to discover if PLAP values might be increased in the inflamed intestinal tissue of patients with IBD and in intestinal tissue of mice with colitis. PATIENTS: Biopsy specimens were taken from patients with ulcerative colitis and Crohn's disease undergoing diagnostic colonoscopy, and normal colonic mucosa was obtained from patients without IBD after surgical resection. METHODS: Immunocytochemistry with affinity purified antibodies to PLAP synthetic peptides was used to locate PLAP antigen in sections of intestinal biopsy specimens from IBD patients compared with that of normal intestinal tissue. Northern blot analysis with a murine [32P] labelled plap cDNA probe was performed on RNA extracted from the colons of mice fed dextran sulphate sodium (DSS) and cultured HT-29 cells exposed to lipopolysaccharide (LPS). RESULTS: PLAP antigen was localised predominantly within monocytes and granulocytes in intestinal tissue sections from IBD patients, and additional deposition of extracellular PLAP antigen was associated with blood vessels and oedema fluid in the inflamed tissues. In contrast, tissue sections from normal human intestine were devoid of PLAP reactive antigen, except for some weak cytoplasmic reaction of luminal intestinal epithelial cells. Similarly, colonic tissue from DSS treated mice contained an increased amount of PLAP antigen compared with controls. The stroma of the lamina propria of the colonic mucosa from the DSS treated mice reacted intensely with antibodies to PLAP synthetic peptides, while no reaction was observed with control

  10. 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.

  11. Angiogenin stimulates endothelial cell prostacyclin secretion by activation of phospholipase A2.

    PubMed Central

    Bicknell, R; Vallee, B L

    1989-01-01

    Angiogenin stimulates capillary and umbilical vein endothelial cell prostacyclin secretion but not that of prostaglandins of the E series. The response was quantitated by radioimmunoassay and by [3H]arachidonate labeling followed by analysis of the secreted prostaglandins. The stimulated secretion lasts for several minutes and is optimal at 2-4 min. The dose-response (peak at 1-10 ng/ml) is similar to that previously observed for activation of endothelial cell phospholipase C. Stimulated secretion was blocked by pretreatment with the inhibitors of prostacyclin synthesis, indomethacin and tranylcypromine, and also the specific inhibitor of phospholipase A2, quinacrine, as well as pertussis toxin and the diglyceryl and monoglyceryl lipase inhibitor RHC 80267. Stimulated secretion was also abolished in cells that were either pretreated for 48 hr with phorbol ester to down-regulate protein kinase C or incubated with the protein kinase inhibitor H7. Hydrolysis of phosphatidylinositol by phospholipase A2 appears to be the source of angiogenin-mobilized arachidonate; angiogenin-induced hydrolysis of phosphatidylcholine was not detected. Activation of phospholipase A2 occurs in the absence of an angiogenin-induced calcium flux. The results are discussed in terms of mechanisms of agonist-induced intracellular arachidonate mobilization and relevance to angiogenesis. PMID:2646638

  12. Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens

    PubMed Central

    Rossignol, Gaelle; Merieau, Annabelle; Guerillon, Josette; Veron, Wilfried; Lesouhaitier, Olivier; Feuilloley, Marc GJ; Orange, Nicole

    2008-01-01

    Background Pseudomonas fluorescens is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions. Results We found that MFN1032 secreted extracellular factors with a lytic potential at least as high as that of MF37, a psychrotrophic strain of P. fluorescens or the mesophilic opportunistic pathogen, Pseudomonas aeruginosa PAO1. We demonstrated the direct, and indirect – through increases in biosurfactant release – involvement of a phospholipase C in the hemolytic activity of this bacterium. Sequence analysis assigned this phospholipase C to a new group of phospholipases C different from those produced by P. aeruginosa. We show that changes in PlcC production have pleiotropic effects and that plcC overexpression and plcC extinction increase MFN1032 toxicity and colonization, respectively. Conclusion This study provides the first demonstration that a PLC is involved in the secreted hemolytic activity of a clinical strain of Pseudomonas fluorescens. Moreover, this phospholipase C seems to belong to a complex biological network associated with the biosurfactant production. PMID:18973676

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

    PubMed

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

    2012-01-01

    Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content. PMID:22024023

  14. INFLUENCE OF CCL4 BIOTRANSFORMATION ON THE ACTIVATION OF RAT LIVER PHOSPHOLIPASE C IN VITRO

    EPA Science Inventory

    The Influence of CCl4 Biotransforrnation on the Activation of Rat Liver Phospholipase C in Vitro. Coleman, J.F., Condie, L.W. AND LAMB, R.G. (1988). Toxicol. Appl Pharmacol. 95, 200-207. Carbon tetrachloride (CCL4) biotransformation and covalent binding was measured in l000g live...

  15. Genes Encoding Phospholipases A2 Mediate Insect Nodulation Reactions to Bacterial Challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We propose that expression of four genes encoding secretory phospholipases A2 (sPLA2) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis...

  16. Carbonothioate phospholipids as substrate for a spectrophotometric assay of phospholipase A2.

    PubMed

    Yu, L; Ternansky, R J; Crisologo, J F; Chang, J; Baker, B L; Coutts, S M

    1998-12-01

    A continuous spectrophotometric assay for phospholipase A2 (PLA2) was developed using novel carbonothioate phospholipids. These phospholipid analogues contain a carbonothioate bond in the place of the sn-2 ester of the natural substrates of phospholipase A2 and were synthesized in a one-pot two-step reaction. Phospholipase A2 from cobra venom (Naja naja atra) hydrolyzes carbonothioate phospholipids and liberates a free thiol, alkylmercaptan, which is reacted with 5,5'-dithiobis(2-nitrobenzoic acid) to yield a product that absorbs at 412 nm. The kinetic studies on PLA2 hydrolysis of carbonothioate phospholipids were carried out in pure phospholipid forms and in Triton X-100 mixed micelles. The hydrolysis of pure carbonothioate phospholipids exhibits an interfacial activation phenomenon. The hydrolysis of phospholipid in mixed Triton X-100 micelles follows classical Michaelis-Menten kinetics. In a mixed micellar system, the catalytic efficiency observed with this series of substrates is two orders of magnitude lower than that of the hydrolysis of the natural substrate dipalmitoyl phosphocholine. However, these substrates bind to the enzyme over 10 times tighter than does the natural substrate. Application of this carbonothioate assay to screen both reversible and irreversible enzyme inhibitors of phospholipase A2 is also demonstrated. PMID:9866705

  17. 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...

  18. Phospholipase C and diacylglycerol lipase in human gallbladder and hepatic bile.

    PubMed

    Pattinson, N R; Willis, K E

    1990-12-01

    A phospholipase C in bile, free of bacterial infection, has recently been identified from cholesterol gallstone patients. Because of the importance of phosphatidylcholine in solubilizing cholesterol in bile, this study further investigates the metabolism of phosphatidylcholine in delipidated gallbladder and common bile duct biles. Phospholipase C activity, as measured by the release of phosphoryl[3H]choline from the substrate 1,2-dipalmitoyl-sn-glycero-3-phospho [N-methyl-3H]choline, was identified in both hepatic and gallbladder biles. Similar levels of activity (nmol.h-1.mg-1 of delipidated protein) were found in common bile duct (11.25 +/- 14.23) and gallbladder bile (19.07 +/- 22.24), although per milliliter of bile, the mean gallbaldder levels were 6.4 times greater than those found in common duct bile. With the tow substrates, 1-palmitoyl-2[9,10-3H] palmitoyl-sn-glycero-3-phosphocholine and 1,2(1-14C) dipalmitoyl-sn-glycero-3-phosphocholine, the majority of organically extracted label, after thin-layer chromatography, was recovered as radiolabeled diglyceride, confirming the presence of phospholipase C. Diglyceride levels were found to be closely correlated with [3H]choline (slope, 0.9820; r = 0.9844). In addition to diglyceride, both radiolabeled free fatty acid and monoglyceride were identified in common bile duct and gallbladder biles, although their levels were an order of magnitude less than measurable phospholipase C activity. To determine whether the free fatty acid release was due to either a diacylglycerol-lipase or a phospholipase A2, the effect of adding unlabeled diglyceride on free fatty acid formation from the substrate [14C]DPPC was examined. As the concentration of unlabeled diglyceride was increased, the amount of free fatty acid and monoglyceride released were both reduced in parallel. Direct measurement of diacylglycerol-lipase activity by incubating the diglyceride, sn-2[3H]dipalmitoyl, resulted in release of both products in a ratio

  19. Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris.

    PubMed

    Borodina, Irina; Jensen, Bettina M; Wagner, Tim; Hachem, Maher A; Søndergaard, Ib; Poulsen, Lars K

    2011-01-01

    Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy. PMID:21731687

  20. Expression of Enzymatically Inactive Wasp Venom Phospholipase A1 in Pichia pastoris

    PubMed Central

    Borodina, Irina; Jensen, Bettina M.; Wagner, Tim; Hachem, Maher A.; Søndergaard, Ib; Poulsen, Lars K.

    2011-01-01

    Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification. Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils. The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy. PMID:21731687

  1. 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}.

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

    SciTech Connect

    Smith, T.L. )

    1990-01-01

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

  3. Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2–DP1 receptor paracrine axis

    PubMed Central

    Taketomi, Yoshitaka; Ueno, Noriko; Kojima, Takumi; Sato, Hiroyasu; Murase, Remi; Yamamoto, Kei; Tanaka, Satoshi; Sakanaka, Mariko; Nakamura, Masanori; Nishito, Yasumasa; Kawana, Momoko; Kambe, Naotomo; Ikeda, Kazutaka; Taguchi, Ryo; Nakamizo, Satoshi; Kabashima, Kenji; Gelb, Michael H.; Arita, Makoto; Yokomizo, Takehiko; Nakamura, Motonao; Watanabe, Kikuko; Hirai, Hiroyuki; Nakamura, Masataka; Okayama, Yoshimichi; Ra, Chisei; Aritake, Kosuke; Urade, Yoshihiro; Morimoto, Kazushi; Sugimoto, Yukihiko; Shimizu, Takao; Narumiya, Shuh; Hara, Shuntaro; Murakami, Makoto

    2014-01-01

    Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell–deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS–ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3–L-PGDS–DP1 loop that drives mast cell maturation. PMID:23624557

  4. Attachment and phospholipase A2-induced lysis of phospholipid bilayer vesicles to plasma-polymerized maleic anhydride/SiO2 multilayers.

    PubMed

    Chifen, Anye N; Förch, Renate; Knoll, Wolfgang; Cameron, Petra J; Khor, Hwei L; Williams, Thomas L; Jenkins, A Toby A

    2007-05-22

    This article describes a method by which intact vesicles can be chemically attached to hydrolyzed maleic anhydride films covalently bound to plasma-polymerized SiO2 on Au substrates. Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) combined with surface plasmon resonance spectroscopy (SPR) was used to monitor the activation of plasma-deposited maleic anhydride (pp-MA) film with EDC/NHS and the subsequent coupling of lipid vesicles. The vesicles were formed from a mixture of phosphatidylcholine and phosphatidylethanolamine lipids, with a water-soluble fluorophore encapsulated within. Vesicle attachment was measured in real time on plasma films formed under different pulse conditions (plasma duty cycle). Optimum vesicle attachment was observed on the pp-MA films containing the highest density of maleic anhydride groups. Phospholipase A2 was used to lyse the surface-bound vesicles and to release the encapsulated fluorophore. PMID:17447800

  5. Light-Dependent Translocation of Arrestin in Rod Photoreceptors is Signaled Through a Phospholipase C Cascade and Requires ATP

    PubMed Central

    Orisme, Wilda; Li, Jian; Goldmann, Tobias; Bolch, Susan; Wolfrum, Uwe; Smith, W. Clay

    2009-01-01

    Partitioning of cellular components is a critical mechanism by which cells can regulate their activity. In rod photoreceptors, light induces a large-scale translocation of arrestin from the inner segments to the outer segments. The purpose of this project is to elucidate the signaling pathway necessary to initiate arrestin translocation to the outer segments and the mechanism for arrestin translocation. Mouse retinal organotypic cultures and eyes from transgenic Xenopus tadpoles expressing a fusion of GFP and rod arrestin were treated with both activators and inhibitors of proteins in the phosphoinositide pathway. Confocal microscopy was used to image the effects of the pharmacological agents on arrestin translocation in rod photoreceptors. Retinas were also depleted of ATP using potassium cyanide to assess the requirement for ATP in arrestin translocation. In this study, we demonstrate that components of the G-protein-linked phospholipase C (PLC) pathway play a role in initiating arrestin translocation. Our results show that arrestin translocation can be stimulated by activators of PLC and protein kinase C (PKC), and by cholera toxin in the absence of light. Arrestin translocation to the outer segments is significantly reduced by inhibitors of PLC and PKC. Importantly, we find that treatment with potassium cyanide inhibits arrestin translocation in response to light. Collectively, our results suggest that arrestin translocation is initiated by a G-protein-coupled cascade through PLC and PKC signaling. Furthermore, our results demonstrate that at least the initiation of arrestin translocation requires energy input. PMID:19887106

  6. Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2.

    PubMed

    Lee, Sung Kyun; Kim, Sang Doo; Kook, Minsoo; Lee, Ha Young; Ghim, Jaewang; Choi, Youngwoo; Zabel, Brian A; Ryu, Sung Ho; Bae, Yoe-Sik

    2015-08-24

    We determined the function of phospholipase D2 (PLD2) in host defense in highly lethal mouse models of sepsis using PLD2(-/-) mice and a PLD2-specific inhibitor. PLD2 deficiency not only increases survival but also decreases vital organ damage during experimental sepsis. Production of several inflammatory cytokines (TNF, IL-1β, IL-17, and IL-23) and the chemokine CXCL1, as well as cellular apoptosis in immune tissues, kidney, and liver, are markedly decreased in PLD2(-/-) mice. Bactericidal activity is significantly increased in PLD2(-/-) mice, which is mediated by increased neutrophil extracellular trap formation and citrullination of histone 3 through peptidylarginine deiminase activation. Recruitment of neutrophils to the lung is markedly increased in PLD2(-/-) mice. Furthermore, LPS-induced induction of G protein-coupled receptor kinase 2 (GRK2) and down-regulation of CXCR2 are markedly attenuated in PLD2(-/-) mice. A CXCR2-selective antagonist abolishes the protection conferred by PLD2 deficiency during experimental sepsis, suggesting that enhanced CXCR2 expression, likely driven by GRK2 down-regulation in neutrophils, promotes survival in PLD2(-/-) mice. Furthermore, adoptively transferred PLD2(-/-) neutrophils significantly protect WT recipients against sepsis-induced death compared with transferred WT neutrophils. We suggest that PLD2 in neutrophils is essential for the pathogenesis of experimental sepsis and that pharmaceutical agents that target PLD2 may prove beneficial for septic patients. PMID:26282875

  7. 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

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

    PubMed

    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

  9. Targeting phospholipase D1 attenuates intestinal tumorigenesis by controlling β-catenin signaling in cancer-initiating cells

    PubMed Central

    Kang, Dong Woo; Choi, Chi Yeol; Cho, Yong-Hee; Tian, Huasong; Di Paolo, Gilbert; Choi, Kang-Yell

    2015-01-01

    Expression of the Wnt target gene phospholipase D1 (PLD1) is up-regulated in various carcinomas, including colorectal cancer (CRC). However, the mechanistic significance of its elevated expression in intestinal tumorigenesis remains unknown. In this study, we show that genetic and pharmacological targeting of PLD1 disrupts spontaneous and colitis-associated intestinal tumorigenesis in ApcMin/+ and azoxymethane/dextran sodium sulfate mice models. Intestinal epithelial cell–specific PLD1 overexpression in ApcMin/+ mice accelerated tumorigenesis with increased proliferation and nuclear β-catenin levels compared with ApcMin/+ mice. Moreover, PLD1 inactivation suppressed the self-renewal capacity of colon cancer–initiating cells (CC-ICs) by decreasing expression of β-catenin via E2F1-induced microRNA (miR)-4496 up-regulation. Ultimately, low expression of PLD1 coupled with a low level of CC-IC markers was predictive of a good prognosis in CRC patients, suggesting in vivo relevance. Collectively, our data reveal that PLD1 has a crucial role in intestinal tumorigenesis via its modulation of the E2F1–miR-4496–β-catenin signaling pathway. Modulation of PLD1 expression and activity represents a promising therapeutic strategy for the treatment of intestinal tumorigenesis. PMID:26122663

  10. Increased phospholipase A2 and decreased lysophospholipase activity in the small intestinal mucosa after ischaemia and revascularisation.

    PubMed Central

    Otamiri, T; Franzén, L; Lindmark, D; Tagesson, C

    1987-01-01

    The influence of ischaemia and revascularisation on lipid peroxidation and phospholipid metabolism in the rat small intestinal mucosa was investigated. Two hours of total ischaemia followed by five minutes of revascularisation caused not only accumulation of malondialdehyde in the mucosa, but also increased activity of phospholipase A2, decreased activity of lysophospholipase, and increased ratio between lysophosphatidylcholine and phosphatidylcholine. Pretreatment with the phospholipase A2 inhibitor, quinacrine, prevented the increases in mucosal phospholipase A2 activity and lysophosphatidylcholine/phosphatidylcholine ratio after ischaemia and morphological examinations revealed that the mucosa was then also protected against ischaemic injury. These findings point to the possibility that activation of phospholipase A2 and accumulation of lysophosphoglycerides could be involved in mediating the mucosal injury caused by small intestinal ischaemia. Images Fig. 7 PMID:3428670

  11. Rapid isolation and partial characterization of two phospholipases from Kenyan Echis carinatus leakeyi (Leakey's saw-scaled viper) venom.

    PubMed

    Desmond, H P; Crampton, J M; Theakston, R D

    1991-01-01

    The purification and partial sequencing of two phospholipase A2 toxins from the venom of Kenyan E. carinatus leakeyi is described. The two proteins exhibit sequence homology with other toxic phospholipases. Both have a molecular weight in the region of 16,000 and are purified to homogeneity from crude venom by a single high performance liquid chromatography. The role of these proteins in the toxicity of the venom remains to be established. PMID:1862528

  12. Purification of an acidic phospholipase A2 from Bothrops lanceolatus (fer de lance) venom: molecular and enzymatic properties.

    PubMed

    de Araújo, A L; Radvanyi, F; Bon, C

    1994-09-01

    The acidic phospholipase A2 from Bothrops lanceolatus venom has been purified by gel filtration on Sephadex G-50 and ion exchange chromatography on CM-cellulose. Analysis by FPLC on Mono-Q column of the purified phospholipase A2 indicated that it is a mixture of several isoenzymes. The two major isoforms consist of a single polypeptide chain with mol. wts of 14,500 and 15,000, which slightly differ in their isoelectric point (4.9 and 5.3) and amino acid composition. However, enzymatic and pharmacological properties of the various isoenzymes are identical. The phospholipase from B. lanceolatus venom is characterized by a progressive increase in the rate of hydrolysis when enzymatic activity is determined with crude egg yolk as substrate in the absence of detergent. This phenomenon, which is not observed with mixed micelles of lecithin-detergent, is not due to the presence of a phospholipase A2 inhibitor in the venom, as previously suggested by several investigators in the case of other Bothrops and Cobra venoms. It is rather a catalytic characteristics of B. lanceolatus venom phospholipase, the enzymatic activity of which depends on the physical state of phospholipids. Bothrops lanceolatus acidic phospholipase A2 is non-toxic. PMID:7801343

  13. Phospholipase A2 activity in platelets. Immuno-purification and localization of the enzyme in rat platelets.

    PubMed

    Aarsman, A J; Leunissen-Bijvelt, J; Van den Koedijk, C D; Neys, F W; Verkleij, A J; Van den Bosch, H

    1989-01-01

    A comparative study on phospholipase A2 activity in platelet lysates from various species was carried out using identical assay conditions with phosphatidylethanolamine as substrate. Platelet phospholipase A2, both when expressed as activity per ml blood and as specific activity in KCl extracts, was low in human, cow, pig and goat. Moderate activities, in increasing order, were found in sheep, horse and rabbit, while rats showed by far the highest activity. In the latter four species total lysate activity was recovered in 1 M KCl extracts, suggesting that the enzyme occurs either in soluble form or as a peripheral membrane-associated protein. Immune cross-reactivity with monoclonal antibodies against rat liver mitochondrial phospholipase A2 was studied in dot-blot and monoclonal antibody-Sepharose binding experiments. Only sheep and rat platelet extracts contained cross-reactive phospholipase(s) A2. Immuno-affinity chromatography of rat platelet extracts indicated virtually complete binding of total phospholipase A2 activity and yielded pure enzyme in a single purification step. Enzyme visualization by immunogold electron microscopy showed a predominant localization in the matrix of alpha-granules. PMID:2519886

  14. Measurement of the phospholipase activity of endothelial lipase in mouse plasma.

    PubMed

    Basu, Debapriya; Lei, Xia; Josekutty, Joby; Hussain, M Mahmood; Jin, Weijun

    2013-01-01

    Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL's hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface. PMID:23103358

  15. Phospholipase A and the interaction of Rickettsia prowazekii and mouse fibroblasts (L-929 cells)

    SciTech Connect

    Winkler, H.H.; Miller, E.T.

    1982-10-01

    L-929 cells were killed when approximately 50 viable Rickettsia prowazekii organisms per L-cell were centrifuged onto a monolayer. The glycerophospholipids of the L-cell were hydrolyzed to lysophosphatides and free fatty acids. Concomitantly, there was a loss of membrane integrity as shown by release of lactate dehydrogenase and 86Rb and permeability to trypan blue dye. No glycerophospholipid hydrolysis or cytotoxicity occurred when the rickettsiae were inactivated by heat, UV irradiation, N-ethylmaleimide, or metabolic inhibitors before their addition to the L-929 cells. On the other hand, treatment of the L929 cells with the cytoskeleton agents colchicine or cytochalasin B or with N-ethylmaleimide inhibited neither the phospholipase A activity nor the loss of membrane integrity. Cytochalasin B-treated cells could be damaged by even small numbers of rickettsiae. We suggest that this phospholipase A activity is used by the rickettsiae to escape from the phagosomes into the cytoplasm of host cells.

  16. 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. PMID:26665171

  17. DNA sequences, recombinant DNA molecules and processes producing human phospholipase inhibitor polypeptides

    SciTech Connect

    Wallner, B.P.; Pepinsky, R.B.; Garwin, J.L.

    1989-11-07

    This patent describes a recombinant DNA molecule. In comprises a DNA sequence coding for a phospholopase inhibitor polypeptide and being selected from the group consisting of: the cDNA insert of ALC, DNA sequences which code on expression for a phospholopase inhibitor, and DNA sequences which are degenerate as a result of the genetic code to either of the foregoing DNA sequences and which code on expression for a phospholipase inhibitor.

  18. Analysis of genetic polymorphism in the phospholipase region of Mycobacterium tuberculosis.

    PubMed Central

    Vera-Cabrera, L; Howard, S T; Laszlo, A; Johnson, W M

    1997-01-01

    mtp40 was originally identified as a short genomic region that was found in strains of Mycobacterium tuberculosis but not in Mycobacterium bovis. Subsequent studies have revealed that the sequence is part of the mpcA gene, which encodes a phospholipase C. To investigate further the distribution of the mtp40 sequence, we analyzed strains of the M. tuberculosis complex by PCR and were able to amplify the mtp40 sequence in 90 of 94 strains of M. tuberculosis and in 2 strains of Mycobacterium microti but not in M. bovis or M. bovis BCG. Based on this, we developed a dot blot assay using genomic DNA which allows M. bovis to be distinguished from the majority of M. tuberculosis strains. We also probed Southern blots of 140 clinical isolates of M. tuberculosis to determine the frequency of strains lacking mtp40. This revealed an unexpected polymorphism in the phospholipase region. Two fragments were detected in 57% of samples. The expected fragment of 0.75 kbp corresponds to the region of mpcA containing mtp40. A 2.1-kbp fragment was observed to belong to a recently discovered second phospholipase gene, mpcB. In addition, some strains appeared to lack both genes, while others showed only the presence of mpcA. A few strains had additional bands, suggesting the existence of other homologs to the two phospholipase genes. We also detected the insertion of IS6110 in the mpcA coding region of one strain. The absence of these genes in some clinical isolates raises questions about their function during infection and in the development of tuberculosis disease in humans. PMID:9114405

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

    PubMed

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

    2010-10-20

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

  20. Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2

    PubMed Central

    2011-01-01

    Background Secretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be highly expressed in the intestine of mammals. However, no study was reported in birds. Results Chicken intestinal group IIA phospholipase A2 (ChPLA2-IIA) was obtained after an acidic treatment (pH.3.0), precipitation by ammonium sulphate, followed by sequential column chromatographies on Sephadex G-50 and mono-S ion exchanger. The enzyme was found to be a monomeric protein with a molecular mass of around 14 kDa. The purified enzyme showed a substrate preference for phosphatidylethanolamine and phosphatidylglycerol, and didn't hydrolyse phosphatidylcholine. Under optimal assay conditions, in the presence of 10 mM NaTDC and 10 mM CaCl2, a specific activity of 160 U.mg-1 for purified ChPLA2-IIA was measured using egg yolk as substrate. The fifteen NH2-terminal amino acid residues of ChPLA2-IIA were sequenced and showed a close homology with known intestinal secreted phospholipases A2. The gene encoding the mature ChPLA2-IIA was cloned and sequenced. To further investigate structure-activity relationship, a 3D model of ChPLA2-IIA was built using the human intestinal phospholipase A2 structure as template. Conclusion ChPLA2-IIA was purified to homogeneity using only two chromatographic colomns. Sequence analysis of the cloned cDNA indicates that the enzyme is highly basic with a pI of 9.0 and has a high degree of homology with mammalian intestinal PLA2-IIA. PMID:21284884

  1. Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom.

    PubMed

    Machado Braga, Marcus Davis; Costa Martins, Alice Maria; Alves, Claudênio Diógenes; de Menezes, Dalgimar Beserra; Martins, René Duarte; Ferreira Barbosa, Paulo Sérgio; de Sousa Oliveira, Isadora Maria; Toyama, Marcos Hikari; Toyama, Daniela Oliveira; Dos Santos Diz Filho, Eduardo Brito; Ramos Fagundes, Fabio Henrique; Fonteles, Manassés Claudino; Azul Monteiro, Helena Serra

    2008-02-01

    Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as IV-1 to IV-5, from which IV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2) ) venom (10 microg/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n=6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa(+)) and chloride tubular reabsorption (%TCl(-)) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. PMID:17953979

  2. Analysis of nociceptive effects of neurotoxic phospholipase A2 from Vipera nikolskii venom in mice

    PubMed Central

    Dyachenko, Igor A; Murashev, Arkadii N; Andreeva, Tatyana V; Tsetlin, Victor I; Utkin, Yuri N

    2013-01-01

    Phospholipases A2 are represented in snake venoms by several types and possess diverse biological activities including neurotoxicity. Previously, we isolated and characterized two neurotoxic phospholipases A2 (HDP-1 and HDP-2) from the venom of Nikolski's viper (Vipera nikolskii), which were heterodimers composed of two non-covalently bound subunits. Each heterodimer consisted of an enzymatically active basic subunit and an inactive acidic subunit. In this work, we studied the in vivo biological activity of HDP-2 in mice. The acute toxicity (LD50 = 0.38 μg/gm) and maximal tolerated dose (0.1 μg/gm) were determined. In the hot plate test, HDP-2 at the maximal tolerated dose, reliably prolonged the time of the mouse staying on the plate. However, taking into account the neurotoxicity of HDP-2, we believe that this effect may be explained by a general intoxication rather than specific decrease of pain sensitivity. In this respect HDP-2 differs from other heterodimeric phospholipases A2 like crotoxin, which possess analgesic activity. This difference can be explained by the dissimilarity in the structure of the acidic subunits, suggesting an important role of this subunit in analgesic activity. PMID:23577231

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  5. In vitro antiplasmodial activity of phospholipases A2 and a phospholipase homologue isolated from the venom of the snake Bothrops asper.

    PubMed

    Castillo, Juan Carlos Quintana; Vargas, Leidy Johana; Segura, Cesar; Gutiérrez, José María; Pérez, Juan Carlos Alarcón

    2012-12-01

    The antimicrobial and antiparasite activity of phospholipase A(2) (PLA(2)) from snakes and bees has been extensively explored. We studied the antiplasmodial effect of the whole venom of the snake Bothrops asper and of two fractions purified by ion-exchange chromatography: one containing catalytically-active phospholipases A(2) (PLA(2)) (fraction V) and another containing a PLA(2) homologue devoid of enzymatic activity (fraction VI). The antiplasmodial effect was assessed on in vitro cultures of Plasmodium falciparum. The whole venom of B. asper, as well as its fractions V and VI, were active against the parasite at 0.13 ± 0.01 µg/mL, 1.42 ± 0.56 µg/mL and 22.89 ± 1.22 µg/mL, respectively. Differences in the cytotoxic activity on peripheral blood mononuclear cells between the whole venom and fractions V and VI were observed, fraction V showing higher toxicity than total venom and fraction VI. Regarding toxicity in mice, the whole venom showed the highest lethal effect in comparison to fractions V and VI. These results suggest that B. asper PLA(2) and its homologue have antiplasmodial potential. PMID:23242318

  6. Generation of lysophosphatidylinositol by DDHD domain containing 1 (DDHD1): Possible involvement of phospholipase D/phosphatidic acid in the activation of DDHD1.

    PubMed

    Yamashita, Atsushi; Kumazawa, Tsukasa; Koga, Hiroki; Suzuki, Naotaka; Oka, Saori; Sugiura, Takayuki

    2010-07-01

    GPR55 is a seven-transmembrane G-protein-coupled receptor that has been proposed as a novel type of cannabinoid receptor. Previously, we identified lysophosphatidylinositol (LPI), in particular 2-arachidonoyl-LPI, as an agonist for GPR55. In the present study, we examined whether intracellular phospholipase A1 (DDHD domain containing 1, or DDHD1), previously identified as phosphatidic acid (PA)-preferring PLA1 (PA-PLA1), is involved in the formation of 2-arachidonoyl-LPI. HEK293 cells expressing DDHD1 produced [(3)H]arachidonic acid-containing LPI after prelabeling with [(3)H]arachidonic acid and subsequent activation by ionomycin; the formation of [(3)H]LPI was inhibited by n-butanol and the overexpression of an inactive PLD1 mutant PLD1K898R. DDHD1 was translocated from the cytosol to membranes upon ionomycin treatment. A purified recombinant DDHD1 formed [(3)H]LPI when incubated with [(3)H]PI; the V(max) and apparent K(m) were 190 micromol/min/mg protein and 10 mol% PI, respectively. DDHD1 binds PA, and the addition of PA to DDHD1 increased the affinity for PI (K(m) ; 3 mol%) and augmented the PI-PLA1 activity. DDHD1 activated by PA was returned to a basal state by its own PA-hydrolytic activity. These results implicate DDHD1 in the formation of 2-arachidonoyl-LPI and indicate that the process is modulated by PA released by phospholipase D. Similar observations for the production of arachidonic acid-containing LPI in neuroblastoma cells suggest the DDHD1-LPI-GPR55 axis to be involved in functions in the brain. PMID:20359546

  7. Description of Loxtox protein family and identification of a new group of Phospholipases D from Loxosceles similis venom gland.

    PubMed

    Dantas, Arthur Estanislau; Carmo, A O; Horta, Carolina Campolina Rebello; Leal, Hortênsia Gomes; Oliveira-Mendes, Bárbara Bruna Ribeiro; Martins, Ana Paula Vimieiro; Chávez-Olórtegui, Carlos; Kalapothakis, Evanguedes

    2016-09-15

    Envenoming resulting from Loxosceles spider bites (loxoscelism) is a recognized public health problem in Brazil. However, the pathophysiology of loxoscelism caused by L. similis bites, which is widespread in Brazil, remains poorly understood. In the present work, the RNA sequencing (RNA-Seq - Next Generation sequencing - NGS) of the L. similis venom gland was performed to identify and analyze the sequences of the key component phospholipase D. The sequences were aligned based on their classical domains, and a phylogenetic tree was constructed. In the bioinformatics analysis, 23 complete sequences of phospholipase D proteins were found and classified as Loxtox proteins, as they contained the characteristic domains of phospholipase D: the active site, the Mg(2+)-binding domain, and the catalytic loop. Three phospholipase D sequences with non-canonical domains were also found in this work. They were analyzed separately and named PLDs from L. similis (PLD-Ls). This study is the first to characterize phospholipase D sequences from Loxosceles spiders by RNA-Seq. These results contribute new knowledge about the composition of L. similis venom, revealing novel tools that could be used for pharmacological, immunological, and biotechnological applications. PMID:27496061

  8. 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.

  9. Inhibition of Nicotinic Acetylcholine Receptors, a Novel Facet in the Pleiotropic Activities of Snake Venom Phospholipases A2

    PubMed Central

    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. PMID:25522251

  10. Phospholipase Cγ1 Connects the Cell Membrane Pathway to the Nuclear Receptor Pathway in Insect Steroid Hormone Signaling*

    PubMed Central

    Liu, Wen; Cai, Mei-Juan; Zheng, Chuan-Chuan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2014-01-01

    In addition to the classical nuclear receptor pathway, there is a nongenomic pathway in the cell membrane that regulates gene expression in animal steroid hormone signaling; however, this mechanism is unclear. Here, we report that the insect steroid hormone 20-hydroxyecdysone (20E) regulates calcium influx via phospholipase Cγ1 (PLCG1) to modulate the protein kinase C phosphorylation of the transcription factor ultraspiracle (USP1) in the lepidopteran insect Helicoverpa armigera. The PLCG1 mRNA levels are increased during the molting and metamorphic stages. The depletion of PLCG1 by RNA interference can block 20E-enhanced pupation, cause larvae death and pupation defects, and repress 20E-induced gene expression. 20E may induce the tyrosine phosphorylation of PLCG1 at the cytosolic tyrosine kinase (Src) homology 2 domains and then determine the migration of PLCG1 toward the plasma membrane. The G-protein-coupled receptor (GPCR) inhibitor suramin, Src family kinase inhibitor PP2, and the depletions of ecdysone-responsible GPCR (ErGPCR) and Gαq restrain the 20E-induced tyrosine phosphorylation of PLCG1. PLCG1 participates in the 20E-induced Ca2+ influx. The inhibition of GPCR, PLC, inositol 1,4,5-trisphosphate receptor, and calcium channels represses the 20E-induced Ca2+ influx. Through calcium signaling, PLCG1 mediates the transcriptional activation driven by the ecdysone-response element. Through PLCG1 and calcium signaling, 20E regulates PKC phosphorylation of USP1 at Ser-21 to determine its ecdysone-response element binding activity. These results suggest that 20E activates PLCG1 via the ErGPCR and Src family kinases to regulate Ca2+ influx and PKC phosphorylation of USP1 to subsequently modulate gene transcription for metamorphosis. PMID:24692553

  11. Metabolic evidence for PtdIns(4,5)P2-directed phospholipase C in permeabilized plant protoplasts.

    PubMed Central

    Brearley, C A; Parmar, P N; Hanke, D E

    1997-01-01

    Comparison of the sequences of the genes encoding phospholipase C (PLC) which have been cloned to date in plants with their mammalian counterparts suggests that plant PLC is similar to PLCdelta of mammalian cells. The physiological role and mechanism of activation of PLCdelta is unclear. It has recently been shown that Ins(1,4,5)P3 may not solely be the product of PtdIns(4,5)P2-directed PLC activity. Enzyme activities capable of producing Ins(1,4,5)P3 from endogenous inositol phosphates are present in Dictyostelium and also in rat liver. Significantly it has not been directly determined whether Ins(1,4,5)P3 present in higher plants is the product of a PtdIns(4, 5)P2-directed PLC activity. Therefore we have developed an experimental strategy for the identification of d-Ins(1,4,5)P3 in higher plants. By the use of a short-term non-equilibrium labelling strategy in permeabilized plant protoplasts, coupled to the use of a 'metabolic trap' to prevent degradation of [32P]Ins(1,4,5)P3, we were able to determine the distribution of 32P in individual phosphate esters of Ins(1,4,5)P3. The [32]Ins(1,4,5)P3 identified showed the same distribution of label in individual phosphate esters as that of [32P]PtdIns(4,5)P2 isolated from the same tissue. We thus provide in vivo evidence for the action of a PtdIns(4,5)P2-directed PLC activity in plant cells which is responsible for the production of Ins(1,4,5)P3 observed here. This observation does not, however, exclude the possibility that in other cells or under different conditions Ins(1,4,5)P3 can be generated by alternative routes. PMID:9164848

  12. Cool-temperature-mediated activation of phospholipase C-γ2 in the human hereditary disease PLAID.

    PubMed

    Schade, Anja; Walliser, Claudia; Wist, Martin; Haas, Jennifer; Vatter, Petra; Kraus, Johann M; Filingeri, Davide; Havenith, George; Kestler, Hans A; Milner, Joshua D; Gierschik, Peter

    2016-09-01

    Deletions in the gene encoding signal-transducing inositol phospholipid-specific phospholipase C-γ2 (PLCγ2) are associated with the novel human hereditary disease PLAID (PLCγ2-associated antibody deficiency and immune dysregulation). PLAID is characterized by a rather puzzling concurrence of augmented and diminished functions of the immune system, such as cold urticaria triggered by only minimal decreases in temperature, autoimmunity, and immunodeficiency. Understanding of the functional effects of the genomic alterations at the level of the affected enzyme, PLCγ2, is currently lacking. PLCγ2 is critically involved in coupling various cell surface receptors to regulation of important functions of immune cells such as mast cells, B cells, monocytes/macrophages, and neutrophils. PLCγ2 is unique by carrying three Src (SH) and one split pleckstrin homology domain (spPH) between the two catalytic subdomains (spPHn-SH2n-SH2c-SH3-spPHc). Prevailing evidence suggests that activation of PLCγ2 is primarily due to loss of SH-region-mediated autoinhibition and/or enhanced plasma membrane translocation. Here, we show that the two PLAID PLCγ2 mutants lacking portions of the SH region are strongly (>100-fold), rapidly, and reversibly activated by cooling by only a few degrees. We found that the mechanism(s) underlying PLCγ2 PLAID mutant activation by cool temperatures is distinct from a mere loss of SH-region-mediated autoinhibition and dependent on both the integrity and the pliability of the spPH domain. The results suggest a new mechanism of PLCγ activation with unique thermodynamic features and assign a novel regulatory role to its spPH domain. Involvement of this mechanism in other human disease states associated with cooling such as exertional asthma and certain acute coronary events appears an intriguing possibility. PMID:27196803

  13. Gqalpha-linked phospholipase Cbeta1 and phospholipase Cgamma are essential components of the pheromone biosynthesis activating neuropeptide (PBAN) signal transduction cascade.

    PubMed

    Hull, J J; Lee, J M; Matsumoto, S

    2010-08-01

    Sex pheromone production for most moths is regulated by pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori, PBAN binding triggers the opening of store-operated Ca(2+) channels, suggesting the involvement of a receptor-activated phospholipase C (PLC). In this study, we found that PLC inhibitors U73122 and compound 48/80 reduced sex pheromone production and that intracellular levels of (3)H-inositol phosphate species increased following PBAN stimulation. In addition, we amplified cDNAs from pheromone glands corresponding to PLCbeta1, PLCbeta4, PLCgamma and two G protein alpha subunits, Go and Gq. In vivo RNA interference-mediated knockdown analyses revealed that BmPLCbeta1, BmGq1, and unexpectedly, BmPLCgamma, are part of the PBAN signal transduction cascade. PMID:20546038

  14. 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. PMID:26699205

  15. The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

    PubMed Central

    Schimmel, R J

    1988-01-01

    Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both

  16. Krabbe disease: psychosine-mediated activation of phospholipase A2 in oligodendrocyte cell death.

    PubMed

    Giri, S; Khan, M; Rattan, R; Singh, I; Singh, A K

    2006-07-01

    Globoid cell leukodystrophy (Krabbe disease) is an inherited neurological disorder caused by the pathogenomic accumulation of psychosine (galactosylsphingosine), a substrate for the deficient enzyme galactocerebroside beta-galactosidase. This study underscores the mechanism of action of psychosine in the regulation of oligodendrocyte cell death via the generation of lysophosphatidylcholine (LPC) and arachidonic acid (AA) by the activation of secretory phospholipase A2 (sPLA2). There was a significant increase in the level of LPC, indicating a phospholipase A2 (PLA2)-dependent pathobiology, in the brains of Krabbe disease patients and those of twitcher mice, an animal model of Krabbe disease. In vitro studies of the treatment of primary oligodendrocytes and the oligodendrocyte MO3.13 cell line with psychosine also showed the generation of LPC and the release of AA in a dose- and time-dependent manner, indicating psychosine-induced activation of PLA2. Studies with various pharmacological inhibitors of cytosolic phospholipase A2 and sPLA2 and psychosine-mediated induction of sPLA2 enzymatic activity in media supernatant suggest that psychosine-induced release of AA and generation of LPC is mainly contributed by sPLA2. An inhibitor of sPLA2, 7,7-dimethyl eicosadienoic acid, completely attenuated the psychosine-mediated accumulation of LPC levels, release of AA, and generation of reactive oxygen species, and blocked oligodendroyte cell death, as evident from cell survival, DNA fragmentation, and caspase 3 activity assays. This study documents for the first time that psychosine-induced cell death is mediated via the sPLA2 signaling pathway and that inhibitors of sPLA2 may hold a therapeutic potential for protection against oligodendrocyte cell death and resulting demyelination in Krabbe disease. PMID:16645197

  17. Endometrial phospholipase A2 activity during the oestrous cycle and early pregnancy in mares.

    PubMed

    Ababneh, M M; Troedsson, M H T

    2013-02-01

    The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare's endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1-palmitoyl-2-[(14) C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μM. Enzyme activity was low in the endometrium of early luteal phase tissue but increased significantly (p < 0.001) during the late luteal phase (5.39 ± 0.16; 3.48 ± 0.33, 6.85 ± 0.59, and 9.96 ± 1.23 nmol oleic acid released/mg protein at oestrus, and Days 3, 8 and 14 after ovulation, respectively). The mean PLA2 activity in endometrial tissue from pregnant mares (4.23 ± 0.74) was significantly lower (p < 0.01) than from cyclic animals during late dioestrus (9.96 ± 1.23). The results indicate that PLA2 activity in equine endometrium changes with the stage of the oestrous cycle and thus may be influenced by systemic hormone concentrations. The inhibitory effects of conceptus products on secretion of prostaglandin during early pregnancy were associated with a competitive inhibitor that decreased endometrial PLA2 activity. PMID:22486770

  18. Phospholipase A2 induced airway hyperreactivity to cooling and acetylcholine in rat trachea: pharmacological modulation.

    PubMed Central

    Chand, N.; Diamantis, W.; Mahoney, T. P.; Sofia, R. D.

    1988-01-01

    1. Rat isolated tracheal smooth muscle preparations respond to phospholipase A2 (PLA2) and phospholipase C (PLC) with contractile responses of highly variable magnitudes. Rat tracheae exposed to PLA2 or PLC for a period of 10-30 min, exhibit airway hyperreactivity (AH) to cooling (10 degrees C), i.e., respond with strong contractile responses. Phospholipase D neither contracted rat tracheae nor induced AH to cooling. 2. PLA2-induced AH to cooling was dependent on the presence of extracellular Ca2+ in the physiological solution. 3. Verapamil, azelastine, diltiazem and TMB-8 (each 10 microM) significantly attenuated PLA2-induced AH. This effect was not shared by nifedipine (10 microM). 4. Bepridil (10 microM), a Ca2+ and calmodulin antagonist, also significantly attenuated AH induced by PLA2. 5. Indomethacin (a cyclo-oxygenase inhibitor), AA-861 (a selective 5-lipoxygenase inhibitor), FPL 55712 (a leukotriene receptor antagonist), methysergide (a 5-hydroxytryptamine D-receptor antagonist) and pyrilamine (a histamine H1-receptor antagonist) exerted little or no effect on PLA2-induced AH to cooling. 6. Atropine significantly attenuated PLA2-induced AH suggesting the participation of acetylcholine. 7. Nordihydroguaiaretic acid (an antioxidant; 5-lipoxygenase inhibitor) and BW 755C (an antioxidant; a dual inhibitor of cyclo-oxygenase and 5-lipoxygenase) significantly attenuated PLA2-induced AH to cooling. 8. In conclusion, these data show that PLA2 (an enzyme involved in the synthesis of Paf-acether, prostaglandins, thromboxanes, leukotrienes, diacylglycerol, superoxide free radicals and lipid peroxides, etc.) induces AH to cooling and acetylcholine in rat trachea. The induction of AH to cooling is dependent on the presence of extracellular Ca2+ and is significantly attenuated by verapamil, diltiazem, bepridil, atropine and azelastine (an antiallergic/antiasthmatic drug). PMID:3207972

  19. 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. PMID:26212074

  20. 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

  1. Dimerization and activation of porcine pancreatic phospholipase A2 via substrate level acylation of lysine 56.

    PubMed

    Tomasselli, A G; Hui, J; Fisher, J; Zürcher-Neely, H; Reardon, I M; Oriaku, E; Kézdy, F J; Heinrikson, R L

    1989-06-15

    The porcine pancreatic phospholipase A2-catalyzed hydrolysis of the water-soluble chromogenic substrate 4-nitro-3-octanoyloxybenzoate shows an initial latency phase similar to the one observed in the hydrolysis of aggregated phospholipids by the same enzyme. We report here that during the latency phase the enzyme undergoes a slow, autocatalytic, substrate-level acylation whereby in a few of the catalytic events the scissile octanoyl group of the substrate, normally transferred to water, is transferred to the epsilon-amino group of lysine 56. The N epsilon 56-octanoylphospholipase shows a strong tendency to dimerize in solution and thus may be separated from the monomeric native enzyme by gel filtration. Octanoylation of Lys-56 activates the enzyme some 180-fold toward 4-nitro-3-octanoyloxybenzoate and more than 100-fold toward monolayers of 1,2-didecanoyl-sn-glycero-3-phosphocholine. Acylation also attends the enzymatic hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with the incorporation of 1 eq of palmitate. Kinetic analysis of the early phase of reaction with 4-nitro-3-octanoyloxybenzoate shows that in this initial step the rate of activation is first order with respect to enzyme and substrate. A much more rapid, autocatalytic activation occurs in the later phases of the reaction where the activation of the enzyme is catalyzed by the activated enzyme itself. These findings with porcine pancreatic phospholipase A2, together with those relative to a snake venom enzyme monomer (Cho, W., Tomasselli, A. G., Heinrikson, R. L., and Kézdy, F. J. (1988) J. Biol. Chem. 263, 11237-11241), strongly support the proposal that interfacial activation of monomeric phospholipases is due to substrate-level autoacylation resulting in fully potentiated dimeric enzymes. PMID:2498336

  2. 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

  3. phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development

    PubMed Central

    Lawson, Nathan D.; Mugford, Joshua W.; Diamond, Brigid A.; Weinstein, Brant M.

    2003-01-01

    In this study, we utilize transgenic zebrafish with fluorescently labeled blood vessels to identify and characterize a mutant (y10) that displays specific defects in the formation of arteries, but not veins. We find that y10 encodes phospholipase C gamma-1 (plcg1), a known effector of receptor tyrosine kinase signaling. We further show that plcg1y10 mutant embryos fail to respond to exogenous Vegf. Our results indicate that Plcg1 functions specifically downstream of the Vegf receptor during embryonic development to govern formation of the arterial system. PMID:12782653

  4. 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

  5. Phytophthora infestans Has a Plethora of Phospholipase D Enzymes Including a Subclass That Has Extracellular Activity

    PubMed Central

    Meijer, Harold J. G.; Hassen, Hussen Harrun; Govers, Francine

    2011-01-01

    In eukaryotes phospholipase D (PLD) is involved in many cellular processes. Currently little is known about PLDs in oomycetes. Here we report that the oomycete plant pathogen Phytophthora infestans has a large repertoire of PLDs divided over six subfamilies: PXPH-PLD, PXTM-PLD, TM-PLD, PLD-likes, and type A and B sPLD-likes. Since the latter have signal peptides we developed a method using metabolically labelled phospholipids to monitor if P. infestans secretes PLD. In extracellular medium of ten P. infestans strains PLD activity was detected as demonstrated by the production of phosphatidic acid and the PLD specific marker phosphatidylalcohol. PMID:21423760

  6. 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. PMID:12217659

  7. Utilization of epidermal phospholipase A2 inhibition to monitor topical steroid action.

    PubMed

    Norris, J F; Ilderton, E; Yardley, H J; Summerly, R; Forster, S

    1984-07-01

    The effect of several steroid creams on epidermal phospholipase A2 (PLA2) activity in symptomless psoriatic and normal epidermis was studied. The magnitude of PLA2 inhibition produced by the steroids was directly proportional to the initial level of the enzyme activity. This differential inhibition resulted in PLA2 activity approaching or attaining the normal range regardless of its initial level. Clobetasol propionate 0.05% (Dermovate) produced more enzyme inhibition than betamethasone valerate 0.1% (Betnovate) but there was no difference in inhibition between this latter steroid and clobetasone butyrate 0.05% (Eumovate). All were more inhibitory than hydrocortisone I% (Efcortelan). PMID:6743552

  8. Proteinase, phospholipase, hyaluronidase and chondroitin-sulphatase production by Malassezia pachydermatis.

    PubMed

    Coutinho, S D; Paula, C R

    2000-02-01

    The production of four functional enzyme categories was investigated in 30 strains of Malassezia pachydermatis isolated from dogs with otitis or dermatitis. The most appropriate reading intervals for these assays were determined with the aid of statistical comparisons. All strains produced proteinase and chondroitin-sulphatase; hyaluronidase and phospholipase were produced by all skin isolates (15/15) and 14 out of 15 ear canal isolates. Strains from ear canals did not differ significantly as a group from skin strains in quantitative production of any of the four enzymes; production of proteinase and chondroitin-sulphatase in particular was markedly uniform. PMID:10746230

  9. OX1 orexin/hypocretin receptor activation of phospholipase D

    PubMed Central

    Jäntti, MH; Putula, J; Somerharju, P; Frohman, MA; Kukkonen, JP

    2012-01-01

    BACKGROUND AND PURPOSE Orexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling. EXPERIMENTAL APPROACH Orexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX1 orexin receptors using [14C]-oleic acid-prelabelling and the transphosphatidylation assay. KEY RESULTS Orexin stimulation strongly increased PLD activity – even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration–response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKCδ. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, Gαi/o nor Gβγ seemed to play an important role in the orexin response. CONCLUSIONS AND IMPLICATIONS Stimulation of OX1 receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKCδ but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKCδ in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKCδ. PMID:21718304

  10. High-affinity cholecystokinin type A receptor/cytosolic phospholipase A2 pathways mediate Ca2+ oscillations via a positive feedback regulation by calmodulin kinase in pancreatic acini.

    PubMed

    Lankisch, T O; Nozu, F; Owyang, C; Tsunoda, Y

    1999-09-01

    In rat pancreatic acini, we previously demonstrated that depending on the agonist used, activation of cholecystokinin type A (CCKA) receptor (CCK-AR) results in the differential involvement of the cytosolic phospholipase A2 (cPLA2), phospholipase Cbeta1 (PLCbeta1) and Src/protein tyrosine kinase (PTK) pathways. The high-affinity CCK-AR appears to be coupled to the Gbeta/cPLA2/arachidonic acid (AA) cascade in mediating Ca2+ oscillations. The low-affinity CCK-AR is coupled to both the Galphaq/11/PLCbeta1/inositol 1,4,5-trisphosphate (IP3) to evoke intracellular Ca2+ release and the Src/PTK pathway to mediate extracellular Ca2+ influx. The objectives of this study were to provide evidence that cPLA2 is present in pancreatic acini and to evaluate the possibility that its activation results in Ca2+ oscillations and amylase secretion. Furthermore, we investigated the mechanism of Ca2+ oscillations mediated by the high-affinity CCK-AR. In rat pancreatic acini, immunoprecipitation studies using an anti-cPLA2 monoclonal antibody, demonstrated a cPLA2 band at the location of 110 kDa. A selective inhibitor of cPLA2, AACOCF3 (100 microM), inhibited production of AA metabolites, Ca2+ oscillations and amylase secretion elicited by the high-affinity CCK-AR agonist, CCK-OPE (10-1000 nM). In addition, through the repetitive release of intracellular Ca2+, CCK-OPE enhanced phosphotransferase activities of Ca2+/calmodulin-dependent protein kinase type IV (CaMK IV), which were inhibited by AACOCF3. The CaMK inhibitor, K252-a (1-3 microM), also abolished basal and CCK-OPE-stimulated CaMK IV activities. The CaM inhibitor, W-7 (100 microM), and K252-a inhibited Ca2+ oscillations and amylase secretion evoked by CCK-OPE without affecting the AA formation. Therefore, it appears that Ca2+ oscillations elicited by the high-affinity CCK-AR/Gbeta/cPLA2/AA pathway activate CaMK IV. Activated CaMK, in turn, regulates Ca2+ oscillations through a positive feedback mechanism to mediate pancreatic

  11. 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. PMID:26516415

  12. 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

  13. A fluorescence-based assay for human type II phospholipase A2.

    PubMed

    Blanchard, S G; Harris, C O; Parks, D J

    1994-11-01

    A fluorescence assay for quantitation of human Type II Phospholipase A2 activity is described. Hydrolysis of 1-Acyl-2-(N-4-nitrobenzo-2-oxo-1,3-diazole)aminododecanoyl Phosphatidylethanolamine is accompanied by an increase in fluorescence intensity that is linearly proportional to enzyme activity. Substrate is prepared in the absence of detergents as a sonicated dispersion in aqueous buffer. Hydrolysis of the corresponding phosphatidylcholine derivative is more than an order of magnitude slower under identical assay conditions. A plot of initial rate versus substrate concentration could be fit to a simple Michaelis-Menten relationship with Km = 13 microM. In contrast to commonly used radiochemical assays for this enzyme, the method described here is continuous and allows estimation of enzyme activity without separation of substrate from product. Thus, the method is suitable for both kinetic analysis and large-scale screening using automated readers for 96-well tissue culture plates. The fluorescence-based assay displays advantages over other continuous assays for human Type II Phospholipase A2 based on (a) high sensitivity and (b) the use of a commercially available substrate. PMID:7864369

  14. A New Phospholipase A₂ from Lachesis muta rhombeata: Purification, Biochemical and Comparative Characterization with Crotoxin B.

    PubMed

    Cordeiro, Francielle A; Perini, Tibério G K; Bregge-Silva, Cristiane; Cremonez, Caroline M; Rodrigues, Renata S; Boldrini-França, Johara; Bordon, Karla de C F; De Souza, Dayane L N; Ache, David C; de M Rodrigues, Veridiana; Dos Santos, Wagner F; Rosa, Jose C; Arantesa, Eliane C

    2015-01-01

    Phospholipases A2 (PLA2s) are enzymes responsible for inflammatory effects, edema formation, myotoxicity, neurotoxicity and other manifestations from envenoming. In this paper we report the isolation and biochemical characterization of Lmr-PLA2, the first acidic PLA2 found in Lachesis muta rhombeata venom. Furthermore, this study compared biological effects of Lmr-PLA2 and crotoxin B (CB), a PLA2 from Crotalus durissus terrificus venom. Lmr-PLA2 was isolated by molecular exclusion and reversed phase chromatography. The purified enzyme showed a molecular mass of 13,975 Da, pI of 5.46 and its partial amino acid sequence showed a high identity with PLA2s already described in the literature. In addition, this enzyme possesses the residue D49 in its amino acid sequence, indicating that it is a catalytically active PLA2. Lmr-PLA2 presented high phospholipase activity and was able to inhibit platelet aggregation. Studies of biochemical characterization of new PLA2s, as Lmr-PLA2, are relevant since they help to clarify the structure-function relationship of this important class of toxins. PMID:26145564

  15. 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.

  16. Immobilized phospholipase A1-catalyzed modification of phosphatidylcholine with n-3 polyunsaturated fatty acid.

    PubMed

    Zhao, TingTing; No, Da Som; Kim, Byung Hee; Garcia, Hugo S; Kim, Yangha; Kim, In-Hwan

    2014-08-15

    n-3 Polyunsaturated fatty acids (n-3 PUFA)-enriched phosphatidylcholine (PC) was successfully produced with fatty acid from fish oil and PC from soybean by immobilized phospholipase A1-catalyzed acidolysis. Detailed studies of immobilization were carried out, and Lewatit VP OC 1600 was selected as a carrier for preparation of immobilized phospholipase A1, which was used for modification of PC by acidolysis. For acidolysis of PC with n-3 PUFA, the effects of several parameters, namely, water content, temperature, and enzyme loading on the reaction time course were investigated to determine optimum conditions. The optimum water content, temperature, and enzyme loading were 1.0%, 55 °C, and 20%, respectively. The highest incorporation (57.4 mol%) of n-3 PUFA into PC was obtained at 24h and the yield of PC was 16.7 mol%. The yield of PC increased significantly by application of vacuum, even though a slight decrease of n-3 PUFA incorporation was observed. PMID:24679762

  17. Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles.

    PubMed

    Ewing, Heather; Fernández-Vega, Virneliz; Spicer, Timothy P; Chase, Peter; Brown, Steven; Scampavia, Louis; Roush, William R; Riley, Sean; Rosen, Hugh; Hodder, Peter; Lambeau, Gerard; Gelb, Michael H

    2016-08-01

    There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate. PMID:27146384

  18. Dual Roles of Group IID Phospholipase A2 in Inflammation and Cancer.

    PubMed

    Miki, Yoshimi; Kidoguchi, Yuh; Sato, Mariko; Taketomi, Yoshitaka; Taya, Choji; Muramatsu, Kazuaki; Gelb, Michael H; Yamamoto, Kei; Murakami, Makoto

    2016-07-22

    Phospholipase A2 enzymes have long been implicated in the promotion of inflammation by mobilizing pro-inflammatory lipid mediators, yet recent evidence suggests that they also contribute to anti-inflammatory or pro-resolving programs. Group IID-secreted phospholipase A2 (sPLA2-IID) is abundantly expressed in dendritic cells in lymphoid tissues and resolves the Th1 immune response by controlling the steady-state levels of anti-inflammatory lipids such as docosahexaenoic acid and its metabolites. Here, we show that psoriasis and contact dermatitis were exacerbated in Pla2g2d-null mice, whereas they were ameliorated in Pla2g2d-overexpressing transgenic mice, relative to littermate wild-type mice. These phenotypes were associated with concomitant alterations in the tissue levels of ω3 polyunsaturated fatty acid (PUFA) metabolites, which had the capacity to reduce the expression of pro-inflammatory and Th1/Th17-type cytokines in dendritic cells or lymph node cells. In the context of cancer, however, Pla2g2d deficiency resulted in marked attenuation of skin carcinogenesis, likely because of the augmented anti-tumor immunity. Altogether, these results underscore a general role of sPLA2-IID as an immunosuppressive sPLA2 that allows the microenvironmental lipid balance toward an anti-inflammatory state, exerting beneficial or detrimental impact depending upon distinct pathophysiological contexts in inflammation and cancer. PMID:27226632

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26665171

  20. Molecular Details of Membrane Fluidity Changes during Apoptosis and Relationship to Phospholipase A2 Activity

    PubMed Central

    Gibbons, Elizabeth; Pickett, Katalyn R.; Streeter, Michael C.; Warcup, Ashley O.; Nelson, Jennifer; Judd, Allan M.; Bell, John D.

    2012-01-01

    Summary Secretory phospholipase A2 exhibits much greater activity toward apoptotic versus healthy cells. Various plasma membrane changes responsible for this phenomenon have been proposed, including biophysical alterations described as “membrane fluidity” and “order.” Understanding of these membrane perturbations was refined by applying studies with model membranes to fluorescence measurements during thapsigargin-induced apoptosis of S49 cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene. Alterations in emission properties of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A2. By applying a quantitative model, additional information was extracted from the kinetics of Patman equilibration with the membrane. Taken together, these data suggested that the phospholipids of apoptotic membranes display greater spacing between adjacent headgroups, reduced interactions between neighboring lipid tails, and increased penetration of water among the heads. The phase transition of artificial bilayers was used to calibrate quantitatively the relationship between probe fluorescence and the energy of interlipid interactions. This analysis was applied to results from apoptotic cells to estimate the frequency with which phospholipids protrude sufficiently at the membrane surface to enter the enzyme’s active site. The data suggested that this frequency increases 50–100-fold as membranes become susceptible to hydrolysis during apoptosis. PMID:22967861

  1. Critical role for cytosolic group IVA phospholipase A2 in early adipocyte differentiation and obesity.

    PubMed

    Peña, Lucía; Meana, Clara; Astudillo, Alma M; Lordén, Gema; Valdearcos, Martín; Sato, Hiroyasu; Murakami, Makoto; Balsinde, Jesús; Balboa, María A

    2016-09-01

    Adipogenesis is the process of differentiation of immature mesenchymal stem cells into adipocytes. Elucidation of the mechanisms that regulate adipocyte differentiation is key for the development of novel therapies for the control of obesity and related comorbidities. Cytosolic group IVA phospholipase A2 (cPLA2α) is the pivotal enzyme in receptor-mediated arachidonic acid (AA) mobilization and attendant eicosanoid production. Using primary multipotent cells and cell lines predetermined to become adipocytes, we show here that cPLA2α displays a proadipogenic function that occurs very early in the adipogenic process. Interestingly, cPLA2α levels decrease during adipogenesis, but cPLA2α-deficient preadipocytes exhibit a reduced capacity to differentiate into adipocytes, which affects early and terminal adipogenic transcription factors. Additionally, the absence of the phospholipase alters proliferation and cell-cycle progression that takes place during adipogenesis. Preconditioning of preadipocytes with AA increases the adipogenic capacity of these cells. Moreover, animals deficient in cPLA2α show resistance to obesity when fed a high fat diet that parallels changes in the expression of adipogenic transcription factors of the adipose tissue. Collectively, these results show that preadipocyte cPLA2α activation is a hitherto unrecognized factor for adipogenesis in vitro and in vivo. PMID:27317983

  2. 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. PMID:25993989

  3. 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

  4. The PNPLA-family phospholipases involved in glycerophospholipid homeostasis of HeLa cells.

    PubMed

    Hermansson, Martin; Hänninen, Satu; Hokynar, Kati; Somerharju, Pentti

    2016-09-01

    Mammalian cells maintain the glycerophospholipid (GPL) compositions of their membranes nearly constant. To achieve this, GPL synthesis and degradation must be coordinated. There is strong evidence that A-type phospholipases (PLAs) are key players in homeostatic degradation of GPLs, but the identities of the PLAs involved have not been established. However, some members of the Patatin-like phospholipase domain-containing proteins (PNPLAs) have been implicated. Accordingly, we knocked down all the PNPLAs significantly expressed in human HeLa cells using RNA interference and then determined whether the turnover of the major glycerophospholipids is affected by using mass spectrometry and metabolic labeling with stable isotope-labeled precursors. Knockdown of PNPLA9, PNPLA6 or PNPLA4 significantly (30-50%) reduced the turnover of phosphatidylcholine, -ethanolamine and -serine. In a notable contrast, turnover of phosphatidylinositol was not significantly affected by the knockdown of any PNPLA. Depletion of PNPLA9 and PNPLA4 also inhibited G0/G1 to S cell cycle progression, which could thus be regulated by GPL turnover. These results strongly suggest that PNPLA9, -6 and -4 play a key role in GPL turnover and homeostasis in human cells. A hypothetical model suggesting how these enzymes could recognize the relative concentration of the different GPLs is proposed. PMID:27317427

  5. Milleporin-1, a new phospholipase A2 active protein from the fire coral Millepora platyphylla nematocysts.

    PubMed

    Radwan, Faisal F Y; Aboul-Dahab, Hosney M

    2004-12-01

    Stings of fire corals, potent hydroids common in the Red Sea, are known to cause severe pain and they develop burns and itching that lasts few hours after contact. Nematocyst venom of Millepora platyphylla (Mp-TX) was isolated according to a recent method developed in our laboratory to conduct a previous investigation on the nematocyst toxicity of Millepora dichotoma and M. platyphylla. In this study, Mp-TX was fractionated by using both gel filtration and ion exchange chromatography. Simultaneous biological and biochemical assays were performed to monitor the hemolytic (using washed human red blood cells, RBCs) and phospholipase A2 (using radiolabeled sn-2 C14-arachidonyl phosphatidylcholine as a substrate) active venom fractions. The magnitude of both hemolysis and phospholipase A2 activity was found in a fraction rich of proteins of molecular masses approximately 30,000-34,000 Daltons. The former fraction was purified by ion exchange chromatography, and a major bioactive protein factor (approx. 32,500 Daltons , here named milleporin-1) was recovered. Milleporin-1 enzymatic activity showed a significant contribution to the overall hemolysis of human RBCs. This activity, however, could not be completely inhibited using phospholipid substrates. Melliporin-1 fraction retained about 30% hemolysis, until totally rendered inactive when boiled for 3 min. The overall mechanism of action of milleporin-1 to impact the cellular membrane was discussed; however, it is pending more biochemical and pharmacological future studies. PMID:15683837

  6. The combined effects of DEHP and PCBs on phospholipase in the livers of mice.

    PubMed

    Lin, Yi; Min, Lingli; Huang, Qiansheng; Chen, Yajie; Fang, Chao; Sun, Xia; Dong, Sijun

    2015-02-01

    Di(2-ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) are two widely distributed pollutants that are of great concern due to their adverse health effects. However, few studies have investigated the combined effects of DEHP and PCBs. In this study, adult mice were continuously exposed to mixtures of DEHP (15 mg/kg bodyweight/day) and Aroclor 1254 (7.5 mg/kg bodyweight/day) for 12 days to investigate the combined effects of these compounds. The results showed that the ratio of the liver weight to the body weight was higher in the treated group than that in the control group. The effects of combined exposure on three important receptors, the proliferator-activated receptor (PPAR), estrogen receptor (ER), and aryl hydrocarbon receptor (AHR), were investigated. The mRNA level of PPARγ was significantly up-regulated after exposure. The expression level of ERα was decreased in the male treated group. In contrast, the expression levels of AHR and related genes (cyp1a1 and cyp1b1) were not markedly affected. The expression level of phospholipase A (PLA) was significantly down-regulated at both the mRNA and protein levels in male mice after combined treatment. In all, our study demonstrated the combined effects of DEHP and PCBs on the expression levels of key receptors in mice. The combined exposure led to a decrease in phospholipase in male mice. PMID:23804495

  7. Listeria monocytogenes listeriolysin O and phosphatidylinositol-specific phospholipase C affect adherence to epithelial cells.

    PubMed

    Krawczyk-Balska, Agata; Bielecki, Jacek

    2005-09-01

    Listeria monocytogenes, a foodborn intracellular animal and human pathogen, produces several exotoxins contributing to virulence. Among these are listeriolysin O (LLO), a pore-forming cholesterol-dependent hemolysin, and a phosphatidylinositol-specific phospholipase C (PI-PLC). LLO is known to play an important role in the escape of bacteria from the primary phagocytic vacuole of macrophages, and PI-PLC supports this process. Evidence is accumulating that LLO and PI-PLC are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. LLO and PI-PLC may induce a number of host cell responses by modulating signal transduction of infected cells via intracellular Ca2+ levels and the metabolism of phospholipids. This would result in the activation of host phospholipase C and protein kinase C. In the present study, using Bacillus sub tilis strains expressing LLO, PI-PLC, and simultaneously LLO and PI-PLC, we show that LLO and PI-PLC enhance bacterial binding to epithelial cells Int407, with LLO being necessary and PI-PLC playing an accessory role. The results of this work suggest that these two listerial proteins act on epithelial cells prior to internalization. PMID:16391652

  8. 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

  9. Lysophospholipid generation and phosphatidylglycerol depletion in phospholipase A(2)-mediated surfactant dysfunction.

    PubMed

    Hite, R Duncan; Seeds, Michael C; Safta, Anca M; Jacinto, Randolph B; Gyves, Julianna I; Bass, David A; Waite, B Moseley

    2005-04-01

    Pulmonary surfactant's complex mixture of phospholipids and proteins reduces the work of breathing by lowering alveolar surface tension during respiration. One mechanism of surfactant damage appears to be the hydrolysis of phospholipid by phospholipases activated in the inflamed lung. Humans have several candidate secretory phospholipase A(2) (sPLA(2)) enzymes in lung cells and infiltrating leukocytes that could damage extracellular surfactant. We considered two mechanisms of surfactant disruption by five human sPLA(2)s, including generation of lysophospholipids and the depletion of specific phospholipids. All five sPLA(2)s studied ultimately caused surfactant dysfunction. Each enzyme exhibited a different pattern of hydrolysis of surfactant phospholipids. Phosphatidylcholine, the major phospholipid in surfactant and the greatest potential source for generation of lysophospholipids, was susceptible to hydrolysis by group IB, group V, and group X sPLA(2)s, but not group IIA or IID. Group IIA hydrolyzed both phosphatidylethanolamine and phosphatidylglycerol, whereas group IID was active against only phosphatidylglycerol. Thus, with groups IB and X, the generation of lysophospholipids corresponded with surfactant dysfunction. However, hydrolysis of and depletion of phosphatidylglycerol had a greater correlation with surfactant dysfunction for groups IIA and IID. Surfactant dysfunction caused by group V sPLA(2) is less clear and may be the combined result of both mechanisms. PMID:15516491

  10. 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

  11. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities.

    PubMed

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Popowycz, Florence; Khatib, Abdallah; Queneau, Yves; Abousalham, Abdelkarim

    2016-08-01

    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. Phospholipase PlaB is a new virulence factor of Legionella pneumophila.

    PubMed

    Schunder, Eva; Adam, Patrick; Higa, Futoshi; Remer, Katharina A; Lorenz, Udo; Bender, Jennifer; Schulz, Tino; Flieger, Antje; Steinert, Michael; Heuner, Klaus

    2010-06-01

    We previously identified Legionella pneumophila PlaB as the major cell-associated phospholipase A/lysophospholipase A with contact-dependent hemolytic activity. In this study, we further characterized this protein and found it to be involved in the virulence of L. pneumophila. PlaB was mainly expressed and active during exponential growth. Active PlaB was outer membrane-associated and at least in parts surface-exposed. Transport to the outer membrane was not dependent on the type I (T1SS), II (T2SS), IVB (T4BSS) or Tat secretion pathways. Furthermore, PlaB activity was not dependent on the presence of the macrophage infectivity potentiator (Mip) or the major secreted zinc metalloproteinase A (MspA). Despite the fact that PlaB is not essential for replication in protozoa or macrophage cell lines, we found that plaB mutants were impaired for replication in the lungs and dissemination to the spleen in the guinea pig infection model. Histological sections monitored less inflammation and destruction of the lung tissue after infection with the plaB mutants compared to L. pneumophila wild type. Taken together, PlaB is the first phospholipase A/lysophospholipase A with a confirmed role in the establishment of Legionnaires' disease. PMID:20153694

  13. Molecular characterization of enterobacterial pldA genes encoding outer membrane phospholipase A.

    PubMed Central

    Brok, R G; Brinkman, E; van Boxtel, R; Bekkers, A C; Verheij, H M; Tommassen, J

    1994-01-01

    The pldA gene of Escherichia coli encodes an outer membrane phospholipase A. A strain carrying the most commonly used mutant pldA allele appeared to express a correctly assembled PldA protein in the outer membrane. Nucleotide sequence analysis revealed that the only difference between the wild type and the mutant is the replacement of the serine residue in position 152 by phenylalanine. Since mutants that lack the pldA gene were normally viable under laboratory conditions and had no apparent phenotype except for the lack of outer membrane phospholipase activity, the exact role of the enzyme remains unknown. Nevertheless, the enzyme seems to be important for the bacteria, since Western blotting (immunoblotting) and enzyme assays showed that it is widely spread among species of the family Enterobacteriaceae. To characterize the PldA protein further, the pldA genes of Salmonella typhimurium, Klebsiella pneumoniae, and Proteus vulgaris were cloned and sequenced. The cloned genes were expressed in E. coli, and their gene products were enzymatically active. Comparison of the predicted PldA primary structures with that of E. coli PldA revealed a high degree of homology, with 79% of the amino acid residues being identical in all four proteins. Implications of the sequence comparison for the structure and the structure-function relationship of PldA protein are discussed. Images PMID:8300539

  14. Phospholipase-catalyzed hydrolysis in an artificial cell membrane in the presence of melittin.

    PubMed

    Lee, Jinyoung; Lee, Joo-Kyung; Busnaina, Ahmed; Park, BaeHo; Lee, HeaYeon

    2013-01-01

    Biomimicry involves the use of the structure and function of biological systems as models for the design and engineering of materials and machines. An artificial cell membrane was developed using biomembrane components, and the membrane, formed by a lipid bilayer, was analyzed using surface plasmon resonance (SPR) to monitor hydrolysis by phospholipase (PL). The simultaneous atomic force microscope (AFM) images show that PL catalyzed the nanometer-scale hydrolysis of the artificial lipid biomembranes through enzymatic hydrolysis. In addition, it was confirmed that the combination of PL and melittin allowed the control of enzyme hydrolysis for the degradation of the lipid bilayer. Regarding the expected activating effect of melittin on hydrolysis, no difference with respect to the non-treated lipid membrane was observed in the AFM images. It is assumed that the partitioning of melittin into the membrane might prevent the binding or hydrolysis of Phospholipase A2 (PLA2). This study provides basic knowledge on a new approach for patterning biomimicking lipid membranes on a nano-scale. PMID:23646709

  15. Phospholipase A2 activation by hydrogen peroxide during in vitro capacitation of buffalo spermatozoa.

    PubMed

    Shit, Sanjoy; Atreja, S K

    2004-05-01

    Progressively motile, washed buffalo spermatozoa (50 x 10(6) cells in 0.5 ml) were in vitro capacitated in HEPES containing Bovine Gamete Medium 3 (BGM3) in presence of heparin (10 microg/ml), and different concentrations of hydrogen peroxide (10 to 100 microM). Spermatozoa (60%) were capacitated in presence of heparin compared to 56% in presence of 25 microM H2O2 (optimally found suitable for capacitation). The extent of capacitation was measured in terms of acrosome reaction (AR) induced by lysophosphatidyl choline (100 microg/ml). The acrosome reacted cells were counted after triple staining. Catalase (100 microg/ml) significantly reduced the sperm capacitation to 16-18% when added with H2O2, or alone in the capacitation medium. Phospholipase A2 activity of spermatozoa increased linearly up to 50 microM H2O2 concentration included in the assay system. Moreover, significant increase in phospholipase A2 activity was observed after capacitation by both, the heparin and 25 microM H2O2. The activity was always higher in acrosome reacted cells. PMID:15233473

  16. 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

  17. Pla2g16 phospholipase mediates gain-of-function activities of mutant p53.

    PubMed

    Xiong, Shunbin; Tu, Huolin; Kollareddy, Madhusudhan; Pant, Vinod; Li, Qin; Zhang, Yun; Jackson, James G; Suh, Young-Ah; Elizondo-Fraire, Ana C; Yang, Peirong; Chau, Gilda; Tashakori, Mehrnoosh; Wasylishen, Amanda R; Ju, Zhenlin; Solomon, Hilla; Rotter, Varda; Liu, Bin; El-Naggar, Adel K; Donehower, Lawrence A; Martinez, Luis Alfonso; Lozano, Guillermina

    2014-07-29

    p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53(R172H/+) osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformation-specific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis. PMID:25024203

  18. 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. PMID:26340512

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

    SciTech Connect

    Oberdisse, E.; Lapetina, E.G.

    1987-05-14

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

  20. THE ROLE OF CCL4 BIOTRANSFORMATION IN THE ACTIVATION OF HEPATOCYTE PHOSPHOLIPASE C IN VIVO AND IN VITRO

    EPA Science Inventory

    The role of CC14 Biotransformation in the Activation of Hepatocyte Phospholipase C in ivo and in Vitro. Coleman, J. B., Condie, L.W. AND LAMB, R.G. (1988). Toxicol. Appl. Pharmacol. 95,208-219. Rats treated with a single 0.5 ml/kg dose (ip) of CCl4 exhibited a threefold increase ...

  1. 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…

  2. 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. PMID:24560778

  3. 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.

  4. Effects of a phospholipase A/sub 2/ inhibitor on uptake and toxicity of liposomes containing plant phosphatidylinositol

    SciTech Connect

    Jett, M.; Alving, C.R.

    1986-05-01

    Plant phosphatidylinositol (PI) has been shown by us to have a direct cytotoxic effect on cultured tumor cells but not on normal cells. Synthetic PI containing /sup 14/C-linoleic acid in the sn-2 position, also showed the same pattern of selective cytotoxicity. When the metabolic fate of synthetic PI was examined with tumor cells, the radioactivity which no longer occurred as PI, was found as either products of phospholipase A/sub 2/ (93%, free fatty acids and phosphatidylcholine) or phospholipase C (7%, diglycerides). Uptake of liposomal PI was directly correlated with cytotoxicity. They tested a variety of inhibitors to see the effect on uptake and/or cytotoxicity of plant PI. General metabolic inhibitors such as metrizamide or sodium azide did not alter cellular uptake of the plant PI liposomes. Inhibitors of lipoxygenase formation, such as indomethacin, also did not alter the uptake or cytotoxicity induced by plant PI. Quinacrine, an inhibitor of phospholipase A/sub 2/, decreased the uptake of the PI containing liposomes to 50% of that seen in the presence or absence of any other inhibitor. Although quinacrine is itself toxic to cells, at low concentrations of quinacrine, plant PI did not show the same degree of cytotoxicity as in the absence of quinacrine. These data are compatible with the hypothesis that plant PI exerts cytotoxicity by serving as a substrate for phospholipase A/sub 2/.

  5. 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.

  6. Phospholipase C in Dictyostelium discoideum. Cyclic AMP surface receptor and G-protein-regulated activity in vitro.

    PubMed

    Bominaar, A A; Kesbeke, F; Van Haastert, P J

    1994-01-01

    The cellular slime mould Dictyostelium discoideum shows several responses after stimulation with the chemoattractant cAMP, including a transient rise in cyclic AMP (cAMP), cGMP and Ins(1,4,5)P3. In this paper the regulation of phospholipase C in vitro is described. Under our experimental conditions commercial PtdIns(4,5)P2 cannot be used to analyse phospholipase C activity in Dictyostelium lysates, because it is hydrolysed mainly to glycerophosphoinositol instead of Ins(1,4,5)P3. Enzyme activity was determined with endogenous unlabelled PtdInsP2 as a substrate. The product was measured by isotope-dilution assay and identified as authentic Ins(1,4,5)P3. Since phospholipase C is strictly Ca(2+)-dependent, with an optimal concentration range of 1-100 microM, cell lysates were prepared in EGTA and the enzyme reaction was started by adding 10 microM free Ca2+. Phospholipase C activity increased 2-fold during Dictyostelium development up to 8 h of starvation, after which the activity declined to less than 10% of the vegetative level. Enzyme activity in vitro increased up to 2-fold after stimulation of cells with the agonist cAMP in vivo. Addition of 10 microM guanosine 5'-[gamma-thio]triphosphate during lysis activated the enzyme to the same extent, and this effect was antagonized by guanosine 5'-[beta-thio]diphosphate. These results strongly suggest that surface cAMP receptors and G-proteins regulate phospholipase C during Dictyostelium development. PMID:8280097

  7. Eicosanoid production by mouse peritoneal macrophages during Toxoplasma gondii penetration: role of parasite and host cell phospholipases.

    PubMed Central

    Thardin, J F; M'Rini, C; Beraud, M; Vandaele, J; Frisach, M F; Bessieres, M H; Seguela, J P; Pipy, B

    1993-01-01

    The metabolism of endogenous arachidonic acid by mouse resident peritoneal macrophages infected in vitro with Toxoplasma gondii was studied. Prelabeling of macrophages with [5,6,8,9,11,12,14,15-3H]arachidonic acid and challenge with tachyzoites for 15 min resulted in a high mobilization of free labeled arachidonic acid (178%) in the culture medium. The parasites also triggered the synthesis of 6-keto-prostaglandin F1 alpha (47%), prostaglandin E2 (44%), leukotrienes C4 and D4 (33%) and 5-, 12-hydroxyeicosatetraenoic acids (155%). The study indicated that during the intracellular development phase of the parasites, 6-keto-prostaglandin F1 alpha (38%), prostaglandin E2 (31%) leukotrienes C4 and D4 (15%), hydroxyeicosatetraenoic acids (43%), and free arachidonic acid (110%) were secreted into the culture medium. Pretreatment of tachyzoites with phospholipase A2 inhibitors (4-p-bromophenacyl bromide and quinacrine) and no calcium in the culture medium resulted in inhibition of tachyzoite penetration into the macrophages and a decrease of the arachidonic acid metabolism. The triggering of the arachidonic acid cascade by T. gondii was dependent on the active penetration of the parasites into the macrophages, whereas preincubation of the macrophages with phospholipase A2 inhibitors did not affect penetration or free arachidonic acid release, thereby supporting a role for parasite phospholipase in the penetration process and in arachidonic acid mobilization from macrophage membrane phospholipids. Moreover, treatment of macrophages with phospholipase A2 inhibitors decreased the activities of the cyclooxygenase and lipoxygenase pathways, also suggesting an activation of host cell phospholipase A2 by the parasite. PMID:8454347

  8. 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

  9. Synthesis of fusogenic lipids through activation of phospholipase D1 by GTPases and the kinase RSK2 is required for calcium-regulated exocytosis in neuroendocrine cells.

    PubMed

    Vitale, Nicolas

    2010-02-01

    Exocytosis of hormones occurs through the fusion of large dense-core secretory vesicles with the plasma membrane. This highly regulated process involves key proteins such as SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) and also specific lipids at the site of membrane fusion. Among the different lipids required for exocytosis, our recent observations have highlighted the crucial role of PA (phosphatidic acid) in the late stages of membrane fusion in various exocytotic events. An RNAi (RNA interference) strategy coupled with the detection of PA in living cells has pointed to plasma membrane-associated PLD1 (phospholipase D(1)) as the main producer of PA in response to secretagogue stimulation. We have identified several GTPases which regulate the activation level of PLD(1) in neuroendocrine cells. Finally, RSK2 (ribosomal S6 kinase 2) appears to phosphorylate and regulate the activity of PLD(1) in a calcium-dependent manner. Altogether our results have unravelled a complex set of regulatory pathways controlling the synthesis of fusogenic lipids at the secretory granule fusion site by PLD(1). PMID:20074053

  10. SCAMP2 Interacts with Arf6 and Phospholipase D1 and Links Their Function to Exocytotic Fusion Pore Formation in PC12 CellsD⃞

    PubMed Central

    Liu, Lixia; Liao, Haini; Castle, Anna; Zhang, Jie; Casanova, James; Szabo, Gabor; Castle, David

    2005-01-01

    SNAP receptor (SNARE)-mediated fusion is regarded as a core event in exocytosis. Exocytosis is supported by other proteins that set up SNARE interactions between secretory vesicle and plasma membranes or facilitate fusion pore formation. Secretory carrier membrane proteins (SCAMPs) are candidate proteins for functioning in these events. In neuroendocrine PC12 cells, SCAMP2 colocalizes on the cell surface with three other proteins required for dense-core vesicle exocytosis: phospholipase D1 (PLD1), the small GTPase Arf6, and Arf6 guanine nucleotide exchange protein ARNO. Arf6 and PLD1 coimmunoprecipitate (coIP) with SCAMP2. These associations have been implicated in exocytosis by observing enhanced coIP of Arf6 with SCAMP2 after cell depolarization and in the presence of guanosine 5′-O-(3-thio)triphosphate and by inhibition of coIP by a SCAMP-derived peptide that inhibits exocytosis. The peptide also suppresses PLD activity associated with exocytosis. Using amperometry to analyze exocytosis, we show that expression of a point mutant of SCAMP2 that exhibits decreased association with Arf6 and of mutant Arf6 deficient in activating PLD1 have the same inhibitory effects on early events in membrane fusion. However, mutant SCAMP2 also uniquely inhibits fusion pore dilation. Thus, SCAMP2 couples Arf6-stimulated PLD activity to exocytosis and links this process to formation of fusion pores. PMID:16030257

  11. 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

  12. Helix coupling

    DOEpatents

    Ginell, W.S.

    1982-03-17

    A coupling for connecting helix members in series, which consists of a pair of U-shaped elements, one of which is attached to each helix end with the U sections of the elements interlocked. The coupling is particularly beneficial for interconnecting helical Nitinol elements utilized in thermal actuators or engines. Each coupling half is attached to the associated helix at two points, thereby providing axial load while being easily removed from the helix, and reusable.

  13. Helix coupling

    DOEpatents

    Ginell, William S.

    1989-04-25

    A coupling for connecting helix members in series, which consists of a pair of U-shaped elements, one of which is attached to each helix end with the "U" sections of the elements interlocked. The coupling is particularly beneficial for interconnecting helical Nitinol elements utilized in thermal actuators or engines. Each coupling half is attached to the associated helix at two points, thereby providing axial load while being easily removed from the helix, and reusable.

  14. Specificity of Lipoprotein-Associated Phospholipase A2 Towards Oxidized Phosphatidylserines: LC-ESI-MS Characterization of Products and Computer Modeling of Interactions

    PubMed Central

    Tyurin, Vladimir A.; Yanamala, Naveena; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Macphee, Colin H.; Kagan, Valerian E.

    2013-01-01

    Ca2+ independent lipoprotein associated phospholipase A2 (Lp-PLA2) is a member of the phospholipase A2 superfamily with a distinguishing characteristic of high specificity for oxidatively modified sn-2 fatty acid residues in phospholipids which has been especially well characterized for peroxidized species of phosphatidylcholines (PC). The ability of Lp-PLA2 to hydrolyze peroxidized species of phosphatidylserine (PS) – acting as a recognition signal for clearance of apoptotic cells by professional phagocytes - as well as the products of the reaction have not been investigated. We performed LC-MS-ESI-based structural characterization of oxygenated/hydrolyzed molecular species of PS - containing linoleic acid in either sn-2 position (C18:0/C18:2) or in both sn-1 and sn-2 positions (C18:2/C18:2) - formed in cytochrome c/ H2O2 driven enzymatic oxidation reaction. Cytochrome c has been chosen as a catalyst of peroxidation reactions due to its likely involvement in PS oxidation in apoptotic cells. We found that Lp-PLA2 catalyzed the hydrolysis of both non-truncated and truncated (oxidatively fragmented) species of oxidized PS species albeit with different efficiencies and performed detailed characterization of the major reaction products – oxygenated derivatives of linoleic acid as well as non-oxygenated and oxygenated species of lyso-PS. Among linoleic acid products, derivatives oxygenated at the C9 position, including 9-hydroxyoctadecadienoic acid (9-HODE) – a potent ligand of G protein-coupled receptor G2A - were the most abundant. Computer modeling of interactions of Lp-PLA2 with different PS oxidized species indicated that they are able to bind in proximity (<5Å) to Ser273 and His351 of the catalytic triad. For 9-hydroxy- and 9-hydroperoxy- derivatives of oxidized PS, the sn-2 ester bond was positioned within the very close proximity (<3Å) from the Ser273 residue - a nucleophile directly attacking the sn-2 bond – thus favoring the hydrolysis reaction. We

  15. Discovery of Desketoraloxifene Analogues as Inhibitors of Mammalian, Pseudomonas aeruginosa, and NAPE Phospholipase D Enzymes

    PubMed Central

    2015-01-01

    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. PMID:25384256

  16. Serum amyloid A protein enhances the activity of secretory non-pancreatic phospholipase A2.

    PubMed Central

    Pruzanski, W; de Beer, F C; de Beer, M C; Stefanski, E; Vadas, P

    1995-01-01

    The acute-phase proteins serum amyloid A protein (SAA) and secretory phospholipase A2 (sPLA2) are simultaneously expressed during inflammatory conditions. SAA associates with high-density lipoprotein (HDL) altering its physicochemical composition. We found that purified acute-phase SAA, but not the constitutive form, markedly enhances the lipolytic activity of sPLA2 in a dose-related manner with phosphatidylcholine/lysophosphatidylcholine or phosphatidylethanolamine/lysophosphatidylethanolamine liposomal substrates. Normal HDL was found to reduce activity of sPLA2 in a dose-dependent manner, but when acute-phase HDL containing 27% SAA was tested, it enhanced sPLA2 activity. Immunopurified monospecific antibodies against SAA completely abolished the enhancing activity of SAA and acute-phase HDL. Given the central role of HDL in lipoprotein metabolism, the interaction between HDL, SAA and sPLA2 may account for changes detected in lipoprotein metabolism during the acute phase. PMID:7542869

  17. Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

    PubMed Central

    Mouneimne, Ghassan; Soon, Lilian; DesMarais, Vera; Sidani, Mazen; Song, Xiaoyan; Yip, Shu-Chin; Ghosh, Mousumi; Eddy, Robert; Backer, Jonathan M.; Condeelis, John

    2004-01-01

    The epidermal growth factor (EGF)–induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF. PMID:15337778

  18. Regulation of retinal angiogenesis by phospholipase C-β3 signaling pathway.

    PubMed

    Ha, Jung Min; Baek, Seung Hoon; Kim, Young Hwan; Jin, Seo Yeon; Lee, Hye Sun; Kim, Sun Ja; Shin, Hwa Kyoung; Lee, Dong Hyung; Song, Sang Heon; Kim, Chi Dae; Bae, Sun Sik

    2016-01-01

    Angiogenesis has an essential role in many pathophysiologies. Here, we show that phospholipase C-β3 (PLC-β3) isoform regulates endothelial cell function and retinal angiogenesis. Silencing of PLC-β3 in human umbilical vein endothelial cells (HUVECs) significantly delayed proliferation, migration and capillary-like tube formation. In addition, mice lacking PLC-β3 showed impaired retinal angiogenesis with delayed endothelial proliferation, reduced endothelial cell activation, abnormal vessel formation and hemorrhage. Finally, tumor formation was significantly reduced in mice lacking PLC-β3 and showed irregular size and shape of blood vessels. These results suggest that regulation of endothelial function by PLC-β3 may contribute to angiogenesis. PMID:27311705

  19. Phospholipase A2 Receptor-Positive Idiopathic Membranous Glomerulonephritis with Onset at 95 Years: Case Report

    PubMed Central

    Kubota, Keiichi; Hoshino, Junichi; Ueno, Toshiharu; Mise, Koki; Hazue, Ryo; Sekine, Akinari; Yabuuchi, Junko; Yamanouchi, Masayuki; Suwabe, Tatsuya; Kikuchi, Koichi; Sumida, Keiichi; Hayami, Noriko; Sawa, Naoki; Takaichi, Kenmei; Fujii, Takeshi; Ohashi, Kenichi; Akiyama, Shinichi; Maruyama, Shoichi; Ubara, Yoshifumi

    2016-01-01

    A 95-year-old woman was admitted to our hospital for evaluation of bilateral lower-limb edema persisting for 3 months. Serum creatinine was 1.55 mg/dl, and urinary protein excretion was 9.1 g/day. Renal biopsy revealed stage 1 membranous glomerulonephritis (MGN) with immunoglobulin G4-dominant staining. This patient did not have any underlying disease such as infection with hepatitis B or C virus or malignancy, and anti-phospholipase A2 receptor (PLA2R) antibody was detected in the serum. Accordingly, idiopathic MGN was diagnosed. Corticosteroid therapy was avoided, but hemodialysis was required to treat generalized edema. The patient is currently doing well. This is the oldest reported case of idiopathic MGN with positivity for anti-PLA2R antibody. PMID:27390744

  20. 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. PMID:22260706

  1. 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. PMID:24009677

  2. 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

  3. 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

    2015-04-01

    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. PMID:25881537

  4. Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys.

    PubMed

    Sugahara, Go; Kamiie, Junichi; Kobayashi, Ryosuke; Mineshige, Takayuki; Shirota, Kinji

    2016-06-01

    Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R. PMID:26854253

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

    PubMed

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

    2015-12-20

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

  6. 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

  7. Characterization of serum phospholipase a(2) activity in three diverse species of west african crocodiles.

    PubMed

    Merchant, Mark; Juneau, Kate; Gemillion, Jared; Falconi, Rodolfo; Doucet, Aaron; Shirley, Matthew H

    2011-01-01

    Secretory phospholipase A(2), 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 PLA(2) inhibitor, confirming that the activity was a direct result of the presence of serum PLA(2). 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 PLA(2) activities but with slightly different thermal profiles. All three crocodilian species showed high levels of activity against eight different species of bacteria. PMID:22110960

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

    PubMed

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

    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

  9. Regulation of retinal angiogenesis by phospholipase C-β3 signaling pathway

    PubMed Central

    Ha, Jung Min; Baek, Seung Hoon; Kim, Young Hwan; Jin, Seo Yeon; Lee, Hye Sun; Kim, Sun Ja; Shin, Hwa Kyoung; Lee, Dong Hyung; Song, Sang Heon; Kim, Chi Dae; Bae, Sun Sik

    2016-01-01

    Angiogenesis has an essential role in many pathophysiologies. Here, we show that phospholipase C-β3 (PLC-β3) isoform regulates endothelial cell function and retinal angiogenesis. Silencing of PLC-β3 in human umbilical vein endothelial cells (HUVECs) significantly delayed proliferation, migration and capillary-like tube formation. In addition, mice lacking PLC-β3 showed impaired retinal angiogenesis with delayed endothelial proliferation, reduced endothelial cell activation, abnormal vessel formation and hemorrhage. Finally, tumor formation was significantly reduced in mice lacking PLC-β3 and showed irregular size and shape of blood vessels. These results suggest that regulation of endothelial function by PLC-β3 may contribute to angiogenesis. PMID:27311705

  10. 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.

  11. Phospholipase Cgamma1 inhibitory principles from the sarcotestas of Ginkgo biloba.

    PubMed

    Lee, J S; Cho, Y S; Park, E J; Kim, J; Oh, W K; Lee, H S; Ahn, J S

    1998-07-01

    Ten phenolic compounds were isolated from the CHCl3 extract of Ginkgo biloba sarcotestas (Ginkgoaceae) as a new class of phosphatidylinositol-specific phospholipase Cgamma1 (PI-PLCgamma1) inhibitors. The substances without the long chain were ineffective. On the other hand, the activities of these compounds were dramatically decreased by acetylation of aromatic hydroxyl groups of cardanol, phenolic acid, and bilobol and by methylation of the aromatic carboxyl group of phenolic acid. The unsaturated long chain as well as the aromatic hydroxyl and carboxyl groups might play a key role for the PI-PLCgamma1 inhibitory activity. These compounds also inhibited the growth of a number of human cancer cell lines, but were less cytotoxic against a human normal colon cell line. PMID:9677265

  12. Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2.

    PubMed

    Peroutka, Raymond J; Elshourbagy, Nabil; Piech, Tara; Butt, Tauseef R

    2008-09-01

    SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases. PMID:18539905

  13. Phospholipase D Toxins of Brown Spider Venom Convert Lysophosphatidylcholine and Sphingomyelin to Cyclic Phosphates

    PubMed Central

    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 31P 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. PMID:24009677

  14. 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. PMID:24862647

  15. Control of phospholipase A2 activities for the treatment of inflammatory conditions.

    PubMed

    Yedgar, Saul; Cohen, Yuval; Shoseyov, David

    2006-11-01

    Phospholipase-A2 (PLA2) enzymes hydrolyze cell membrane phospholipids to produce arachidonic acid (AA) and lyso-phospholipids (LysoPL), playing a key role in the production of inflammatory lipid mediators, mainly eicosanoids. They are therefore considered pro-inflammatory enzymes and their inhibition has long been recognized as a desirable therapeutic target. However, attempts to develop suitable PLA2 inhibitors for the treatment of inflammatory diseases have yet to succeed. This is due to their functional and structural diversity, and their homeostatic and even anti-inflammatory roles in certain circumstances. In the present review we outline the diversity and functions of PLA2 isoforms, and their interplay in the induction and inhibition of inflammatory processes, with emphasis on discussing approaches for therapeutic manipulation of PLA2 activities. PMID:16978919

  16. 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

  17. Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys

    PubMed Central

    SUGAHARA, Go; KAMIIE, Junichi; KOBAYASHI, Ryosuke; MINESHIGE, Takayuki; SHIROTA, Kinji

    2016-01-01

    Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R. PMID:26854253

  18. 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. PMID:24813107

  19. 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

  20. 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. PMID:26031599

  1. 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

  2. Comparative structural studies on Lys49-phospholipases A(2) from Bothrops genus reveal their myotoxic site.

    PubMed

    dos Santos, Juliana I; Soares, Andreimar Martins; Fontes, Marcos R M

    2009-08-01

    Phospholipases A(2) (PLA(2)s) are membrane-associated enzymes that hydrolyze phospholipids at the sn-2 position, releasing lysophospholipids and free fatty acids. Phospholipase A(2) homologues (Lys49-PLA(2)s) are highly myotoxic and cause extensive tissue damage despite not showing measurable catalytic activity. They are found in different snake venoms and represent one third of bothropic venom composition. The importance of these toxins during envenomation is related to the pronounced local myotoxic effect they induce since this effect is not neutralized by serum therapy. We present herein three structures of Lys49-PLA(2)s from Bothrops genus snake venom crystallized under the same conditions, two of which were grown in the presence of alpha-tocopherol (vitamin E). Comparative structural analysis of these and other Lys49-PLA(2)s showed two different patterns of oligomeric conformation that are related to the presence or absence of ligands in the hydrophobic channel. This work also confirms the biological dimer indicated by recent studies in which both C-termini are in the dimeric interface. In this configuration, we propose that the myotoxic site of these toxins is composed by the Lys 20, Lys115 and Arg118 residues. For the first time, a residue from the short-helix (Lys20) is suggested as a member of this site and the importance of Tyr119 residue to myotoxicity of bothropic Lys49-PLA(2)s is also discussed. These results support a complete hypothesis for these PLA(2)s myotoxic activity consistent with all findings on bothropic Lys49-PLA(2)s studied up to this moment, including crystallographic, bioinformatics, biochemical and biophysical data. PMID:19401234

  3. 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

  4. 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

  5. Human cardiac phospholipase D activity is tightly controlled by phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Kurz, Thomas; Kemken, Dorit; Mier, Kenneth; Weber, Isabel; Richardt, Gert

    2004-02-01

    Phospholipase D (PLD) plays a central role in receptor-mediated breakdown of choline phospholipids and formation of phosphatidic acid (PA), an important regulator of cardiac function. However, specific mechanisms that regulate myocardial PLD activity remain largely unknown, particularly in the human heart. We hypothesized that phosphatidylinositol 4,5-bisphosphate (PIP2), best known as substrate for phospholipase C (PLC) isozymes, plays a critical role in regulating myocardial PLD activity. We examined the effect of PIP2 on human myocardial PLD activity in vitro by utilizing a fluorescence HPLC assay. PIP2 increased 10-fold the maximal activity of a partially solubilized PLD from human atrial myocardium. PIP2-stimulated PLD activity was accompanied by a consecutive increase in diacylglycerol, indicating dephosphorylation of PA by PA phosphohydrolase. Likewise, phosphatidylinositol 3,4,5-trisphosphate, which is produced from PIP2 by phosphatidylinositol 3-kinase, increased PLD activity with about the same potency but with somewhat lower efficacy. In contrast, other phospholipids were ineffective, indicating that the action of PIP2 on PLD is highly specific. Neomycin, a high-affinity ligand of PIP2, inhibited PLD activity in human atrial myocardium, but had no effect on the activity of partially solubilized enzyme. The addition of PIP2 restored the sensitivity of solubilized PLD to neomycin inhibition, indicating that neomycin inhibits PLD activity by binding to endogenous PIP2. Our results demonstrate a critical role for PIP2 in human cardiac PLD activity and suggest that PIP2 synthesis (by phosphatidylinositol 4-phosphate 5-kinase) and hydrolysis (by PIP2-specific PLC) could be important determinants in regulating PLD signal transduction in the human heart. PMID:14871550

  6. Citicoline decreases phospholipase A2 stimulation and hydroxyl radical generation in transient cerebral ischemia.

    PubMed

    Adibhatla, Rao Muralikrishna; Hatcher, James F

    2003-08-01

    Neuroprotection by citicoline (CDP-choline) in transient cerebral ischemia has been demonstrated previously. Citicoline has undergone several Phase III clinical trials for stroke, and is being evaluated for treatment of Alzheimer's and Parkinson's diseases. Phospholipid degradation and generation of reactive oxygen species (ROS) are major factors causing neuronal injury in CNS trauma and neurodegenerative diseases. Oxidative metabolism of arachidonic acid (released by the action of phospholipases) contributes to ROS generation. We examined the effect of citicoline on phospholipase A(2) (PLA(2)) activity in relation to the attenuation of hydroxyl radical (OH.) generation after transient forebrain ischemia of gerbil. PLA(2) activity (requires mM Ca(2+)) increased significantly (P < 0.05) in both membrane (50.2 +/- 2.2 pmol/min/mg protein compared to sham 35.9 +/- 3.2) and mitochondrial fractions (77.0 +/- 1.2 pmol/min/mg protein compared to sham 33.9 +/- 1.2) after cerebral ischemia and 2 hr reperfusion in gerbil, which was significantly attenuated (P < 0.01) by citicoline (membrane, 39.9. +/- 2.2 and mitochondria, 41.9 +/- 3.2 pmol/min/mg protein). In vitro, citicoline and its components cytidine and choline had no effect on PLA(2) activity, and thus citicoline as such is not a PLA(2) inhibitor. Ischemia/reperfusion resulted in significant OH. generation (P < 0.01) and citicoline significantly (P < 0.01) attenuated their formation (expressed as 2,3-dihydroxybenzoic acid/salicylate ratio; ischemia/24 hr reperfusion, 6.30 +/- 0.23; sham, 2.56 +/- 0.27; ischemia/24 hr reperfusion + citicoline, 4.85 +/- 0.35). These results suggest that citicoline affects PLA(2) stimulation and decreases OH. generation after transient cerebral ischemia. PMID:12868064

  7. 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. PMID:27473012

  8. Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation

    PubMed Central

    Zavaleta-Pastor, Maritza; Sohlenkamp, Christian; Gao, Jun-Lian; Guan, Ziqiang; Zaheer, Rahat; Finan, Turlough M.; Raetz, Christian R. H.; López-Lara, Isabel M.; Geiger, Otto

    2009-01-01

    Rhizobia are Gram-negative soil bacteria able to establish nitrogen-fixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithine-containing lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse–chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria. PMID:20018679

  9. 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

  10. Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA)

    PubMed Central

    2005-01-01

    Envenomation by the brown recluse spider (Loxosceles reclusa) may cause local dermonecrosis and, rarely, coagulopathies, kidney failure and death. A venom phospholipase, SMaseD (sphingomyelinase D), is responsible for the pathological manifestations of envenomation. Recently, the recombinant SMaseD from Loxosceles laeta was demonstrated to hydrolyse LPC (lysophosphatidylcholine) to produce LPA (lysophosphatidic acid) and choline. Therefore activation of LPA signalling pathways may be involved in some manifestations of Loxosceles envenomation. To begin investigating this idea, we cloned a full-length cDNA encoding L. reclusa SMaseD. The 305 amino acid sequence of the L. reclusa enzyme is 87, 85 and 60% identical with those of L. arizonica, L. intermedia and L. laeta respectively. The recombinant enzyme expressed in bacteria had broad substrate specificity. The lysophospholipids LPC, LPI (18:1-1-oleyol lysophosphatidylinositol), LPS, LPG (18:1-1-oleoyl-lysophosphatidylglycerol), LBPA (18:1-1-oleoyl-lysobisphosphatidic acid) (all with various acyl chains), lyso-platelet-activating factor (C16:0), cyclic phosphatidic acid and sphingomyelin were hydrolysed, whereas sphingosylphosphorylcholine, PC (phosphatidylcholine; C22:6, C20:4 and C6:0), oxidized PCs and PAF (platelet-activating factor; C16:0) were not hydrolysed. The PAF analogue, edelfosine, inhibited enzyme activity. Recombinant enzyme plus LPC (C18:1) induced the migration of A2058 melanoma cells, and this activity was blocked by the LPA receptor antagonist, VPC32183. The recombinant spider enzyme was haemolytic, but this activity was absent from catalytically inactive H37N (His37→Asn) and H73N mutants. Our results demonstrate that Loxosceles phospholipase D hydrolyses a wider range of lysophospholipids than previously supposed, and thus the term ‘SMaseD’ is too limited in describing this enzyme. PMID:15926888

  11. Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA).

    PubMed

    Lee, Sangderk; Lynch, Kevin R

    2005-10-15

    Envenomation by the brown recluse spider (Loxosceles reclusa) may cause local dermonecrosis and, rarely, coagulopathies, kidney failure and death. A venom phospholipase, SMaseD (sphingomyelinase D), is responsible for the pathological manifestations of envenomation. Recently, the recombinant SMaseD from Loxosceles laeta was demonstrated to hydrolyse LPC (lysophosphatidylcholine) to produce LPA (lysophosphatidic acid) and choline. Therefore activation of LPA signalling pathways may be involved in some manifestations of Loxosceles envenomation. To begin investigating this idea, we cloned a full-length cDNA encoding L. reclusa SMaseD. The 305 amino acid sequence of the L. reclusa enzyme is 87, 85 and 60% identical with those of L. arizonica, L. intermedia and L. laeta respectively. The recombinant enzyme expressed in bacteria had broad substrate specificity. The lysophospholipids LPC, LPI (18:1-1-oleyol lysophosphatidylinositol), LPS, LPG (18:1-1-oleoyl-lysophosphatidylglycerol), LBPA (18:1-1-oleoyl-lysobisphosphatidic acid) (all with various acyl chains), lyso-platelet-activating factor (C16:0), cyclic phosphatidic acid and sphingomyelin were hydrolysed, whereas sphingosylphosphorylcholine, PC (phosphatidylcholine; C22:6, C20:4 and C6:0), oxidized PCs and PAF (platelet-activating factor; C16:0) were not hydrolysed. The PAF analogue, edelfosine, inhibited enzyme activity. Recombinant enzyme plus LPC (C18:1) induced the migration of A2058 melanoma cells, and this activity was blocked by the LPA receptor antagonist, VPC32183. The recombinant spider enzyme was haemolytic, but this activity was absent from catalytically inactive H37N (His37-->Asn) and H73N mutants. Our results demonstrate that Loxosceles phospholipase D hydrolyses a wider range of lysophospholipids than previously supposed, and thus the term 'SMaseD' is too limited in describing this enzyme. PMID:15926888

  12. Purification and analysis of a phospholipase A2-like lytic factor of Trichomonas vaginalis.

    PubMed

    Lubick, Kirk J; Burgess, Donald E

    2004-03-01

    Trichomonas vaginalis produces soluble factors that have been reported to have the ability to damage target cells in vitro, and it has been hypothesized that these factors may play a role in the pathogenesis of human trichomoniasis. A lytic factor (LF) was purified from T. vaginalis, and the molecular characteristics of LF were determined. T. vaginalis extract was subjected to hydrophobic chromatography with a 10 to 60% N-propanol gradient in 0.1 M ammonium acetate, resulting in the elution of LF from the column at 30% N-propanol. Cytotoxicity assays revealed that LF was cytotoxic to WEHI 164 cells and bovine red blood cells, and inactivation of LF by treatment with trypsin suggested that the active component of LF was a protein. Size exclusion chromatography of LF produced two fractions at 144 and 168 kDa, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LF under reducing conditions revealed two subunits of 57 and 60 kDa. Results of a fluorescence assay of LF on carboxyfluorescein-labeled liposomes composed of phosphatidylcholine-cholesterol showed that liposomes were hydrolyzed, suggesting that LF had phospholipase activity. Thin-layer chromatography analysis of BODIPY (4,4-difluoro-3a,4adiaza-s-indacene)-labeled phosphatidylcholine treated with LF demonstrated products that migrated identically to the products produced by treatment with phospholipase A(2) (PLA(2)). These results suggest that LF is a PLA(2) and may be an important virulence factor of T. vaginalis mediating the destruction of host cells and contributing to tissue damage and inflammation in trichomoniasis. PMID:14977929

  13. Prognostic Utility of Secretory Phospholipase A2 in Patients with Stable Coronary Artery Disease

    PubMed Central

    O’Donoghue, Michelle; Mallat, Ziad; Morrow, David A; Benessiano, Joelle; Sloan, Sarah; Omland, Torbjørn; Solomon, Scott D.; Braunwald, Eugene; Tedgui, Alain; Sabatine, Marc S

    2011-01-01

    Background Secretory phospholipase A2 (sPLA2) may contribute to atherogenesis. To date, few prospective studies have examined the utility of sPLA2 for risk stratification in coronary artery disease (CAD). Methods Plasma sPLA2 activity was measured at baseline in 3708 subjects in the PEACE randomized trial of trandolapril versus placebo in stable CAD. Median follow-up was 4.8 years. Cox regression was used to adjust for demographics, clinical risk factors, apolipoprotein B, apolipoprotein A1, and medications. Results After multivariable adjustment, sPLA2 was associated with an increased risk of cardiovascular death, myocardial infarction or stroke (adjusted hazard ratio quartile 4:quartile 1 1.55, 95% CI 1.13–2.14) and cardiovascular death or heart failure (adjusted hazard ratio quartile 4:quartile 1 1.91, 95% CI 1.20–3.03). In further multivariable assessment, increased activities of sPLA2 were associated with the risk of cardiovascular death, myocardial infarction or stroke (adjusted hazard ratio 1.47, 95% CI 1.06–2.04) independent of lipoprotein-associated phospholipase A2 mass and C-reactive protein, and modestly improved the area under the curve (AUC) beyond established clinical risk factors (AUC 0.668 to 0.675, P=0.01). sPLA2, NT-pro B-type natriuretic peptide and high-sensitivity cardiac troponin T were all independently associated with cardiovascular death or heart failure and each improved risk discrimination (P=0.02, P<0.001, P<0.001, respectively). Conclusion sPLA2 activity provides independent prognostic information beyond established risk markers in patients with stable CAD. These data are encouraging for studies designed to evaluate the role of sPLA2 as a therapeutic target. PMID:21784767

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

    PubMed

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

    2006-02-01

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

  15. 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.

  16. Human group II 14 kDa phospholipase A2 activates human platelets.

    PubMed Central

    Polgár, J; Kramer, R M; Um, S L; Jakubowski, J A; Clemetson, K J

    1997-01-01

    Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier. PMID:9355761

  17. Phylogenetic and structural analysis of the phospholipase A2 gene family in vertebrates

    PubMed Central

    HUANG, QI; WU, YUAN; QIN, CHAO; HE, WENWU; WEI, XING

    2015-01-01

    The phospholipase A (PLA)2 family is the most complex gene family of phospholipases and plays a crucial role in a number of physiological activities. However, the phylogenetic background of the PLA2 gene family and the amino acid residues of the PLA2G7 gene following positive selection gene remain undetermined. In this study, we downloaded 49 genomic data sets of PLA from different species, including the human, house mouse, Norway rat, pig, dog, chicken, cattle, African clawed frog, Sumatran orangutan and the zebrafish species. Phylogenetic relationships were determined using the neighbor-joining (NJ), minimum evolution (ME) and maximum parsimony (MP) methods, as well as the Bayesian information criterion. The results were then presented as phylogenetic trees. Positive selection sites were detected using site, branch and branch-site models. These methods led us to the following assumptions: i) closer lineages were observed between PLA2G16 and PLA2G6, PLA2G7 and PLA2G4, PLA2G3 and PLA2G12, as well as among PLA2G10, PLA2G5 and PLA2G15; ii) PLA2G5 appeared to be the origin of the PLA2 family, and PLA2G7 was one of the most evolutionarily distant PLA2 proteins; iii) 16 positive-selection sites were detected and were marked in the PLA2G7 protein sequence as 327D, 257Q, 276G, 34s, 66G, 67C, 319S, 28N, 50S, 54T, 58R, 75T, 88Q, 92R, 179H and 191K. PMID:25543670

  18. Phospholipase A2 Activity Triggers the Wound-Activated Chemical Defense in the Diatom Thalassiosira rotula

    PubMed Central

    Pohnert, Georg

    2002-01-01

    The activation of oxylipin-based chemical defense in the diatom Thalassiosira rotula is initiated by phospholipases that act immediately after cell damage. This lipase activity is responsible for the preferential release of free mono- and polyunsaturated fatty acids. Among these, eicosatetraenoic- and eicosapentaenoic acid are further converted by lipoxygenases to reactive defensive metabolites such as the antiproliferative α,β,γ,δ-unsaturated aldehydes 2,4-decadienal and 2,4,7-decatrienal. We show that mainly saturated free fatty acids are present in the intact diatom T. rotula, whereas the amount of free polyunsaturated eicosanoids is drastically increased in the first minutes after wounding. Using fluorescent probes, the main enzyme activity responsible for initiation of the aldehyde-generating lipase/lipoxygenase/hydroperoxide lyase cascade was characterized as a phospholipase A2. All enzymes involved in this specific defensive reaction are active in seawater over several minutes. Thus, the mechanism allows the unicellular algae to overcome restrictions arising out of potential dilution of defensive metabolites. Only upon predation are high local concentrations of aldehydes formed in the vicinity of the herbivores, whereas in times of low stress, cellular resources can be invested in the formation of eicosanoid-rich phospholipids. In contrast to higher plants, which use lipases acting on galactolipids to release C18 fatty acids for production of leaf-volatile aldehydes, diatoms rely on phospholipids and the transformation of C20 fatty acids to form 2,4-decadienal and 2,4,7-decatrienal as an activated defense. PMID:12011342

  19. Cell-Associated Hemolysis Induced by Helicobacter pylori Is Mediated by Phospholipases with Mitogen-Activated Protein Kinase-Activating Properties

    PubMed Central

    Sitaraman, Ramakrishnan; Israel, Dawn A.; Romero-Gallo, Judith

    2012-01-01

    Pathogenic Helicobacter pylori strains can selectively activate epithelial mitogen-activated protein kinase (MAPK) signaling pathways linked with disease. We now demonstrate that H. pylori-induced hemolysis is strain specific and is mediated by phospholipases PldA1 and PldD. Inactivation of PldD inhibited activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), indicating that H. pylori hemolytic phospholipases also harbor MAPK-activating properties. PMID:22205825

  20. Cell-associated hemolysis induced by Helicobacter pylori is mediated by phospholipases with mitogen-activated protein kinase-activating properties.

    PubMed

    Sitaraman, Ramakrishnan; Israel, Dawn A; Romero-Gallo, Judith; Peek, Richard M

    2012-03-01

    Pathogenic Helicobacter pylori strains can selectively activate epithelial mitogen-activated protein kinase (MAPK) signaling pathways linked with disease. We now demonstrate that H. pylori-induced hemolysis is strain specific and is mediated by phospholipases PldA1 and PldD. Inactivation of PldD inhibited activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), indicating that H. pylori hemolytic phospholipases also harbor MAPK-activating properties. PMID:22205825

  1. Phospholipase D activation mediates cobalamin-induced downregulation of Multidrug Resistance-1 gene and increase in sensitivity to vinblastine in HepG2 cells.

    PubMed

    Marguerite, Véronique; Gkikopoulou, Effrosyni; Alberto, Jean-Marc; Guéant, Jean-Louis; Merten, Marc

    2013-02-01

    Failure of cancer chemotherapy due to multidrug resistance is often associated with altered Multidrug Resistance-1 gene expression. Cobalamin is the cofactor of methionine synthase, a key enzyme of the methionine cycle which synthesizes methionine, the precursor of cell S-adenosyl-methionine synthesis. We previously showed that cobalamin was able to down-regulate Multidrug Resistance-1 gene expression. Herein we report that this effect occurs through cobalamin-activation of phospholipase D activity in HepG2 cells. Cobalamin-induced down-regulation of Multidrug Resistance-1 gene expression was similar to that induced by the phospholipase D activator oleic acid and was negatively modulated by the phospholipase D inhibitor n-butanol. Cobalamin increased cell S-adenosyl-methionine content, which is the substrate for phosphatidylethanolamine-methyltransferase-dependent phosphatidylcholine production. We showed that cobalamin-induced increase in cell phosphatidylcholine production was phosphatidylethanolamine-methyltransferase-dependent. Oleic acid-dependent activation of phospholipase D was accompanied by an increased sensitivity to vinblastine of HepG2 cells while n-butanol enhanced the resistance of the cells to vinblastine. These data indicate that cobalamin mediates down-regulation of Multidrug Resistance-1 gene expression through increased S-adenosyl-methionine and phosphatidylcholine productions and phospholipase D activation. This points out phospholipase D as a potential target to down-regulate Multidrug Resistance-1 gene expression for improving chemotherapy efficacy. PMID:23032700

  2. The stimulation by transmitter substances and putative transmitter substances of the net activity of phospholipase A2 of synaptic membranes of cortex of guinea-pig brain.

    PubMed Central

    Gullis, R J; Rowe, C E

    1975-01-01

    1. The distribution of the hydrolyses of phosphatidylcholine by phospholipase A2 and phospholipase A1, and the hydrolysis of lysophosphatidylcholine by lysophospholipase, in subcellular and subsynaptosomal fractions of cerebral cortices of guinea-pig brain, was determined. 2. Noradrenaline stimulated hydrolysis by phospholipase A2 in whole synaptosomes, synaptic membranes and fractions containing synaptic vesicles. 3. Stimulation of hydrolysis by phospholipase A2 in synaptic membranes by noradrenaline was enhanced by CaCl2, and by a mixture of ATP and MgCl2. The optimum concentration of CaCl2, in the presence of ATP and MgCl2, for stimulation by 10 muM-noradrenaline was in the range 1-10muM. The optimum concentration for ATP-2MgCl2 in the presence of 1 muM-CaCl2 was in the range 0.1-1mM. 4. Hydrolysis by phospholipase A2 of synaptic membranes was also stimulated by acetylcholine, carbamoylcholine, 5-hydroxytryptamine, dopamine (3,4-dihydroxyphenethylamine), histamine, psi-aminobutyric acid, glutamic acid and aspartic acid. With appropriate concentrations of cofactors, sigmoidal dose-response curves were obtained, half-maximum stimulations being obtained with concentrations of stimulant in the range 0.1-1muM. 5. Taurine also stimulated hydrolysis of phosphatidylcholine by phospholipase A2. There were only slight stimulations with methylamine, ethylenediamine or spermidine. No stimulation was obtained with glucagon. PMID:239707

  3. Optical coupling

    NASA Astrophysics Data System (ADS)

    Bock, J. J.; Gundersen, J.; Lee, A. T.; Richards, P. L.; Wollack, E.

    2009-03-01

    This paper describes contributions to the CMBpol Technology Study Workshop concerning optical coupling structures. These are structures in or near the focal plane which convert the free space wave to a superconducting microstrip on a SI wafer, or to the waveguide input to a HEMT receiver. In addition to an introduction and conclusions by the editor, this paper includes independent contributions by Bock on 'Planar Antenna-Coupled Bolometers for CMB Polarimetry', by Gunderson and Wollack on 'Millimeter-Wave Platlet Feeds', and by Lee on 'Multi-band Dual-Polarization Lens-coupled Planar Antennas for Bolometric CMB polarimetry.'

  4. Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

    PubMed

    Babon, Aurélie; Wurceldorf, Thibault; Almunia, Christine; Pichard, Sylvain; Chenal, Alexandre; Buhot, Cécile; Beaumelle, Bruno; Gillet, Daniel

    2016-06-15

    We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes. PMID:26253725

  5. Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled

    SciTech Connect

    De Nucci, G.; Gryglewski, R.J.; Warner, T.D.; Vane, J.R. )

    1988-04-01

    Bovine aortic endothelial cells were grown on microcarrier beads and were perfused with Krebs-Ringer solution. Endothelium-derived relaxing factor (EDRF) was bioassayed on a cascade of four strips of rabbit aorta, and prostacyclin was analyzed by RIA of 6-oxo-prostaglandin F{sub 1{alpha}}. The endothelial cells released EDRF and prostacyclin when stimulated with bradykinin and its analogues, or with ADP, ATP, arachidonic acid, and phospholipase C. The detection of EDRF was potentiated by superoxide dismutase, and the relaxation of rabbit aortic strips induced by EDRF was antagonized by methylene blue. The release of EDRF and prostacyclin was inhibited by phorbol myristate acetate, R59022 (a diacylglycerol kinase inhibitor), and gentamycin. The authors suggest that the release of EDRF and prostacyclin is coupled and the initial common step is activation of a phospholipase C.

  6. 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

  7. 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. PMID:26853495

  8. Development of a Direct and Continuous Phospholipase D Assay Based on the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline.

    PubMed

    Rahier, Renaud; Noiriel, Alexandre; Abousalham, Abdelkarim

    2016-01-01

    Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca(2+) complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca(2+)/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 μM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant Vigna unguiculata PLD, as well as the PLD from Streptomyces chromofuscus, cabbage, or peanuts, and no PA production could be detected using phospholipase A1, phospholipase A2, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale. PMID:26636829

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

    SciTech Connect

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

    1989-04-04

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

  10. 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. PMID:26346287

  11. The effects of phorbol ester, diacylglycerol, phospholipase C and Ca2+ ionophore on protein phosphorylation in human and sheep erythrocytes.

    PubMed Central

    Raval, P J; Allan, D

    1985-01-01

    Treatment of human or sheep erythrocytes with PMA (phorbol myristate acetate) enhanced [32P]phosphate labelling of membrane polypeptides of approx. 100, 80 and 46 kDa. The 80 kDa and 46 kDa polypeptides coincided with bands 4.1 and 4.9 respectively on Coomassie-Blue-stained gels. Similar but smaller effects were obtained by treating human cells with 1-oleoyl-2-acetyl-rac-glycerol (OAG), exogenous bacterial phospholipase C or ionophore A23187 + Ca2+, each of which treatments would be expected to raise the concentration of membrane diacylglycerol. In contrast, sheep cells, which do not increase their content of diacylglycerol when treated with phospholipase C or A23187 + Ca2+, only showed enhanced phosphorylation with OAG. Neither human nor sheep cells showed any enhanced [32P]phosphate labelling of phosphoproteins when treated with 1-mono-oleoyl-rac-glycerol. It is concluded that diacylglycerol from a variety of sources can activate erythrocyte protein kinase C, but that the most effective diacylglycerol is that derived from endogenous polyphosphoinositides. In contrast with bacterial phospholipase C and A23187, which stimulate synthesis of phosphatidate by increasing the cell-membrane content of diacylglycerol in human erythrocytes, PMA, OAG or 1-mono-oleoyl-rac-glycerol caused no change in phospholipid metabolism. Images PMID:4084238

  12. Antimicrobial activity of apitoxin, melittin and phospholipase A₂ of honey bee (Apis mellifera) venom against oral pathogens.

    PubMed

    Leandro, Luís F; Mendes, Carlos A; Casemiro, Luciana A; Vinholis, Adriana H C; Cunha, Wilson R; de Almeida, Rosana; Martins, Carlos H G

    2015-03-01

    In this work, we used the Minimum Inhibitory Concentration (MIC) technique to evaluate the antibacterial potential of the apitoxin produced by Apis mellifera bees against the causative agents of tooth decay. Apitoxin was assayed in natura and in the commercially available form. The antibacterial actions of the main components of this apitoxin, phospholipase A2, and melittin were also assessed, alone and in combination. The following bacteria were tested: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis, Lactobacillus casei, and Enterococcus faecalis. The MIC results obtained for the commercially available apitoxin and for the apitoxin in natura were close and lay between 20 and 40 µg / mL, which indicated good antibacterial activity. Melittin was the most active component in apitoxin; it displayed very promising MIC values, from 4 to 40 µg / mL. Phospholipase A2 presented MIC values higher than 400 µg / mL. Association of mellitin with phospholipase A2 yielded MIC values ranging between 6 and 80 µg / mL. Considering that tooth decay affects people's health, apitoxin and its component melittin have potential application against oral pathogens. PMID:25806982

  13. Modulation of oxidative stress, inflammation, and atherosclerosis by lipoprotein-associated phospholipase A2

    PubMed Central

    Rosenson, Robert S.; Stafforini, Diana M.

    2012-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a unique member of the phospholipase A2 superfamily. This enzyme is characterized by its ability to specifically hydrolyze PAF as well as glycerophospholipids containing short, truncated, and/or oxidized fatty acyl groups at the sn-2 position of the glycerol backbone. In humans, Lp-PLA2 circulates in active form as a complex with low- and high-density lipoproteins. Clinical studies have reported that plasma Lp-PLA2 activity and mass are strongly associated with atherogenic lipids and vascular risk. These observations led to the hypothesis that Lp-PLA2 activity and/or mass levels could be used as biomarkers of cardiovascular disease and that inhibition of the activity could offer an attractive therapeutic strategy. Darapladib, a compound that inhibits Lp-PLA2 activity, is anti-atherogenic in mice and other animals, and it decreases atherosclerotic plaque expansion in humans. However, disagreement continues to exist regarding the validity of Lp-PLA2 as an independent marker of atherosclerosis and a scientifically justified target for intervention. Circulating Lp-PLA2 mass and activity are associated with vascular risk, but the strength of the association is reduced after adjustment for basal concentrations of the lipoprotein carriers with which the enzyme associates. Genetic studies in humans harboring an inactivating mutation at this locus indicate that loss of Lp-PLA2 function is a risk factor for inflammatory and vascular conditions in Japanese cohorts. Consistently, overexpression of Lp-PLA2 has anti-inflammatory and anti-atherogenic properties in animal models. This thematic review critically discusses results from laboratory and animal studies, analyzes genetic evidence, reviews clinical work demonstrating associations between Lp-PLA2 and vascular disease, and summarizes results from animal and human clinical trials in which administration of

  14. Interferon-gamma induces the synthesis and activation of cytosolic phospholipase A2.

    PubMed Central

    Wu, T; Levine, S J; Lawrence, M G; Logun, C; Angus, C W; Shelhamer, J H

    1994-01-01

    Both IFN-alpha/beta and IFN-gamma have recently been demonstrated to induce a rapid but transient activation of phospholipase A2 (PLA2) in BALB/c 3T3 fibroblasts and a human neuroblastoma cell line. We report that IFN-gamma induces the synthesis and prolonged activation of cytosolic phospholipase A2 (cPLA2) in a human bronchial epithelial cell line (BEAS 2B). Treatment of the cells with IFN-gamma (300 U/ml) increased the release of [3H]arachidonic acid (AA) from prelabeled cells with a maximal effect at 12 h after stimulation. The increased [3H]AA release was inhibited by the PLA2 inhibitor p-bromophenacyl bromide (10(-5) M). Calcium ionophore A23187 (10(-5) M) further increased the [3H]AA release from the IFN-gamma-treated cells. Subcellular enzyme activity assay revealed that IFN-gamma increased PLA2 activity in both the cytosol and membrane fractions with a translocation of the cPLA2 to cell membranes in a Ca(2+)-free cell lysing buffer. Treatment with IFN-gamma also induced the release of 15-HETE, an arachidonic acid metabolite. Immunoblot showed that IFN-gamma induced the synthesis of cPLA2 protein. Nuclear run-on assay demonstrated that IFN-gamma initiated cPLA2 gene transcription within 15 min, and this effect was sustained at 4 h and returned to near control level at 12 h. The cPLA2 mRNA level was assayed by reverse transcription and PCR. IFN-gamma was found to increase the cPLA2 mRNA after 2-24 h treatment. Furthermore, the IFN-gamma induced cPLA2 mRNA increase was blocked by inhibitors of protein kinase C and calcium/calmodulin-dependent protein kinases, suggesting the involvement of these protein kinases in IFN-gamma-induced gene expression of cPLA2. This study shows that IFN-gamma induces the synthesis and prolonged activation of cPLA2. Images PMID:8113394

  15. 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

  16. Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

    PubMed Central

    Cruchaga, Carlos; Benitez, Bruno A.; Cai, Yefei; Guerreiro, Rita; Harari, Oscar; Norton, Joanne; Budde, John; Bertelsen, Sarah; Jeng, Amanda T.; Cooper, Breanna; Skorupa, Tara; Carrell, David; Levitch, Denise; Hsu, Simon; Choi, Jiyoon; Ryten, Mina; Sassi, Celeste; Bras, Jose; Gibbs, Raphael J.; Hernandez, Dena G.; Lupton, Michelle K.; Powell, John; Forabosco, Paola; Ridge, Perry G.; Corcoran, Christopher D.; Tschanz, JoAnn T.; Norton, Maria C.; Munger, Ronald G.; Schmutz, Cameron; Leary, Maegan; Demirci, F. Yesim; Bamne, Mikhil N.; Wang, Xingbin; Lopez, Oscar L.; Ganguli, Mary; Medway, Christopher; Turton, James; Lord, Jenny; Braae, Anne; Barber, Imelda; Brown, Kristelle; Pastor, Pau; Lorenzo-Betancor, Oswaldo; Brkanac, Zoran; Scott, Erick; Topol, Eric; Morgan, Kevin; Rogaeva, Ekaterina; Singleton, Andy; Hardy, John; Kamboh, M. Ilyas; George-Hyslop, Peter St; Cairns, Nigel; Morris, John C.; Kauwe, John S.K.; Goate, Alison M.

    2014-01-01

    Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD)1,2. These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low frequency coding variants with large effects on LOAD risk, we performed whole exome-sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large case-control datasets. A rare variant in PLD3 (phospholipase-D family, member 3, rs145999145; V232M) segregated with disease status in two independent families and doubled risk for AD in seven independent case-control series (V232M meta-analysis; OR= 2.10, CI=1.47-2.99; p= 2.93×10-5, 11,354 cases and controls of European-descent). Gene-based burden analyses in 4,387 cases and controls of European-descent and 302 African American cases and controls, with complete sequence data for PLD3, indicate that several variants in this gene increase risk for AD in both populations (EA: OR= 2.75, CI=2.05-3.68; p=1.44×10-11, AA: OR= 5.48, CI=1.77-16.92; p=1.40×10-3). PLD3 is highly expressed in brain regions vulnerable to AD pathology, including hippocampus and cortex, and is expressed at lower levels in neurons from AD brains compared to control brains (p=8.10×10-10). Over-expression of PLD3 leads to a significant decrease in intracellular APP and extracellular Aβ42 and Aβ40, while knock-down of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a two-fold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may be used to identify rare variants with large effects on risk for disease or other complex traits. PMID

  17. Prosthesis coupling

    NASA Technical Reports Server (NTRS)

    Reswick, J. B.; Mooney, V.; Bright, C. W.; Owens, L. J. (Inventor)

    1979-01-01

    A coupling for use in an apparatus for connecting a prosthesis to the bone of a stump of an amputated limb is described which permits a bio-compatible carbon sleeve forming a part of the prosthesis connector to float so as to prevent disturbing the skin seal around the carbon sleeve. The coupling includes a flexible member interposed between a socket that is inserted within an intermedullary cavity of the bone and the sleeve. A lock pin is carried by the prosthesis and has a stem portion which is adapted to be coaxially disposed and slideably within the tubular female socket for securing the prosthesis to the stump. The skin around the percutaneous carbon sleeve is able to move as a result of the flexing coupling so as to reduce stresses caused by changes in the stump shape and/or movement between the bone and the flesh portion of the stump.

  18. FLEXIBLE COUPLING

    DOEpatents

    Babelay, E.F.

    1962-02-13

    A flexible shaft coupling for operation at speeds in excess of 14,000 rpm is designed which requires no lubrication. A driving sleeve member and a driven sleeve member are placed in concentric spaced relationship. A torque force is transmitted to the driven member from the driving member through a plurality of nylon balls symmetrically disposed between the spaced sleeves. The balls extend into races and recesses within the respective sleeve members. The sleeve members have a suitable clearance therebetween and the balls have a suitable radial clearance during operation of the coupling to provide a relatively loose coupling. These clearances accommodate for both parallel and/or angular misalignments and avoid metal-tometal contact between the sleeve members during operation. Thus, no lubrication is needed, and a minimum of vibrations is transmitted between the sleeve members. (AEC)

  19. Differing roles for members of the phospholipase A2 superfamily in experimental autoimmune encephalomyelitis

    PubMed Central

    Kalyvas, Athena; Baskakis, Constantinos; Magrioti, Victoria; Constantinou-Kokotou, Violetta; Stephens, Daren; López-Vales, Rubèn; Lu, Jian-Qiang; Yong, V. Wee; Dennis, Edward A.; Kokotos, George

    2009-01-01

    The phospholipase A2 (PLA2) superfamily hydrolyzes phospholipids to release free fatty acids and lysophospholipids, some of which can mediate inflammation and demyelination, hallmarks of the CNS autoimmune disease multiple sclerosis. The expression of two of the intracellular PLA2s (cPLA2 GIVA and iPLA2 GVIA) and two of the secreted PLA2s (sPLA2 GIIA and sPLA2 GV) are increased in different stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We show using small molecule inhibitors, that cPLA2 GIVA plays a role in the onset, and iPLA2 GVIA in the onset and progression of EAE. We also show a potential role for sPLA2 in the later remission phase. These studies demonstrate that selective inhibition of iPLA2 can ameliorate disease progression when treatment is started before or after the onset of symptoms. The effects of these inhibitors on lesion burden, chemokine and cytokine expression as well as on the lipid profile provide insights into their potential modes of action. iPLA2 is also expressed by macrophages and other immune cells in multiple sclerosis lesions. Our results therefore suggest that iPLA2 might be an excellent target to block for the treatment of CNS autoimmune diseases, such as multiple sclerosis. PMID:19218359

  20. A Model for the Interfacial Kinetics of Phospholipase D Activity on Long-Chain Lipids

    PubMed Central

    Majd, Sheereen; Yusko, Erik C.; Yang, Jerry; Sept, David; Mayer, Michael

    2013-01-01

    The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics. PMID:23823233

  1. 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. PMID:26260942

  2. Group III secreted phospholipase A2 transgenic mice spontaneously develop inflammation

    PubMed Central

    Sato, Hiroyasu; Taketomi, Yoshitaka; Isogai, Yuki; Masuda, Seiko; Kobayashi, Tetsuyuki; Yamamoto, Kei; Murakami, Makoto

    2009-01-01

    PLA2 (phospholipase A2) group III is an atypical sPLA2 (secretory PLA2) that is homologous with bee venom PLA2 rather than with other mammalian sPLA2s. In the present paper, we show that endogenous group III sPLA2 (PLA2G3) is expressed in mouse skin and that Tg (transgenic) mice overexpressing human PLA2G3 spontaneously develop skin inflammation. Pla2g3-Tg mice over 9 months of age frequently developed dermatitis with hyperkeratosis, acanthosis, parakeratosis, erosion, ulcer and sebaceous gland hyperplasia. The dermatitis was accompanied by infiltration of neutrophils and macrophages and by elevated levels of pro-inflammatory cytokines, chemokines and prostaglandin E2. In addition, Pla2g3-Tg mice had increased lymph aggregates and mucus in the airway, lymphocytic sialadenitis, hepatic extramedullary haemopoiesis, splenomegaly with increased populations of granulocytes and monocytes/macrophages, and increased serum IgG1. Collectively, these observations provide the first demonstration of spontaneous development of inflammation in mice with Tg overexpression of mammalian sPLA2. PMID:19371233

  3. Plant phospholipase C family: Regulation and functional role in lipid signaling.

    PubMed

    Singh, Amarjeet; Bhatnagar, Nikita; Pandey, Amita; Pandey, Girdhar K

    2015-08-01

    Phospholipase C (PLC), a major membrane phospholipid hydrolyzing enzyme generates signaling messengers such as diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) in animals, and their phosphorylated forms such as phosphatidic acid (PA) and inositol hexakisphosphate (IP6) are thought to regulate various cellular processes in plants. Based on substrate specificity, plant PLC family is sub-divided into phosphatidylinositol-PLC (PI-PLC) and phosphatidylcholine-PLC (PC-PLC) groups. The activity of plant PLCs is regulated by various factors and the major ones include, Ca(2+) concentration, phospholipid substrate, post-translational modifications and interacting proteins. Most of the PLC members have been localized at the plasma membrane, suited for their function of membrane lipid hydrolysis. Several PLC members have been implicated in various cellular processes and signaling networks, triggered in response to a number of environmental cues and developmental events in different plant species, which makes them potential candidates for genetically engineering the crop plants for stress tolerance and enhancing the crop productivity. In this review article, we are focusing mainly on the plant PLC signaling and regulation, potential cellular and physiological role in different abiotic and biotic stresses, nutrient deficiency, growth and development. PMID:25933832

  4. [DIACYLGLYCEROL ACCUMULATION IMPAIRS SHORT-TERM ACTIVATION OF PHOSPHOLIPASE D BY THYROXINE IN THE LIVER CELLS].

    PubMed

    Hassouneh, Loay Kh M

    2015-01-01

    Thyroid hormones (TG) are known modulators of signal transduction. Phospholipase D (PLD) is one of the targets of TG in the stimulated cells. Response of cells to the short-term TG action significantly reduces at old age. Taking into account that diacylglycerol (DAG) accumulation induces the resistance of cells to some of regulatory factors in the target cells the aim of the present study was to determine if DAG content increase in hepatocytes impairs the L-thyroxine (L-T4) short-term action. The experiments were performed in either the [14C]palmitic acid- labeled hepatocytes or [14C]oleic acid-pre-labeled liver cells of 3- and 24-month-old rats. To study the short-term L-T4 action on cells the PLD activation was determined. The DAG production and content in hepatocytes significantly increased at old age and in the young cells pre-treated with palmitic acid. The reduction of DAG level in cells by means of DAG-kinase activator, alfa-tocoferol acetate, or long-term L-T4 treatment improved the short-term hormone action. The above data have indicated that DAG play important role in the L-T4 PLD regulation. The cross-talk between classic and non-genomic pathways of TG regulation of lipid metabolism has been determined. PMID:26387163

  5. Predominant role of cytosolic phospholipase A2α in dioxin-induced neonatal hydronephrosis in mice.

    PubMed

    Yoshioka, Wataru; Kawaguchi, Tatsuya; Fujisawa, Nozomi; Aida-Yasuoka, Keiko; Shimizu, Takao; Matsumura, Fumio; Tohyama, Chiharu

    2014-01-01

    Hydronephrosis is a common disease characterized by dilation of the renal pelvis and calices, resulting in loss of kidney function in the most severe cases. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces nonobstructive hydronephrosis in mouse neonates through upregulation of prostaglandin E2 (PGE2) synthesis pathway consisting of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) by a yet unknown mechanism. We here studied possible involvement of cytosolic phospholipase A2α (cPLA2α) in this mechanism. To this end, we used a cPLA2α-null mouse model and found that cPLA2α has a significant role in the upregulation of the PGE2 synthesis pathway through a noncanonical pathway of aryl hydrocarbon receptor. This study is the first to demonstrate the predominant role of cPLA2α in hydronephrosis. Elucidation of the pathway leading to the onset of hydronephrosis using the TCDD-exposed mouse model will deepen our understanding of the molecular basis of nonobstructive hydronephrosis in humans. PMID:24509627

  6. Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight.

    PubMed

    Trujillo Viera, Jonathan; El-Merahbi, Rabih; 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

  7. 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

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

    PubMed

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

    2015-01-01

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

  9. Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus.

    PubMed

    Xin, Yu; Choo, Young Moo; Hu, Zhigang; Lee, Kwang Sik; Yoon, Hyung Joo; Cui, Zheng; Sohn, Hung Dae; Jin, Byung Rae

    2009-10-01

    Phospholipase A(2) (PLA(2)) is one of the main components of bee venom. Here, we identify a venom PLA(2) from the bumblebee, Bombus ignitus. Bumblebee venom PLA(2) (Bi-PLA(2)) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA(2) gene. Bi-PLA(2) is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA(2) (136 amino acids) possesses features consistent with other bee PLA(2)s, including ten conserved cysteine residues, as well as a highly conserved Ca(2+)-binding site and active site. Phylogenetic analysis of bee PLA(2)s separated the bumblebee and honeybee PLA(2) proteins into two groups. The mature Bi-PLA(2) purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA(2). Immunofluorescence staining of Bi-PLA(2)-treated insect Sf9 cells revealed that Bi-PLA(2) binds at the cell membrane and induces apoptotic cell death. PMID:19539776

  10. Emergence of a metalloproteinase / phospholipase A2 axis of systemic inflammation

    PubMed Central

    Fernandez-Patron, Carlos; Leung, Dickson

    2015-01-01

    We review select aspects of the biology of matrix metalloproteinases (MMPs) with a focus on the modulation of inflammatory responses by MMP-2. MMP-2 is a zinc- and calcium-dependent endoprotease with substrates including extracellular matrix proteins, vasoactive peptides and chemokines. Humans and mice with MMP-2 deficiency exhibit a predominantly inflammatory phenotype. Recent research shows that MMP-2 deficient mice display elevated activity of a secreted phospholipase A2 in the heart. Additionally, MMP-2 deficient mice exhibit abnormally high prostaglandin E2 levels in various organs (i.e., the heart, brain and liver), signs of inflammation and exacerbated lipopolysaccharide-induced fever. We briefly review the biology of sPLA2 enzymes to propose the existence of a heart-centric MMP-2/sPLA2 axis of systemic inflammation. Moreover, we postulate that PLA2 activation is induced by chemokines, whose ability to signal inflammation is regulated in a tissue-specific fashion by MMPs. Thus, genetic and pharmacologically induced MMP-deficiencies can be expected to perturb PLA2-mediated inflammatory mechanisms. PMID:26491703

  11. Identification of hyaluronidase and phospholipase B in Lachesis muta rhombeata venom.

    PubMed

    Wiezel, Gisele A; dos Santos, Patty K; Cordeiro, Francielle A; Bordon, Karla C F; Selistre-de-Araújo, Heloisa S; Ueberheide, Beatrix; Arantes, Eliane C

    2015-12-01

    Hyaluronidases contribute to local and systemic damages after envenoming, since they act as spreading factors cleaving the hyaluronan presents in the connective tissues of the victim, facilitating the diffusion of venom components. Although hyaluronidases are ubiquitous in snake venoms, they still have not been detected in transcriptomic analysis of the Lachesis venom gland and neither in the proteome of its venom performed previously. This work purified a hyaluronidase from Lachesis muta rhombeata venom whose molecular mass was estimated by SDS-PAGE to be 60 kDa. The hyaluronidase was more active at pH 6 and 37 °C when salt concentration was kept constant and more active in the presence of 0.15 M monovalent ions when the pH was kept at 6. Venom was fractionated by reversed-phase liquid chromatography (RPLC). Edman sequencing after RPLC failed to detect hyaluronidase, but identified a new serine proteinase isoform. The hyaluronidase was identified by mass spectrometry analysis of the protein bands in SDS-PAGE. Additionally, phospholipase B was identified for the first time in Lachesis genus venom. The discovery of new bioactive molecules might contribute to the design of novel drugs and biotechnology products as well as to development of more effective treatments against the envenoming. PMID:26335358

  12. Novel Translocation Responses of Cytosolic Phospholipase A2α Fluorescent Proteins

    PubMed Central

    Wooten, Rhonda E.; Willingham, Mark C.; Daniel, Larry W.; Leslie, Christina C.; Rogers, LeAnn C.; Sergeant, Susan; O’Flaherty, Joseph T.

    2008-01-01

    Cytosolic phospholipase A2 (cPLA2)α responds to the rise in cytosolic Ca2+ ([Ca2+]i) attending cell stimulation by moving to intracellular membranes, releasing arachidonic acid (AA) from these membranes, and thereby initiating the synthesis of various lipid mediators. Under some conditions, however, cPLA2α translocation occurs without any corresponding changes in [Ca2+]i. The signal for such responses has not been identified. Using confocal microscopy to track fluorescent proteins fused to cPLA2α or cPLA2α’s C2 domain, we find that AA mimics Ca2+ ionophores in stimulating cPLA2α translocations to the perinuclear ER and to a novel site, the lipid body. Unlike the ionophores, AA acted independently of [Ca2+]i rises and did not translocate the proteins to the Golgi. AA’s action did not involve its metabolism to eicosanoids or acylation into cellular lipids. Receptor agonists also stimulated translocations targeting lipid bodies. We propose that AA is a signal for Ca2+-independent cPLA2α translocation and that lipid bodies are common targets of cPLA2α and contributors to stimulus-induced lipid mediator synthesis. PMID:18406359

  13. Novel translocation responses of cytosolic phospholipase A2alpha fluorescent proteins.

    PubMed

    Wooten, Rhonda E; Willingham, Mark C; Daniel, Larry W; Leslie, Christina C; Rogers, LeAnn C; Sergeant, Susan; O'Flaherty, Joseph T

    2008-08-01

    Cytosolic phospholipase A2 (cPLA2)alpha responds to the rise in cytosolic Ca2+ ([Ca2+]i) attending cell stimulation by moving to intracellular membranes, releasing arachidonic acid (AA) from these membranes, and thereby initiating the synthesis of various lipid mediators. Under some conditions, however, cPLA2alpha translocation occurs without any corresponding changes in [Ca2+]i. The signal for such responses has not been identified. Using confocal microscopy to track fluorescent proteins fused to cPLA2alpha or cPLA2alpha's C2 domain, we find that AA mimics Ca2+ ionophores in stimulating cPLA(2)alpha translocations to the perinuclear ER and to a novel site, the lipid body. Unlike the ionophores, AA acted independently of [Ca2+](i) rises and did not translocate the proteins to the Golgi. AA's action did not involve its metabolism to eicosanoids or acylation into cellular lipids. Receptor agonists also stimulated translocations targeting lipid bodies. We propose that AA is a signal for Ca2+-independent cPLA2alpha translocation and that lipid bodies are common targets of cPLA2alpha and contributors to stimulus-induced lipid mediator synthesis. PMID:18406359

  14. Oxidative stress and redox regulation of phospholipase D in myocardial disease.

    PubMed

    Tappia, Paramjit S; Dent, Melissa R; Dhalla, Naranjan S

    2006-08-01

    Oxidative stress may be viewed as an imbalance between reactive oxygen species (ROS) and oxidant production and the state of glutathione redox buffer and antioxidant defense system. Recently, a new paradigm of redox signaling has emerged whereby ROS and oxidants can function as intracellular signaling molecules, where ROS- and oxidant-induced death signal is converted into a survival signal. It is now known that oxidative stress is involved in cardiac hypertrophy and in the pathogenesis of cardiomyopathies, ischemic heart disease and congestive heart failure. Phospholipase D (PLD) is an important signaling enzyme in mammalian cells, including cardiomyocytes. PLD catalyzes the hydrolysis of phosphatidylcholine to produce phosphatidic acid (PA). Two mammalian PLD isozymes, PLD1 and PLD2 have been identified, characterized and cloned. The importance of PA in heart function is evident from its ability to stimulate cardiac sarcolemmal membrane and sarcoplasmic reticular Ca2+-related transport systems and to increase the intracellular concentration of free Ca2+ in adult cardiomyocytes and augment cardiac contractile activity of the normal heart. In addition, PA is also considered an important signal transducer in cardiac hypertrophy. Accordingly, this review discusses a role for redox signaling mediated via PLD in ischemic preconditioning and examines how oxidative stress affects PLD in normal hearts and during different myocardial diseases. In addition, the review provides a comparative account on the regulation of PLD activities in vascular smooth muscle cells under conditions of oxidative stress. PMID:16843818

  15. 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

  16. Differential changes in phospholipase D and phosphatidate phosphohydrolase activities in ischemia-reperfusion of rat heart.

    PubMed

    Asemu, Girma; Dent, Melissa R; Singal, Tushi; Dhalla, Naranjan S; Tappia, Paramjit S

    2005-04-01

    Phospholipase D (PLD2) produces phosphatidic acid (PA), which is converted to 1,2 diacylglycerol (DAG) by phosphatidate phosphohydrolase (PAP2). Since PA and DAG regulate Ca(2+) movements, we examined PLD2 and PAP2 in the sarcolemma (SL) and sarcoplasmic reticular (SR) membranes from hearts subjected to ischemia and reperfusion (I-R). Although SL and SR PLD2 activities were unaltered after 30 min ischemia, 5 min reperfusion resulted in a 36% increase in SL PLD2 activity, whereas 30 min reperfusion resulted in a 30% decrease in SL PLD2 activity, as compared to the control value. SR PLD2 activity was decreased (39%) after 5 min reperfusion, but returned to control levels after 30 min reperfusion. Ischemia for 60 min resulted in depressed SL and SR PLD2 activities, characterized with reduced V(max) and increased K(m) values, which were not reversed during reperfusion. Although the SL PAP2 activity was decreased (31%) during ischemia and at 30 min reperfusion (28%), the SR PAP2 activity was unchanged after 30 min ischemia, but was decreased after 5 min reperfusion (25%) and almost completely recovered after 30 min reperfusion. A 60 min period of ischemia followed by reperfusion caused an irreversible depression of SL and SR PAP2 activities. Our results indicate that I-R induced cardiac dysfunction is associated with subcellular changes in PLD2 and PAP2 activities. PMID:15752718

  17. 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 myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes. PMID:25428992

  18. 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. PMID:15616193

  19. Purification and characterization of an anticoagulant phospholipase A(2) from Indian monocled cobra (Naja kaouthia) venom.

    PubMed

    Doley, Robin; Mukherjee, Ashis Kumar

    2003-01-01

    An anticoagulant, non-toxic phospholipase A(2) was isolated from the venom of Indian monocled cobra (Naja kaouthia) by a combination of ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. This purified protein named NK-PLA(2)-I, had a subunit molecular mass of 13.6 kDa and migrated as a dimer under non-reduced condition in SDS-PAGE. NK-PLA(2)-I was a highly thermostable protein requiring basic pH optima for its catalytic activity and showed preferential hydrolysis of phosphotidylcholine. This protein exhibited higher anticoagulant, indirect hemolysis, liver and heart tissue damaging activity but exerted less toxicity, direct hemolysis, edema and lung tissue damaging activity as compared to whole venom. Treatment of NK-PLA(2)-I with rho-BPB, TPCK, PMSF, antivenom and heating had almost equal effect on PLA(2), and other pharmacological properties except in vitro tissue damaging activity. Current investigation provides a fairly good indication that NK-PLA(2)-I induces various pharmacological effects by mechanisms, which are either dependent or independent of its catalytic activity. PMID:12467665

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

    PubMed

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

    2014-07-01

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

  1. Kinetic Scaffolding Mediated by a Phospholipase C–β and Gq Signaling Complex

    PubMed Central

    Waldo, Gary L.; Ricks, Tiffany K.; Hicks, Stephanie N.; Cheever, Matthew L.; Kawano, Takeharu; Tsuboi, Kazuhito; Wang, Xiaoyue; Montell, Craig; Kozasa, Tohru; Sondek, John; Harden, T. Kendall

    2011-01-01

    Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)–dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide–binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gαq reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein–dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gαq, causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs. PMID:20966218

  2. Promoter analysis and expression of a phospholipase D gene from castor bean.

    PubMed Central

    Xu, L; Zheng, S; Zheng, L; Wang, X

    1997-01-01

    The expression of a castor bean (Ricinus communis L.) phospholipase D (PLD; EC 3.1.4.4) gene has been studied by examining its promoter activity in transgenic tobacco (Nicotiana tabacum) carrying a PLD promoter-glucuronidase transgene and by monitoring the levels of PLD mRNA in castor bean. Sequence and the 5' truncation analyses revealed that the 5' flanking region from nucleotide -1200 to -730 is required for the regulation and basal function of the PLD promoter. The PLD promoter in vegetative tissues is highly active in the rapidly growing regions such as the shoot apex and the secondary meristem producing axillary buds and vascular tissues of young leaves and stems. The PLD promoter activity in floral tissues was high in stigma, ovary, and pollen grains, but low in petals, sepals, the epidermis of anthers, styles, and filaments. The PLD promoter activity was enhanced by abscisic acid. Northern-blot analysis of PLD in castor bean showed that the PLD mRNA levels were high in young and metabolically more active tissues such as expanding leaves, hypocotyl hooks, developing seeds, and young seedlings, and they decreased in mature tissues such as fully expanded leaves and developed seeds. These patterns of expression suggest a role of PLD in rapid cell growth, proliferation, and reproduction. PMID:9342861

  3. Inhibition of phospholipase A/sub 2/ from human plasma by sodium bisulfite

    SciTech Connect

    Wiggins, C.W.; Franson, R.C.

    1987-05-01

    The anti-oxidant sodium bisulfite has been shown to inhibit acid active(lysosomal), non-Ca/sup + +/-dependent phospholipase A/sub 2/ (PLA/sub 2/), and to interact reversibly with unsaturated fatty acids, altering their chromatographic mobility. The authors examined the effect of bisulfite on neutral active, Ca/sup + +/-dependent PLA/sub 2/ from human plasma. Using (1-/sup 14/C)oleate-labelled autoclaved E. coli as substrate, PLA/sub 2/ activity was inhibited in a dose-dependent manner by bisulfite. Maximal inhibition occurred at 100..mu..M bisulfite. Preincubation of plasma for 0-30 minutes with bisulfite resulted in a time-dependent increase in PLA/sub 2/ inhibition. Preincubation of substrate with bisulfite had no such effect. When the plasma PLA/sub 2/ was purified 25-fold by SP-Sephadex chromatography it was no longer inhibited by bisulfite. The SP-Sephadex wash through fraction, which contained greater than 95% of the applied protein but not PLA/sub 2/ activity, did not inhibit the purified enzyme. When incubated with bisulfite however, the SP-wash through fraction produced dose-dependent inhibition of the purified enzyme. These results indicate that sodium bisulfite inhibits human plasma PLA/sub 2/, in vitro, indirectly by interaction with a factor(s) present in plasma and suggests that anti-oxidants may similarly influence expression of extracellular PLA/sub 2/ in vivo.

  4. 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

  5. Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

    PubMed Central

    Plihtari, Riia; Hurt-Camejo, Eva; Öörni, Katariina; Kovanen, Petri T.

    2010-01-01

    LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A2 (sPLA2-IIa and sPLA2-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA2-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA2-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA2-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis. PMID:20124257

  6. MALDI-TOF MS to monitor the kinetics of phospholipase A2-digestion of oxidized phospholipids.

    PubMed

    Schröter, Jenny; Süß, Rosmarie; Schiller, Jürgen

    2016-07-15

    Free fatty acids (FFA) are released through phospholipase A2 (PLA2), which cleaves the fatty acyl residue at the sn-2 position of phospholipids (PL). During inflammatory diseases, reactive oxygen species (such as HOCl) lead to the formation of oxidatively modified PL (e.g., chlorohydrin generation). It is still widely unknown to which extent the oxidation of PL influences their digestibility by PLA2. Additionally, investigations on the impact of the position of the unsaturated fatty acyl residue (sn-1 versus sn-2 position) and modifications of the headgroup (for instance phosphatidylcholine (PC) versus phosphatidylethanolamine (PE)) are also lacking. Therefore, the aim of this study is the investigation of these aspects using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to elucidate the PL/lysophospholipid (LPL) ratios as measures of the PLA2 digestibility. We will show that oxidative modifications of PL by HOCl have a considerable impact on the PLA2 digestibility, i.e., oxidation of the unsaturated fatty acyl residues leads to a reduced digestibility of both PC and PE. Besides, it will be shown that MALDI MS is a convenient and reliable tool to investigate the related changes. PMID:26721598

  7. Phosphatidylcholine enrichment with medium chain fatty acids by immobilized phospholipase A(1) -catalyzed acidolysis.

    PubMed

    Ochoa, Angélica A; Hernández-Becerra, Josafat A; Cavazos-Garduño, Adriana; García, Hugo S; Vernon-Carter, Eduardo J

    2013-01-01

    Phospholipids are a biologically and industrially important class of compounds whose physical properties can be improved for diverse applications by substitution of medium-chain fatty acids for their native fatty acid chains. In this study, phosphatidylcholine (PC) was enriched with medium-chain fatty acids (MCFAs) by acidolysis with phospholipase A(1) (PLA(1) ) immobilized on Duolite A568. Response surface methodology was employed to evaluate the effects of the molar ratio of substrates (PC to free MCFAs), enzyme loading, and reaction temperature on the incorporation of free MCFAs into PC and on PC recovery. Enzyme loading and molar ratio of substrates contributed positively, but temperature negatively, to the incorporation of free MCFAs into PC. Increases in enzyme loading and the molar ratio of PC to free MCFAs led to increased incorporation of the latter into the former, but increased temperature had the opposite effect. By contrast, an increase in enzyme loading led to decreased PC recovery. Increased temperature had also a negative effect on PC recovery. Optimal conditions for maximum incorporation and PC recovery were molar ratio of PC to free MCFAs of 1:16, enzyme loading of 16%, and 50°C. Under these conditions, the incorporation of free MCFAs was 41% and the PC recovery was 53%. PMID:23074091

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

    SciTech Connect

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

    1986-03-05

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

  9. Varespladib inhibits secretory phospholipase A2 in bronchoalveolar lavage of different types of neonatal lung injury.

    PubMed

    De Luca, Daniele; Minucci, Angelo; Trias, Joaquim; Tripodi, Domenico; Conti, Giorgio; Zuppi, Cecilia; Capoluongo, Ettore

    2012-05-01

    Secretory phospholipase A2 (sPLA2), which links surfactant catabolism and lung inflammation, is associated with lung stiffness, surfactant dysfunction, and degree of respiratory support in acute respiratory distress syndrome and in some forms of neonatal lung injury. Varespladib potently inhibits sPLA2 in animal models. The authors investigate varespladib ex vivo efficacy in different forms of neonatal lung injury. Bronchoalveolar lavage fluid was obtained from 40 neonates affected by hyaline membrane disease, infections, or meconium aspiration and divided in 4 aliquots added with increasing varespladib or saline. sPLA2 activity, proteins, and albumin were measured. Dilution was corrected with the urea ratio. Varespladib was also tested in vitro against pancreatic sPLA2 mixed with different albumin concentration. Varespladib was able to inhibit sPLA2 in the types of neonatal lung injury investigated. sPLA2 activity was reduced in hyaline membrane disease (P < .0001), infections (P = .003), and meconium aspiration (P = .04) using 40 µM varespladib; 10 µM was able to lower enzyme activity (P = .001), with an IC(50) of 87 µM. An inverse relationship existed between protein level and activity reduction (r = 0.5; P = .029). The activity reduction/protein ratio tended to be higher in hyaline membrane disease. Varespladib efficacy was higher in vitro than in lavage fluids obtained from neonates (P < .001). PMID:21602519

  10. The Molecular Basis of Leukocyte Adhesion Involving Phosphatidic Acid and Phospholipase D*

    PubMed Central

    Speranza, Francis; Mahankali, Madhu; Henkels, Karen M.; Gomez-Cambronero, Julian

    2014-01-01

    Defining how leukocytes adhere to solid surfaces, such as capillary beds, and the subsequent migration through the extracellular matrix, is a central biological issue. We show here that phospholipase D (PLD) and its enzymatic reaction product, phosphatidic acid (PA), regulate cell adhesion of immune cells (macrophages and neutrophils) to collagen and have defined the underlying molecular mechanism in a spatio-temporal manner that coincides with PLD activity timing. A rapid (t½ = 4 min) and transient activation of the PLD1 isoform occurs upon adhesion, and a slower (t½ = 7.5 min) but prolonged (>30 min) activation occurs for PLD2. Importantly, PA directly binds to actin-related protein 3 (Arp3) at EC50 = 22 nm, whereas control phosphatidylcholine did not bind. PA-activated Arp3 hastens actin nucleation with a kinetics of t½ = 3 min at 300 nm (compared with controls of no PA, t½ = 5 min). Thus, PLD and PA are intrinsic components of cell adhesion, which reinforce each other in a positive feedback loop and react from cues from their respective solid substrates. In nascent adhesion, PLD1 is key, whereas a sustained adhesion in mature or established focal points is dependent upon PLD2, PA, and Arp3. A prolonged adhesion could effectively counteract the reversible intrinsic nature of this cellular process and constitute a key player in chronic inflammation. PMID:25187519

  11. 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).

  12. Cytosolic phospholipase A{sub 2} gene in human and rat: Chromosomal localization and polymorphic markers

    SciTech Connect

    Tay, A.; Simon, J.S.; Jacob, H.J.

    1995-03-01

    The authors report the chromosomal localization and a simple sequence repeat (SSR) in the cytosolic phospholipase A{sub 2} (cPLA{sub 2}) gene in both human and rat. A (CA){sub 18} repeat in the promoter of the rat gene was determined to exhibit length polymorphism when analyzed using the polymerase chain reaction (PCR) in 19 different inbred rat strains. Genotyping for this marker in 234 F{sub 2} progeny of a SHRXBN intercross mapped the gene to rat chromosome 13. Using a PCR strategy, a fragment of the promoter for the human gene was isolated, and a (CA){sub 18} repeat was identified. Since this marker displayed a low heterozygosity index, they also identified a mononucleotide repeat in the promoter for cPLA{sub 2} that displayed a polymorphism information content value of 0.76. The human gene was mapped using fluorescence in situ hybridization (FISH) to chromosome 1q25. Of interest, the gene encoding the enzyme prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2), which acts on the arachidonic acid product of cPLA{sub 2}, was previously localized to this same chromosomal region, raising the possibility of coordinate regulation. Identification of intragenic markers may facilitate studies of polymorphic variants of these genes as candidates for disorders in which perturbations of the eicosanoid cascade may play a role. 20 refs., 3 figs., 2 tabs.

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

    PubMed Central

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

    2015-01-01

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

  14. 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

  15. A facile assay to monitor secretory phospholipase A₂ using 8-anilino-1-naphthalenesulfonic acid.

    PubMed

    Vivek, Hamse K; Swamy, Supritha G; Priya, Babu S; Sethi, Gautam; Rangappa, Kanchugarakoppal S; Swamy, S Nanjunda

    2014-09-15

    Secretory phospholipases A2 (sPLA2s) are present in snake venoms, serum, and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Lipid mediators in the inflammatory processes have potential value for controlling phospholipid metabolism through sPLA2 inhibition. Thus, it demands the need for screening of potential leads for sPLA2 inhibition. To date, sPLA2 activity has been assayed using expensive radioactive or chromogenic substrates, thereby limiting a large number of assays. In this study, a simple and sensitive NanoDrop assay was developed using non-fluorogenic and non-chromogenic phospholipid substrate 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 8-anilino-1-naphthalenesulfonic acid (ANS) as interfacial hydrophobic probe. The modified assay required a 10ng concentration of sPLA2. ANS, as a strong anion, binds predominantly to cationic group of choline head of DMPC through ion pair formation, imparting hydrophobicity and lipophilicity and resulting in an increase in fluorescence. Triton X-100 imparts correct geometrical space during sPLA2 catalyzing DMPC, releasing lysophospholipid and acidic myristoyl acid, which in turn alters the hydrophobic environment prevailing around ANS-DMPC, which leads to weakening of the electrostatic ion pair interaction between DMPC and ANS ensuing decrease in fluorescence. These characteristic fluorescence changes between DMPC and ANS in response to sPLA2 catalysis are well documented and validated in this study. PMID:24915638

  16. Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

    PubMed Central

    Bennett, C F; Chiang, M Y; Wilson-Lingardo, L; Wyatt, J R

    1994-01-01

    Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner. PMID:8065936

  17. Phospholipase A2 up-regulation during mycorrhiza formation in Tuber borchii.

    PubMed

    Miozzi, Laura; Balestrini, Raffaella; Bolchi, Angelo; Novero, Mara; Ottonello, Simone; Bonfante, Paola

    2005-07-01

    TbSP1 is a secreted and surface-associated phospholipase A(2) previously found to be up-regulated in C- or N-deprived free-living mycelia from the ectomycorrhizal ascomycete Tuber borchii. As nutrient limitation is considered an important environmental factor favouring the transition to symbiotic status, TbSP1 was suggested to be involved in the formation of mycorrhizas. An in vitro symbiosis system between Cistus incanus and T. borchii was set up: TbSP1 mRNA levels in free-living mycelia and in mycorrhizas sampled in different districts of the plant-fungus interaction were examined. In the same samples, TbSP1 protein expression was analysed by immunoelectron microscopy. A substantially enhanced TbSP1 mRNA expression, compared with nutrient-limited but free-living mycelia, was detected in the presence of the plant and reached maximal levels in fully developed mycorrhizas. A similar expression trend was revealed by immunolocalization experiments. We have shown that TbSP1 appears to respond to two partially overlapping yet distinct stimuli: nutrient starvation and mycorrhiza formation. PMID:15948845

  18. Tumor suppressor role of phospholipase Cε in Ras-triggered cancers

    PubMed Central

    Martins, Marta; McCarthy, Afshan; Baxendale, Rhona; Guichard, Sabrina; Magno, Lorenza; Kessaris, Nicoletta; El-Bahrawy, Mona; Yu, Philipp; Katan, Matilda

    2014-01-01

    Phospholipase Cε (PLCε) has been characterized as a direct effector of Ras in vitro and in cellular systems; however, the role of PLCε in tumorigenesis and its link to Ras in this context remain unclear. To assess the role of PLCε in Ras-driven cancers, we generated two new mouse strains: one carrying a targeted deletion of Plce (Plce−/−) and the other carrying mutant alleles of Plce unable to bind to Ras (PlceRAm/RAm). The Plce−/− and, to a lesser degree, PlceRAm/RAm transgenic mice exhibited increased susceptibility to tumor formation in the two-stage skin carcinogenesis protocol, revealing a tumor suppressor function for this PLC. This result also suggests that in this context Ras binding in part regulates functions of PLCε. Although significant differences were not seen in the LSL-KrasG12D nonsmall cell lung carcinoma model, down-regulation of PLCε was found in animal tumors and in cellular systems following expression of the oncogenic Ras. An inhibitory impact of PLCε on cell growth requires intact lipase activity and is likely mediated by protein kinase C enzymes. Further cellular studies suggest involvement of histone deacetylase in the mechanism of PLCε down-regulation. Taken together, our results show a previously unidentified tumor suppressor role for this PLC in animal models and, together with observations of marked down-regulation in colorectal, lung, and skin tumors, suggest its use as a biological marker in cancer. PMID:24591640

  19. The expression of phospholipase A2 group X is inversely associated with metastasis in colorectal cancer

    PubMed Central

    HIYOSHI, MASAYA; KITAYAMA, JOJI; KAZAMA, SHINSUKE; TAKETOMI, YOSHITAKA; MURAKAMI, MAKOTO; TSUNO, NELSON H.; HONGO, KUMIKO; KANEKO, MANABU; SUNAMI, EIJI; WATANABE, TOSHIAKI

    2013-01-01

    Among the secretory phospholipase A2s (sPLA2), sPLA2 group X (PLA2GX) has the most potent hydrolyzing activity toward phosphatidylcholine, and has recently been shown to be implicated in chronic inflammatory diseases. The aim of the present study was to investigate PLA2GX expression in colorectal cancer (CRC) and its correlation with patient clinicopathological features. The present study comprises a series of 158 patients who underwent surgical resection for primary CRC. PLA2GX expression in CRC tissues was examined by immunohistochemistry and compared with patient clinicopathological findings and survival. A total of 64% of the tumors expressed PLA2GX at high levels. Statistical analysis revealed that PLA2GX expression was inversely correlated with hematogenous metastasis (P=0.005). In the subgroup analysis, left-sided tumors with high PLA2GX expression showed an inverse correlation with lymph node metastasis (P=0.018) and hematogenous metastasis (P=0.017). Patients with high PLA2GX expression tended to have a longer disease-specific survival compared with those with low PLA2GX expression in left-sided, but not right-sided, CRC (P=0.08). In light of the present results, we suggest that PLA2GX has an inhibitory effect on the progression of CRC. PMID:23420493

  20. Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity.

    PubMed

    Chemburu, Sireesha; Ji, Eunkyung; Casana, Yosune; Wu, Yang; Buranda, Tione; Schanze, Kirk S; Lopez, Gabriel P; Whitten, David G

    2008-11-20

    A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range. PMID:18808092

  1. On-Tissue Phospholipase C Digestion for Enhanced MALDI-MS Imaging of Neutral Glycosphingolipids.

    PubMed

    Vens-Cappell, Simeon; Kouzel, Ivan U; Kettling, Hans; Soltwisch, Jens; Bauwens, Andreas; Porubsky, Stefan; Müthing, Johannes; Dreisewerd, Klaus

    2016-06-01

    Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here, we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections. The ion signal intensities of mono-, di-, and oligohexosylceramides were enhanced by up to 10-fold. In addition, visualization of Shiga toxin receptor globotriaosylceramide (Gb3Cer) in the kidneys of wild-type and α-galactosidase A-knockout (Fabry) mice was possible at about ten micrometer resolution. Importantly, the PLC treatment did not decrease the high lateral resolution of the MS imaging analysis. PMID:27212679

  2. Kinetic Analysis of Phospholipase C from Catharanthus roseus Transformed Roots Using Different Assays1

    PubMed Central

    Hernández-Sotomayor, S.M. Teresa; De Los Santos-Briones, César; Muñoz-Sánchez, J. Armando; Loyola-Vargas, Victor M.

    1999-01-01

    The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [33P]phosphatidylinositol 4,5-bisphosphate (PIP2) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP2 hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP2 vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP2 and PIP2 surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 μm. These findings will add to our understanding of the mechanisms of regulation of plant PLC. PMID:10444091

  3. Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography.

    PubMed

    Tran, Benjamin; Grosskopf, Vanessa; Wang, Xiangdan; Yang, Jihong; Walker, Don; Yu, Christopher; McDonald, Paul

    2016-03-18

    Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies. PMID:26896920

  4. The role of group IIF-secreted phospholipase A2 in epidermal homeostasis and hyperplasia

    PubMed Central

    Yamamoto, Kei; Miki, Yoshimi; Sato, Mariko; Taketomi, Yoshitaka; Nishito, Yasumasa; Taya, Choji; Muramatsu, Kazuaki; Ikeda, Kazutaka; Nakanishi, Hiroki; Taguchi, Ryo; Kambe, Naotomo; Kabashima, Kenji; Lambeau, Gérard; Gelb, Michael H.

    2015-01-01

    Epidermal lipids are important for skin homeostasis. However, the entire picture of the roles of lipids, particularly nonceramide lipid species, in epidermal biology still remains obscure. Here, we report that PLA2G2F, a functionally orphan-secreted phospholipase A2 expressed in the suprabasal epidermis, regulates skin homeostasis and hyperplasic disorders. Pla2g2f−/− mice had a fragile stratum corneum and were strikingly protected from psoriasis, contact dermatitis, and skin cancer. Conversely, Pla2g2f-overexpressing transgenic mice displayed psoriasis-like epidermal hyperplasia. Primary keratinocytes from Pla2g2f−/− mice showed defective differentiation and activation. PLA2G2F was induced by calcium or IL-22 in keratinocytes and preferentially hydrolyzed ethanolamine plasmalogen-bearing docosahexaenoic acid secreted from keratinocytes to give rise to unique bioactive lipids (i.e., protectin D1 and 9S-hydroxyoctadecadienoic acid) that were distinct from canonical arachidonate metabolites (prostaglandins and leukotrienes). Ethanolamine lysoplasmalogen, a PLA2G2F-derived marker product, rescued defective activation of Pla2g2f−/− keratinocytes both in vitro and in vivo. Our results highlight PLA2G2F as a previously unrecognized regulator of skin pathophysiology and point to this enzyme as a novel drug target for epidermal-hyperplasic diseases. PMID:26438362

  5. Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex Trunculus digestive cells

    PubMed Central

    2011-01-01

    Background Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. Results The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. Conclusion The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids. PMID:21631952

  6. Knockout of phospholipase Cε attenuates N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder tumorigenesis

    PubMed Central

    JIANG, TAIMAO; LIU, TAO; LI, LIN; YANG, ZHIJUN; BAI, YUNFENG; LIU, DONGYE; KONG, CHUIZE

    2016-01-01

    Bladder cancer frequently shows mutational activation of the oncogene Ras, which is associated with bladder carcinogenesis. However, the signaling pathway downstream of Ras remains to be fully elucidated. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) is able to induce bladder cancer by driving the clonal expansion of initiated cells carrying the activated form of Ras. Phospholipase Cε (PLCε) is the main target of BBN, while the tumor promoting role of PLCε remains controversial. The present study examined the role of PLCε in BBN-induced bladder carcinogenesis of mice with genetically inactivated PLCε. Using light and electron microscopy, the present study demonstrated that PLCε−/− mice were resistant to BBN-induced bladder carcinogenesis. Furthermore, it was demonstrated that cyclooxygenase 2 and vascular endothelial growth factor-A were affected by the PLCε background of the mice, suggesting that the role of PLCε in tumor promotion may be ascribed to augmentation of inflammatory responses and angiogenesis. These results indicated that PLCε is crucial for BBN-induced bladder carcinogenesis as well as signaling downstream of Ras, and that PLCε is a candidate molecular target for the development of anti-cancer drugs. PMID:26782701

  7. Phospholipase D is a target for inhibition of astroglial proliferation by ethanol.

    PubMed

    Burkhardt, Ute; Wojcik, Bartosch; Zimmermann, Martina; Klein, Jochen

    2014-04-01

    The proliferation of astrocytes during early brain development is driven by growth factors and is accompanied by the activation of phospholipase D (PLD). Ethanol disrupts PLD signaling in astrocytes, a process which may contribute to delayed brain growth of fetuses exposed to alcohol during pregnancy. We here report that insulin-like growth factor 1 (IGF-1) is a strong mitogen for rat astrocytes (EC50 0.2 μg/ml) and a strong stimulator of astroglial PLD activity; both effects are inhibited by ethanol and 1-butanol, but not t-butanol, suggesting participation of PLD. Downregulation of PLD1 and exposure to the PLD1 inhibitor VU0359595 attenuated PLD activity and strongly reduced the mitogenic activity of serum and IGF-1. The PLD2 inhibitor VU0285655-1 also reduced PLD activity but had lesser effects on IGF-1-driven proliferation. PLD2 down-regulation affected serum - but not IGF-1-induced proliferation. In separate experiments, alcohol treatment of murine astrocytes taken from PLD-deficient animals revealed an insensitivity of PLD1(-/-) cells to 1-butanol whereas PLD2(-/-) cells were not affected. We conclude that astroglial proliferation induced by IGF-1 is critically dependent on the PLD signaling pathway, with a stronger contribution from PLD1 than PLD2. The teratogenic effects of ethanol may be explained, at least in part, by disruption of the IGF1-PLD signaling pathway. PMID:24262632

  8. Characterization of opiate receptor heterogeneity using affinity ligands and phospholipase A/sub 2/

    SciTech Connect

    Reichman, M.

    1985-01-01

    The primary aim of the dissertation was to study the heterogeneity of opiate receptors by utilizing affinity ligands, and by modification of the receptor lipid-microenvironment with phospholipase A/sub 2/ (PLA/sub 2/). The affinity ligands, 14-bromacetamidomorphine (BAM) and 14-chloroacetylnaltrexone (CAN), selectively inactivated high affinity dihydromorphine binding sites in an apparently irreversible manner (the inhibition was resistant to extensive washes of treated neural membrane homogenates). The inhibitory effect of PLA/sub 2/ (10 ng/ml) on opiate receptor subtypes was determined using (/sup 3/H)-dihydromorphine (..mu..-type agonist), (/sup 3/H)-enkephalin (delta agonist) and (/sup 3/H)-naloxone (..mu.. antagonist). PLA/sub 2/ abolished the high affinity antagonist binding site, whereas it inhibited high and low affinity agonist binding sites similarly. The results suggest that high affinity antagonist binding sites are different from high affinity agonist binding sites. Indirect binding assays demonstrated that the selectivities of ..mu..- and delta receptors are not affected significantly by PLA/sub 2/ treatment.

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

    PubMed Central

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

    2015-01-01

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

  10. An Effector Domain Mutant of Arf6 Implicates Phospholipase D in Endosomal Membrane RecyclingD⃞

    PubMed Central

    Jovanovic, Olivera A.; Brown, Fraser D.; Donaldson, Julie G.

    2006-01-01

    In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment. PMID:16280360

  11. Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages.

    PubMed

    Setúbal, S S; Pontes, A S; Furtado, J L; Xavier, C V; Silva, F L; Kayano, A M; Izidoro, L F M; Soares, A M; Calderon, L A; Stábeli, R G; Zuliani, J P

    2013-02-01

    The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A2 (PLA2), and BaltTX-II, an Asp49 catalytically active PLA2 isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA2 BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA2, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA2s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages. PMID:23581990

  12. Phospholipase D affects translocation of NPR1 to the nucleus in Arabidopsis thaliana

    PubMed Central

    Janda, Martin; Šašek, Vladimír; Chmelařová, Hana; Andrejch, Jan; Nováková, Miroslava; Hajšlová, Jana; Burketová, Lenka; Valentová, Olga

    2015-01-01

    Phytohormone salicylic acid (SA) is a crucial component of plant-induced defense against biotrophic pathogens. Although the key players of the SA pathway are known, there are still gaps in the understanding of the molecular mechanism and the regulation of particular steps. In our previous research, we showed in Arabidopsis suspension cells that n-butanol, which specifically modulates phospholipase D activity, significantly suppresses the transcription of the pathogenesis related (PR-1) gene, which is generally accepted as the SA pathway marker. In the presented study, we have investigated the site of n-butanol action in the SA pathway. We were able to show in Arabidopsis plants treated with SA that n-butanol inhibits the transcription of defense genes (PR-1, WRKY38). Fluorescence microscopy of Arabidopsis thaliana mutants expressing 35S::NPR1-GFP (nonexpressor pathogenesis related 1) revealed significantly decreased nuclear localization of NPR1 in the presence of n-butanol. On the other hand, n-butanol did not decrease the nuclear localization of NPR1 in 35S::npr1C82A-GFP and 35S::npr1C216A-GFP mutants constitutively expressing NPR1 monomers. Mass spectrometric analysis of plant extracts showed that n-butanol significantly changes the metabolic fingerprinting while t-butanol had no effect. We found groups of the plant metabolites, influenced differently by SA and n-butanol treatment. Thus, we proposed several metabolites as markers for n-butanol action. PMID:25741350

  13. Structural and phylogenetic basis for the classification of group III phospholipase A2.

    PubMed

    Hariprasad, Gururao; Srinivasan, Alagiri; Singh, Reema

    2013-09-01

    Secretory phospholipase A2 (PLA2) catalyses the hydrolysis of the sn-2 position of glycerophospholipids to liberate arachidonic acid, a precursor of eicosanoids, that are known mediators of inflammation. The group III PLA2 enzymes are present in a wide array of organisms across many species with completely different functions. A detailed understanding of the structure and evolutionary proximity amongst the enzymes was carried out for a meaningful classification of this group. Fifty protein sequences from different species of the group were considered for a detailed sequence, structural and phylogenetic studies. In addition to the conservation of calcium binding motif and the catalytic histidine, the sequences exhibit specific 'amino acid signatures'. Structural analysis reveals that these enzymes have a conserved globular structure with species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C-terminal domain and the quaternary conformational state. Character and distance based phylogenetic analysis of these sequences are in accordance with the structural features. The outcomes of the structural and phylogenetic analysis lays a convincing platform for the classification the group III PLA2s into (1A) venomous insects; (IB) non-venomous insects; (II) mammals; (IIIA) gila monsters; (IIIB) reptiles, amphibians, fishes, sea anemones and liver fluke, and (IV) scorpions. This classification also helps to understand structure-function relationship, enzyme-substrate specificity and designing of potent inhibitors against the drug target isoforms. PMID:23793742

  14. Modulation of Insulin Sensitivity of Hepatocytes by the Pharmacological Downregulation of Phospholipase D

    PubMed Central

    Babenko, Nataliya A.; Kharchenko, Vitalina S.

    2015-01-01

    Background. The role of phospholipase D (PLD) as a positive modulator of glucose uptake activation by insulin in muscle and adipose cells has been demonstrated. The role of PLD in the regulation of glucose metabolism by insulin in the primary hepatocytes has been determined in this study. Methods. For this purpose, we studied effects of inhibitors of PLD on glucose uptake and glycogen synthesis stimulation by insulin. To determine the PLD activity, the method based on determination of products of transphosphatidylation reaction, phosphatidylethanol or phosphatidylbutanol, was used. Results. Inhibition of PLD by a general antagonist (1-butanol) or specific inhibitor, halopemide, or N-hexanoylsphingosine, or by cellular ceramides accumulated in doxorubicin-treated hepatocytes decreased insulin-stimulated glucose metabolism. Doxorubicin-induced hepatocytes resistance to insulin action could be abolished by inhibition of ceramide production. Halopemide could nullify this effect. Addition of propranolol, as well as inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) (wortmannin, LY294002) or suppressors of Akt phosphorylation/activity, luteolin-7-O-glucoside or apigenin-7-O-glucoside, to the culture media could block cell response to insulin action. Conclusion. PLD plays an important role in the insulin signaling in the hepatocytes. PLD is activated downstream of PI3-kinase and Akt and is highly sensitive to ceramide content in the liver cells. PMID:26089893

  15. 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

    2016-07-01

    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. PMID:27220631

  16. The role of group IIF-secreted phospholipase A2 in epidermal homeostasis and hyperplasia.

    PubMed

    Yamamoto, Kei; Miki, Yoshimi; Sato, Mariko; Taketomi, Yoshitaka; Nishito, Yasumasa; Taya, Choji; Muramatsu, Kazuaki; Ikeda, Kazutaka; Nakanishi, Hiroki; Taguchi, Ryo; Kambe, Naotomo; Kabashima, Kenji; Lambeau, Gérard; Gelb, Michael H; Murakami, Makoto

    2015-10-19

    Epidermal lipids are important for skin homeostasis. However, the entire picture of the roles of lipids, particularly nonceramide lipid species, in epidermal biology still remains obscure. Here, we report that PLA2G2F, a functionally orphan-secreted phospholipase A2 expressed in the suprabasal epidermis, regulates skin homeostasis and hyperplasic disorders. Pla2g2f(-/-) mice had a fragile stratum corneum and were strikingly protected from psoriasis, contact dermatitis, and skin cancer. Conversely, Pla2g2f-overexpressing transgenic mice displayed psoriasis-like epidermal hyperplasia. Primary keratinocytes from Pla2g2f(-) (/-) mice showed defective differentiation and activation. PLA2G2F was induced by calcium or IL-22 in keratinocytes and preferentially hydrolyzed ethanolamine plasmalogen-bearing docosahexaenoic acid secreted from keratinocytes to give rise to unique bioactive lipids (i.e., protectin D1 and 9S-hydroxyoctadecadienoic acid) that were distinct from canonical arachidonate metabolites (prostaglandins and leukotrienes). Ethanolamine lysoplasmalogen, a PLA2G2F-derived marker product, rescued defective activation of Pla2g2f(-/-) keratinocytes both in vitro and in vivo. Our results highlight PLA2G2F as a previously unrecognized regulator of skin pathophysiology and point to this enzyme as a novel drug target for epidermal-hyperplasic diseases. PMID:26438362

  17. Structure of a king cobra phospholipase A2 determined from a hemihedrally twinned crystal.

    PubMed

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

    2003-09-01

    An acidic PLA(2) (OH APLA(2)-II) from the venom of Ophiophagus hannah (king cobra) shows greater phospholipase A(2) activity and weaker cardiotoxic and myotoxic activity than a homologous acidic PLA(2) from the same venom. The crystal of the enzyme belongs to space group P6(3). The crystals are invariably hemihedrally twinned, exhibiting perfect 622 Laue symmetry. The structure was determined by molecular replacement and refined using a hemihedral twinning program at 2.1 A resolution. The final model has reasonable stereochemistry and a crystallographic R factor of 19.5% (R(free) = 21.5%). The structure reveals the molecular arrangement and the mode of twinning. There are six independent molecules in the asymmetric unit. Owing to the presence of a non-crystallographic twofold parallel to the hemihedral twinning twofold, the molecular packing in the twinned crystal is extremely similar to that in an untwinned crystal for four of the molecules. This unique molecular arrangement may be related to the difficulty in recognizing the twinning. The structure was compared with the previously determined structure of a homologous acidic PLA(2) from the same source. The comparison shows structural changes that might be implicated in the increased catalytic activity and weakened toxicity. PMID:12925787

  18. 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. PMID:24802400

  19. 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.

  20. 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

  1. Regulation of macrophage differentiation and polarization by group IVC phospholipase A₂.

    PubMed

    Ishihara, Keiichi; Kuroda, Asuka; Sugihara, Kanako; Kanai, Shiho; Nabe, Takeshi; Akiba, Satoshi

    2011-12-16

    Although the cellular function of group IVC phospholipase A(2) (IVC-PLA(2)) remains to be understood, the expression of IVC-PLA(2) in human monocytic THP-1 cells was increased during phorbol ester-induced macrophage differentiation. We therefore examined the role of IVC-PLA(2) in macrophage differentiation using THP-1 cells. Two THP-1 cell lines stably expressing IVC-PLA(2)-specific shRNA were established. Differentiation and maturation into macrophages on treatment with phorbol ester were facilitated by knockdown of IVC-PLA(2) without the compensatory induction of mRNA expression for other group IV and VI PLA(2)s. Furthermore, the enhancement of macrophage differentiation by IVC-PLA(2)-knockdown were abolished by treatment with lysophosphatidylcholine, a metabolite of phospholipids generated by PLA(2)-mediated hydrolysis, indicating that PLA(2) activity is necessary for the inhibition of macrophage differentiation by IVC-PLA(2). Additionally, we found that the differentiation into classically activated M1 macrophage was superior in IVC-PLA(2)-knockdown cells, whereas the differentiation into alternatively activated M2 macrophage was suppressed by IVC-PLA(2)-knockdown. These findings suggest that IVC-PLA(2) is involved in regulations of macrophage differentiation and macrophage polarization. PMID:22108055

  2. Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling.

    PubMed

    Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I; Nienhaus, G Ulrich; Gierschik, Peter

    2015-07-10

    The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca(2+). The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca(2+) and regulation of Ca(2+)-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca(2+) release from intracellular stores; (iii) Ca(2+) entry from the extracellular compartment; and (iv) nuclear translocation of the Ca(2+)-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca(2+) signaling. PMID:25903139

  3. Potent and selective fluoroketone inhibitors of group VIA calcium-independent phospholipase A2.

    PubMed

    Kokotos, George; Hsu, Yuan-Hao; Burke, John E; Baskakis, Constantinos; Kokotos, Christoforos G; Magrioti, Victoria; Dennis, Edward A

    2010-05-13

    Group VIA calcium-independent phospholipase A(2) (GVIA iPLA(2)) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA(2) inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA(2) than for GIVA cPLA(2) and GV sPLA(2), respectively, makes it a valuable tool to explore the role of GVIA iPLA(2) in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited GVIA iPLA(2) with a X(I)(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA(2) and GV sPLA(2) at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA(2); however, they are not selective. PMID:20369880

  4. Potent and Selective Fluoroketone Inhibitors of Group VIA Calcium-Independent Phospholipase A2

    PubMed Central

    Kokotos, George; Hsu, Yuan-Hao; Burke, John E.; Baskakis, Constantinos; Kokotos, Christoforos G.; Magrioti, Victoria; Dennis, Edward A.

    2010-01-01

    Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA2 inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA2 (XI(50) 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA2 than for GIVA cPLA2 and GV sPLA2, respectively, makes it a valuable tool to explore the role of GVIA iPLA2 in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl) octan-4-one inhibited GVIA iPLA2 with a XI(50) value of 0.001, while inhibiting the other intracellular GIVA cPLA2 and GV sPLA2 at least 90-times less potently. Hexa- and octa-fluoro ketones were also found to be potent inhibitors of GVIA iPLA2; however they are not selective. PMID:20369880

  5. Cytosolic Phospholipase A2 Protein as a Novel Therapeutic Target for Spinal Cord Injury

    PubMed Central

    Liu, Nai-Kui; Deng, Ling-Xiao; Zhang, Yi Ping; Lu, Qing-Bo; Wang, Xiao-Fei; Hu, Jian-Guo; Oakes, Eddie; Bonventre, Joseph V; Shields, Christopher B; Xu, Xiao-Ming

    2014-01-01

    Objective The objective of this study was to investigate whether cytosolic phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). Methods A combination of molecular, histological, immunohistochemical, and behavioral assessments were used to test whether blocking cPLA2 activation pharmacologically or genetically reduced cell death, protected spinal cord tissue, and improved behavioral recovery after a contusive SCI performed at the 10th thoracic level in adult mice. Results SCI significantly increased cPLA2 expression and activation. Activated cPLA2 was localized mainly in neurons and oligodendrocytes. Notably, the SCI-induced cPLA2 activation was mediated by the extracellular signal-regulated kinase signaling pathway. In vitro, activation of cPLA2 by ceramide-1-phosphate or A23187 induced spinal neuronal death, which was substantially reversed by arachidonyl trifluoromethyl ketone, a cPLA2 inhibitor. Remarkably, blocking cPLA2 pharmacologically at 30 minutes postinjury or genetically deleting cPLA2 in mice ameliorated motor deficits, and reduced cell loss and tissue damage after SCI. Interpretation cPLA2 may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary tissue damage and promoting recovery of function after SCI. PMID:24623140

  6. Lipoprotein-associated phospholipase A2: a novel marker of cardiovascular risk and potential therapeutic target.

    PubMed

    Macphee, Colin; Benson, G Martin; Shi, Yi; Zalewski, Andrew

    2005-06-01

    Although the clinical benefit of statins is well established, these agents reduce the risk of cardiovascular events by only 20 - 40%, and the residual risk for high-risk patients is considerable. The recognition of atherosclerosis as an inflammatory disease has opened the door to numerous complementary therapeutic approaches to further reduce risk and the overall burden of cardiovascular disease. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a novel inflammatory marker of cardiovascular risk that is being evaluated as a potential therapeutic target. The biological function of this enzyme in atherosclerosis has been controversial but recent evidence supports its pro-atherogenic role. The enzyme is predominantly bound to low-density lipoprotein cholesterol particles in humans, and its activity produces bioactive lipid mediators that promote inflammatory processes present at every stage of atherogenesis, from atheroma initiation to plaque destabilisation and rupture. Initial clinical studies suggest that the inhibitors of Lp-PLA(2) can block enzyme activity in plasma and within atherosclerotic plaques. However, more studies are needed to determine the potential clinical benefits of inhibiting Lp-PLA(2). PMID:16004595

  7. Effects of smoke inhalation on surfactant phospholipids and phospholipase A2 activity in the mouse lung.

    PubMed Central

    Oulton, M.; Moores, H. K.; Scott, J. E.; Janigan, D. T.; Hajela, R.

    1991-01-01

    The effects of smoke inhalation on the pulmonary surfactant system were examined in mice exposed for 30 minutes to smoke generated from the burning of polyurethane foam. At 8 or 12 hours after exposure, surfactants were isolated separately from lung lavage (extracellular surfactant) and residual lung tissue (intracellular surfactant) for phospholipid analysis. Calcium-dependent phospholipase A2 (PLA2) was measured on a microsomal fraction prepared from the tissue homogenate. Smoke inhalation produced a twofold increase in extracellular surfactant total phospholipid. While there was no change in the total phospholipid or phosphatidylcholine (PC) content of the intracellular surfactant, smoke inhalation significantly decreased the disaturated species of PC (DSPC). The specific activity of PLA2 was reduced by more than 50% in both groups of exposed mice. Smoke inhalation appears to result in selective depletion of the DSPC of intracellular surfactant and PLA2 involved in its synthesis. This depletion may be compensated for by increased secretion or slower breakdown of the material present in the extracellular compartment. Images Figure 1 PMID:1987765

  8. CNS myelin structural modification induced in vitro by phospholipases A2.

    PubMed

    Yunes Quartino, Pablo J; Pusterla, Julio M; Galván Josa, Victor M; Fidelio, Gerardo D; Oliveira, Rafael G

    2016-01-01

    Myelin is the self-stacked membrane surrounding axons; it is also the target of several pathological and/or neurodegenerative processes like multiple sclerosis. These processes involve, among others, the hydrolytic attack by phospholipases. In this work we describe the changes in isolated myelin structure after treatment with several secreted PLA2 (sPLA2), by using small angle x-ray scattering (SAXS) measurements. It was observed that myelin treated with all the tested sPLA2s (from cobra and bee venoms and from pig pancreas) preserved the lamellar structure but displayed an enlarged separation between membranes in certain zones. Additionally, the peak due to membrane asymmetry was clearly enhanced. The coherence length was also lower than the non-treated myelin, indicating increased disorder. These SAXS results were complemented by Langmuir film experiments to follow myelin monolayer hydrolysis at the air/water interface by a decrease in electric surface potential at different surface pressures. All enzymes produced hydrolysis with no major qualitative difference between the isoforms tested. PMID:26514604

  9. Preventive Effects of Bee Venom Derived Phospholipase A₂ on Oxaliplatin-Induced Neuropathic Pain in Mice.

    PubMed

    Li, Dongxing; Kim, Woojin; Shin, Dasom; Jung, Yongjae; Bae, Hyunsu; Kim, Sun Kwang

    2016-01-01

    Oxaliplatin, a chemotherapy drug used to treat colorectal cancer, induces specific sensory neurotoxicity signs that are aggravated by cold and mechanical stimuli. Here we examined the preventive effects of Bee Venom (BV) derived phospholipase A₂ (bvPLA₂) on oxaliplatin-induced neuropathic pain in mice and its immunological mechanism. The cold and mechanical allodynia signs were evaluated by acetone and von Frey hair test on the hind paw, respectively. The most significant allodynia signs were observed at three days after an injection of oxaliplatin (6 mg/kg, i.p.) and then decreased gradually to a normal level on days 7-9. The oxaliplatin injection also induced infiltration of macrophages and upregulated levels of the pro-inflammatory cytokine interleukin (IL)-1β in the lumbar dorsal root ganglia (DRG). Daily treatment with bvPLA₂ (0.2 mg/kg, i.p.) for five consecutive days prior to the oxaliplatin injection markedly inhibited the development of cold and mechanical allodynia, and suppressed infiltration of macrophages and the increase of IL-1β level in the DRG. Such preventive effects of bvPLA₂ were completely blocked by depleting regulatory T cells (Tregs) with CD25 antibody pre-treatments. These results suggest that bvPLA₂ may prevent oxaliplatin-induced neuropathic pain by suppressing immune responses in the DRG by Tregs. PMID:26797636

  10. 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

  11. Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice.

    PubMed

    Elimam, Hanan; Papillon, Joan; Kaufman, Daniel R; Guillemette, Julie; Aoudjit, Lamine; Gross, Richard W; Takano, Tomoko; Cybulsky, Andrey V

    2016-07-01

    Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria. PMID:27226532

  12. Phospholipase D affects translocation of NPR1 to the nucleus in Arabidopsis thaliana.

    PubMed

    Janda, Martin; Šašek, Vladimír; Chmelařová, Hana; Andrejch, Jan; Nováková, Miroslava; Hajšlová, Jana; Burketová, Lenka; Valentová, Olga

    2015-01-01

    Phytohormone salicylic acid (SA) is a crucial component of plant-induced defense against biotrophic pathogens. Although the key players of the SA pathway are known, there are still gaps in the understanding of the molecular mechanism and the regulation of particular steps. In our previous research, we showed in Arabidopsis suspension cells that n-butanol, which specifically modulates phospholipase D activity, significantly suppresses the transcription of the pathogenesis related (PR-1) gene, which is generally accepted as the SA pathway marker. In the presented study, we have investigated the site of n-butanol action in the SA pathway. We were able to show in Arabidopsis plants treated with SA that n-butanol inhibits the transcription of defense genes (PR-1, WRKY38). Fluorescence microscopy of Arabidopsis thaliana mutants expressing 35S::NPR1-GFP (nonexpressor pathogenesis related 1) revealed significantly decreased nuclear localization of NPR1 in the presence of n-butanol. On the other hand, n-butanol did not decrease the nuclear localization of NPR1 in 35S::npr1C82A-GFP and 35S::npr1C216A-GFP mutants constitutively expressing NPR1 monomers. Mass spectrometric analysis of plant extracts showed that n-butanol significantly changes the metabolic fingerprinting while t-butanol had no effect. We found groups of the plant metabolites, influenced differently by SA and n-butanol treatment. Thus, we proposed several metabolites as markers for n-butanol action. PMID:25741350

  13. High-affinity selective inhibitor against phospholipase A2 (PLA2): a computational study.

    PubMed

    Chinnasamy, Sathishkumar; Chinnasamy, Selvakkumar; Muthusamy, Karthikeyan

    2016-04-01

    Phospholipase A2 (PLA2) is the most abundant protein found in snake venom. PLA2 induces a variety of pharmacological effects such as neurotoxicity, myotoxicity and cardiotoxicity as well as anticoagulant, hemolytic, anti-platelet, hypertensive, hemorrhagic and edema inducing effects. In this study, the three dimensional structure of PLA2 of Naja sputatrix (Malayan spitting cobra) was modeled by I-TASSER, SWISS-MODEL, PRIME and MODELLER programs. The best model was selected based on overall stereo-chemical quality. Further, molecular dynamics simulation was performed to know the stability of the modeled protein using Gromacs software. Average structure was generated during the simulation period of 10 ns. High throughput virtual screening was employed through different databases (Asinex, Hit finder, Maybridge, TOSLab and ZINC databases) against PLA2. The top seven compounds were selected based on the docking score and free energy binding calculations. These compounds were analyzed by quantum polarized ligand docking, induced fit docking and density functional theory calculation. Furthermore, the stability of lead molecules in the active site of PLA2 was employed by MD simulation. The results show that selected lead molecules were highly stable in the active site of PLA2. PMID:26422703

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

    PubMed Central

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

    2012-01-01

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

  15. Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development

    PubMed Central

    Wilensky, Robert L; Shi, Yi; Mohler, Emile R; Hamamdzic, Damir; Burgert, Mark E; Li, Jun; Postle, Anthony; Fenning, Robert S; Bollinger, James G; Hoffman, Bryan E; Pelchovitz, Daniel J; Yang, Jisheng; Mirabile, Rosanna C; Webb, Christine L; Zhang, LeFeng; Zhang, Ping; Gelb, Michael H; Walker, Max C; Zalewski, Andrew; Macphee, Colin H

    2010-01-01

    Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA2 is a causative agent. Here we show that selective inhibition of Lp-PLA2 with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA2 activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA2 inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke. PMID:18806801

  16. Two FGF Receptor Kinase Molecules Act in Concert to Recruit and Transphosphorylate Phospholipase Cγ.

    PubMed

    Huang, Zhifeng; Marsiglia, William M; Basu Roy, Upal; Rahimi, Nader; Ilghari, Dariush; Wang, Huiyan; Chen, Huaibin; Gai, Weiming; Blais, Steven; Neubert, Thomas A; Mansukhani, Alka; Traaseth, Nathaniel J; Li, Xiaokun; Mohammadi, Moosa

    2016-01-01

    The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation. PMID:26687682

  17. 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

  18. Evolutionary conservation of physical and functional interactions between phospholipase D and actin.

    PubMed

    Kusner, David J; Barton, James A; Qin, Chunbo; Wang, Xuemin; Iyer, Shankar S

    2003-04-15

    Phospholipase D (PLD) enzymes from bacteria to mammals exhibit a highly conserved core structure and catalytic mechanism, but whether protein-protein interactions exhibit similar commonality is unknown. Our objective was to determine whether the physical and functional interactions of mammalian PLDs with actin are evolutionarily conserved among bacterial and plant PLDs. Highly purified bacterial and plant PLDs cosedimented with mammalian skeletal muscle alpha-actin, indicating direct interaction with F-actin. The binding of bacterial PLD to G-actin exhibited two affinity states, with dissociation constants of 1.13 pM and 0.58 microM. The effects of actin on the activities of bacterial and plant PLDs were polymerization dependent; monomeric G-actin inhibited PLD activity, whereas polymerized F-actin augmented PLD activity. Actin modulation of bacterial and plant PLDs demonstrated kinetic characteristics, efficacies, and potencies similar to those of human PLD1. Thus, physical and functional interactions between PLD and actin in PLD family members from bacteria to mammals are highly conserved throughout evolution. PMID:12667487

  19. Genome-wide analysis and expression profiling of the phospholipase D gene family in Gossypium arboreum.

    PubMed

    Tang, Kai; Dong, Chunjuan; Liu, Jinyuan

    2016-02-01

    The plant phospholipase D (PLD) plays versatile functions in multiple aspects of plant growth, development, and stress responses. However, until now, our knowledge concerning the PLD gene family members and their expression patterns in cotton has been limited. In this study, we performed for the first time the genome-wide analysis and expression profiling of PLD gene family in Gossypium arboretum, and finally, a total of 19 non-redundant PLD genes (GaPLDs) were identified. Based on the phylogenetic analysis, they were divided into six well-supported clades (α, β/γ, δ, ε, ζ and φ). Most of the GaPLD genes within the same clade showed the similar exon-intron organization and highly conserved motif structures. Additionally, the chromosomal distribution pattern revealed that GaPLD genes were unevenly distributed across 10 of the 13 cotton chromosomes. Segmental duplication is the major contributor to the expansion of GaPLD gene family and estimated to have occurred from 19.61 to 20.44 million years ago when a recent large-scale genome duplication occurred in cotton. Moreover, the expression profiling provides the functional divergence of GaPLD genes in cotton and provides some new light on the molecular mechanisms of GaPLDα1 and GaPLDδ2 in fiber development. PMID:26718354

  20. 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

  1. Modulation of the Activity of Secretory Phospholipase A2 by Antimicrobial Peptides

    PubMed Central

    Zhao, Hongxia; Kinnunen, Paavo K. J.

    2003-01-01

    The antimicrobial peptides magainin 2, indolicidin, and temporins B and L were found to modulate the hydrolytic activity of secretory phospholipase A2 (sPLA2) from bee venom and in human lacrimal fluid. More specifically, hydrolysis of phosphatidylcholine (PC) liposomes by bee venom sPLA2 at 10 μM Ca2+ was attenuated by these peptides while augmented product formation was observed in the presence of 5 mM Ca2+. The activity of sPLA2 towards anionic liposomes was significantly enhanced by the antimicrobial peptides at low [Ca2+] and was further enhanced in the presence of 5 mM Ca2+. Similarly, with 5 mM Ca2+ the hydrolysis of anionic liposomes was enhanced significantly by human lacrimal fluid sPLA2, while that of PC liposomes was attenuated. These results indicate that concerted action of antimicrobial peptides and sPLA2 could improve the efficiency of the innate response to infections. Interestingly, inclusion of a cationic gemini surfactant in the vesicles showed an essentially similar pattern on sPLA2 activity, suggesting that the modulation of the enzyme activity by the antimicrobial peptides may involve also charge properties of the substrate surface. PMID:12604528

  2. Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation.

    PubMed

    Inaba, Takehiko; Kishimoto, Takuma; Murate, Motohide; Tajima, Takuya; Sakai, Shota; Abe, Mitsuhiro; Makino, Asami; Tomishige, Nario; Ishitsuka, Reiko; Ikeda, Yasuo; Takeoka, Shinji; Kobayashi, Toshihide

    2016-07-12

    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

  3. 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

  4. Bromophenacyl bromide, a phospholipase A2 inhibitor attenuates chemically induced gastroduodenal ulcers in rats

    PubMed Central

    Tariq, Mohammad; Elfaki, Ibrahim; Khan, Haseeb Ahmad; Arshaduddin, Mohammad; Sobki, Samia; Moutaery, Meshal Al

    2006-01-01

    AIM: To study the effect of bromophenacyl bromide (BPB), a phospholipase A2 inhibitor on gastric secretion and to protect chemically induced gastric and duodenal ulcers in rats. METHODS: Acid secretion studies were undertaken in pylorus-ligated rats with BPB treatment (0, 5, 15 and 45 mg/kg). Gastric and duodenal lesions in the rats were induced by ethanol and cysteamine respectively. The levels of gastric wall mucus, nonprotein sulfhydryls (NP-SH) and myeloperoxidase (MPO) were also measured in the glandular stomach of rats following ethanol induced gastric lesions. RESULTS: BPB produced a dose-dependent inhibition of gastric acid secretion and acidity in rats. Pretreatment with BPB significantly attenuated the formation of ethanol induced gastric lesion. BPB also protected intestinal mucosa against cysteamine-induced duodenal ulcers. The antiulcer activity of BPB was associated with significant inhibition of ethanol-induced depletion of gastric wall mucus, NP-SH and MPO. These findings pointed towards the mediation of sulfhydryls in BPB induced gastrointestinal cytoprotection. CONCLUSION: BPB possesses significant antiulcer and cytoprotective activity against experimentally induced gastroduodenal lesions. PMID:17007045

  5. Inhibition of human platelet phospholipase A/sub 2/ by mono(2-ethylhexyl)phthalate

    SciTech Connect

    Labow, R.S.; Meek, E.; Adams, G.A.; Rock, G.

    1988-06-01

    There is evidence that the carcinogenic and teratogenic effects attributed to the plasticizer di(2-ethylhexyl)phthalate (DEHP) are due to its major metabolite mono(2-ethylhexyl)phthalate (MEHP). MEHP is also formed ex vivo by a plasma enzyme in blood products stored in polyvinyl chloride (PVC) DEHP plastic containers. People who receive large amounts of blood products, such as hemophiliacs or patients undergoing hemodialysis, cardiopulmonary bypass, or massive transfusion, are exposed to significant levels of plasticizer. In this study, the platelet was used to show that MEHP inhibits phospholipase A/sub 2/ (PLA/sub 2/), one of the enzymes important in the release of arachidonic acid from membrane phospholipids. PLA/sub 2/ was measured by the liberation of /sup 14/C-arachidonic acid from 1-stearoyl-2-(1-/sup 14/C)arachidonyl-L-3-phosphatidylcholine. MEHP inhibits PLA/sub 2/ activity noncompetitively in intact human platelets and lysates with a K/sub i/ of 3.7 x 10/sup -4/ M. DEHP does not inhibit PLA/sub 2/ in whole platelets. Inhibition of PLA/sub 2/ by MEHP occurs at only three times the circulating level of MEHP measured in neonates undergoing exchange transfusion and 20-fold the levels experienced by patients during cardiopulmonary bypass. Therefore, infants and adult patients with multisystem failure who accumulate MEHP in their blood may be at risk for decreased platelet function.

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

    SciTech Connect

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

    2005-05-13

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

  7. 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

  8. 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

  9. Regulation of NADPH Oxidase in Vascular Endothelium: The Role of Phospholipases, Protein Kinases, and Cytoskeletal Proteins

    PubMed Central

    Pendyala, Srikanth; Usatyuk, Peter V.; Gorshkova, Irina A.; Garcia, Joe G.N.

    2009-01-01

    The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47phox, p67phox, and Rac1) and membrane-associated components (Noxes and p22phox). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase–derived ROS in the pathobiology of lung diseases. Antioxid. Redox Signal. 11, 841–860. PMID:18828698

  10. A thermoactive secreted phospholipase A₂ purified from the venom glands of Scorpio maurus: relation between the kinetic properties and the hemolytic activity.

    PubMed

    Louati, Hanen; Krayem, Najeh; Fendri, Ahmed; Aissa, Imen; Sellami, Mohamed; Bezzine, Sofiane; Gargouri, Youssef

    2013-09-01

    A lipolytic activity was located in the scorpion venom glands (telsons), from which a phospholipase A₂ (Sm-PLVG) was purified. Like known phospholipases A₂ from scorpion venom, which are 14-18 kDa proteins, the purified Scorpio maurus-Phospholipase from Venom Glands (Sm-PLVG) has a molecular mass of 17 kDa containing long and short chains linked by disulfide bridge. It has a specific activity of 5500 U/mg measured at 47 °C and pH 8.5 using phosphatidylcholine as a substrate in presence of 8 mM NaTDC and 12 mM CaCl₂. The NH₂-terminal amino acid sequences of the purified Sm-PLVG showed similarities with those of long and short chains of some previously purified phospholipases from venom scorpions. Moreover, the Sm-PLVG exhibits hemolytic activity toward human, rabbit or rat erythrocytes. This hemolytic activity was related to its ability to interact with phospholipids' monolayer at high surface pressure. These properties are similar to those of phospholipases isolated from snake venoms. PMID:23831286

  11. Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A{sub 2}

    SciTech Connect

    Santos, K. F.; Murakami, M. T.; Toyama, M. H.; Marangoni, S.; Forrer, V. P.; Brandão Neto, J. R.; Polikarpov, I.; Arni, R. K.

    2007-04-01

    Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A{sub 2} and a catalytically inactive acidic phospholipase A{sub 2} analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A{sub 2} and a catalytically inactive acidic phospholipase A{sub 2} analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 Å, and diffracted to 1.75 Å resolution. The crystal of the phospholipase A{sub 2} domain belongs to the hexagonal space group P6{sub 1}22 (or its enantiomorph P6{sub 5}22), with unit-cell parameters a = b = 38.7, c = 286.7 Å, and diffracted to 2.6 Å resolution. The crotapotin crystal diffracted to 2.3 Å resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

  12. Phospholipases and the Network of Auxin Signal Transduction with ABP1 and TIR1 as Two Receptors: A Comprehensive and Provocative Model

    PubMed Central

    Scherer, Günther F. E.; Labusch, Corinna; Effendi, Yunus

    2012-01-01

    Three types of phospholipases, phospholipase D, secreted phospholipase A2, and patatin-related phospholipase A (pPLA) have functions in auxin signal transduction. Potential linkage to auxin receptors ABP1 or TIR1, their rapid activation or post-translational activation mechanisms, and downstream functions regulated by these phospholipases is reviewed and discussed. Only for pPLA all aspects are known at least to some detail. Evidence is gathered that all these signal reactions are located in the cytosol and seem to merge on regulation of PIN-catalyzed auxin efflux transport proteins. As a consequence, auxin concentration in the nucleus is also affected and this regulates the E3 activity of this auxin receptor. We showed that ABP1, PIN2, and pPLA, all outside the nucleus, have an impact on regulation of auxin-induced genes within 30 min. We propose that regulation of PIN protein activities and of auxin efflux transport are the means to coordinate ABP1 and TIR1 activity and that no physical contact between components of the ABP1-triggered cytosolic pathways and TIR1-triggered nuclear pathways of signaling is necessary to perform this. PMID:22629277

  13. AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs

    PubMed Central

    El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

    2015-01-01

    Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs. PMID:26162096

  14. Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels.

    PubMed

    Thakur, Dhananjay P; Tian, Jin-bin; Jeon, Jaepyo; Xiong, Jian; Huang, Yu; Flockerzi, Veit; Zhu, Michael X

    2016-01-26

    Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCβ pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems. PMID:26755577

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

    PubMed

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

    2016-07-01

    Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca(2+) release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors. PMID:27167772

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

    PubMed Central

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

    2016-01-01

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

  17. Inhibition of P2Y6 receptor-mediated phospholipase C activation and Ca(2+) signalling by prostaglandin E2 in J774 murine macrophages.

    PubMed

    Ito, Masaaki; Matsuoka, Isao

    2015-02-15

    Extracellular nucleotides act as inflammatory mediators through activation of multiple purinoceptors. Under inflammatory conditions, the purinergic signalling is affected by various inflammatory mediators. We previously showed that prostaglandin (PG) E2 suppressed the elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) stimulated by P2X4, P2Y2, and P2Y6 receptors in J774 murine macrophages. In this study, we examined the mechanism of PGE2 inhibitory effects on P2Y6 receptor-mediated function in J774 cells. The P2Y6 receptor agonist UDP induced a sustained elevation of [Ca(2+)]i by stimulating the phospholipase C (PLC) signalling pathway. PGE2 inhibited [Ca(2+)]i elevation and phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. J774 cells highly expressed the E-type prostanoid 2 (EP2) receptor subtype, a Gs-coupled receptor. PGE2 and a selective EP2 receptor agonist caused cyclic AMP (cAMP) accumulation in J774 cells. The inhibitory effects of PGE2 on P2Y6 receptor-mediated responses were mimicked by the selective EP2 receptor agonist. Although EP2 receptor is linked to adenylyl cyclase activation, PGE2-induced inhibition of Ca(2+) response and PI hydrolysis could not be mimicked by a lipophilic cAMP derivative, dibutyryl cAMP, or an adenylyl cyclase activator, forskolin. The inhibition of UDP-induced PLC activation by PGE2 was not affected by down-regulation of protein kinase C by phorbol-12-myristate-13-acetate treatment. PGE2 inhibited PLC activation induced by aluminium fluoride, but not by the Ca(2+)-ionophore, ionomycin. Finally, the inhibition of UDP-induced PLC activation by PGE2 was impaired by Gs knockdown using siRNA. These results suggest that EP2 receptor activation in macrophages negatively controls the Gq/11-PLC signalling through a Gs-mediated, but cAMP-independent signalling mechanism. PMID:25614334

  18. Lysophospholipids and their G protein-coupled receptors in atherosclerosis.

    PubMed

    Li, Ya-Feng; Li, Rong-Shan; Samuel, Sonia B; Cueto, Ramon; Li, Xin-Yuan; Wang, Hong; Yang, Xiao-Feng

    2016-01-01

    Lysophospholipids (LPLs) are bioactive lipid-derived signaling molecules generated by the enzymatic and chemical processes of regiospecific phospholipases on substrates such as membrane phospholipids (PLs) and sphingolipids (SLs). They play a major role as extracellular mediators by activating G-protein coupled receptors (GPCRs) and stimulating diverse cellular responses from their signaling pathways. LPLs are involved in various pathologies of the vasculature system including coronary heart disease and hypertension. Many studies suggest the importance of LPLs in their association with the development of atherosclerosis, a chronic and severe vascular disease. This paper focuses on the pathophysiological effects of different lysophospholipids on atherosclerosis, which may promote the pathogenesis of myocardial infarction and strokes. Their atherogenic biological activities take place in vascular endothelial cells, vascular smooth muscle cells, fibroblasts, monocytes and macrophages, dendritic cells, T-lymphocytes, platelets, etc. PMID:26709762

  19. Crystallization and preliminary X-ray diffraction analysis of myotoxin I, a Lys49-phospholipase A{sub 2} from Bothrops moojeni

    SciTech Connect

    Marchi-Salvador, D. P.; Silveira, L. B.; Soares, A. M.

    2005-10-01

    A new myotoxic Lys49-phospholipase from B. moojeni has been crystallized and X-ray diffraction data were collected to 2.18 Å resolution. Preliminary analysis indicates the presence of four molecules in the asymmetric unit, leading to a possible new oligomeric structure for Lys49-PLA{sub 2}s. A new myotoxic Lys49-phospholipase A{sub 2} isolated from Bothrops moojeni snake venom has been crystallized. The crystals diffracted to 2.18 Å resolution using a synchrotron-radiation source and belong to space group C2. The unit-cell parameters are a = 56.8, b = 125.0, c = 64.7 Å, β = 105.5°. Preliminary analysis indicates the presence of four molecules in the asymmetric unit. This may suggest a new quaternary structure for this Lys49-phospholipase A{sub 2} in contrast to the dimeric and monomeric structures solved so far for this class of proteins.

  20. Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A{sub 2}-induced degranulation in mast cells

    SciTech Connect

    Nishikawa, Hirofumi; Kitani, Seiichi

    2011-05-01

    Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of {beta}-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G{sub M1}), di-sialoganglioside (G{sub D1a}) and tri-sialoganglioside (G{sub T1b}). In contrast, honeybee venom-derived phospholipase A{sub 2} induced the net degranulation directly without cytotoxicity, which was not inhibited by G{sub M1}, G{sub D1a} and G{sub T1b}. For analysis of distribution of G{alpha}{sub q} and G{alpha}{sub i} protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of G{alpha}{sub q} and G{alpha}{sub i} at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A{sub 2}-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A{sub 2}-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

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

    PubMed

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

    1995-01-15

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

  2. Identification of a new binding protein for crotoxin and other neurotoxic phospholipase A sub 2 s on brain synaptic membranes

    SciTech Connect

    Chonho Yen ); Muchin Tzeng Academia Sinica, Taipei )

    1991-12-03

    Crotoxin and other neurotoxic phospholipase A{sub 2}s exert neurotoxicity by acting primarily at the presynaptic level. Strong binding of crotoxin and several other to synaptic membranes has been demonstrated previously. In this study the authors used simple chemical cross-linking techniques to identify the neuronal membrane molecules involved in the binding of these toxins. After {sup 125}I-crotoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate, disuccinimidyl dithiobis(propionate) or ethylene glycol bis(succinimidyl succinate) resulted in the formation of a predominant radioactive conjugate of {approximately}60 kDa, which was different from the conjugate formed by photoaffinity labeling technique in a previous report. The membrane component in the conjugate was shown to be a single-chain protein {approximately}45 kDa. In subfractions of synaptosomes, this binding protein was mostly found in the synaptic membrane fraction and was not present in the mitochondrial fraction. Plasma membranes from several nonneural tissues also did not contain this binding protein. Unmodified crotoxin inhibited the formation o this adduct with an IC{sub 50} of around 1 {times} 10{sup {minus}8} M. Mojave toxin and some other phospholipase A{sub 2}s were also highly inhibitory to this conjugation, and notexin and others were less effective, while {beta}-bungarotoxin and pancreatic PLA{sub 2} were totally ineffective. They concluded that a new protein of 45 kDa specifically present in neuronal membranes is another major molecule responsible for the binding of crotoxin and other phospholipase A{sub 2}s.

  3. Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

    PubMed Central

    Wang, He; Chen, Xiaole; Zhou, Mei; Wang, Lei; Chen, Tianbao; Shaw, Chris

    2016-01-01

    Secretory phospholipase A2 (sPLA2) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization. PMID:27258312

  4. 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. PMID:26978518

  5. Edema Toxin Impairs Anthracidal Phospholipase A2 Expression by Alveolar Macrophages

    PubMed Central

    Raymond, Benoit; Leduc, Dominique; Ravaux, Lucas; Goffic, Ronan Le; Candela, Thomas; Raymondjean, Michel; Goossens, Pierre Louis; Touqui, Lhousseine

    2007-01-01

    Bacillus anthracis, the etiological agent of anthrax, is a spore-forming Gram-positive bacterium. Infection with this pathogen results in multisystem dysfunction and death. The pathogenicity of B. anthracis is due to the production of virulence factors, including edema toxin (ET). Recently, we established the protective role of type-IIA secreted phospholipase A2 (sPLA2-IIA) against B. anthracis. A component of innate immunity produced by alveolar macrophages (AMs), sPLA2-IIA is found in human and animal bronchoalveolar lavages at sufficient levels to kill B. anthracis. However, pulmonary anthrax is almost always fatal, suggesting the potential impairment of sPLA2-IIA synthesis and/or action by B. anthracis factors. We investigated the effect of purified ET and ET-deficient B. anthracis strains on sPLA2-IIA expression in primary guinea pig AMs. We report that ET inhibits sPLA2-IIA expression in AMs at the transcriptional level via a cAMP/protein kinase A–dependent process. Moreover, we show that live B. anthracis strains expressing functional ET inhibit sPLA2-IIA expression, whereas ET-deficient strains induced this expression. This stimulatory effect, mediated partly by the cell wall peptidoglycan, can be counterbalanced by ET. We conclude that B. anthracis down-regulates sPLA2-IIA expression in AMs through a process involving ET. Our study, therefore, describes a new molecular mechanism implemented by B. anthracis to escape innate host defense. These pioneering data will provide new molecular targets for future intervention against this deathly pathogen. PMID:18069891

  6. Secretory phospholipase A2 inhibitor PX-18 preserves microvascular reactivity after cerebral ischemia in piglets.

    PubMed

    Domoki, Ferenc; Zimmermann, Alíz; Lenti, Laura; Tóth-Szuki, Valéria; Pardeike, Jana; Müller, Rainer H; Bari, Ferenc

    2009-09-01

    Cerebral ischemia/reperfusion (I/R) results in cellular energy failure and dysfunction of the neurovascular unit that contribute to subsequent neuronal cell death in the neonate. PX-18 is a putative neuroprotective inhibitor of secretory phospholipase A(2) (sPLA(2)) but its in vivo testing has been limited by its poor solubility. Our purpose was to assess whether PX-18 preserved neuronal-vascular reactivity to I/R-sensitive endothelium-dependent (hypercapnia, bradykinin) and/or neuron-dependent (N-methyl-D-aspartate; NMDA) stimuli. To make the drug available for in vivo studies, PX-18 was formulated as a 3% nanosuspension applying high pressure homogenization. Newborn piglets (1-day old, n=40) were anesthetized and ventilated, and cerebrovascular reactivity to the above stimuli was determined by measuring changes in pial arteriolar diameters using the closed cranial window/intravital videomicroscopy technique. Intravenous infusion of PX-18 nanosuspension (6 mg/kg, 20 min) did not affect baseline arteriolar diameters, or hypercapnia-, bradykinin-, or NMDA-induced pial arteriolar vasodilation under normoxic conditions. Global cerebral ischemia (10 min) followed by 1 h of reperfusion significantly attenuated hypercapnia-, bradykinin-, and NMDA-induced vasodilation in untreated or vehicle-treated controls. However, PX-18 resulted in nearly full preservation of cerebrovascular reactivity to all these stimuli. In conclusion, inhibition of sPLA(2) by PX-18 improves neurovascular function both at the neuronal and the microvascular level following I/R. This effect of PX-18 likely contributes to its neuroprotective effect. PMID:19555699

  7. Phospholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells

    PubMed Central

    Satow, Reiko; Hirano, Tamaki; Batori, Ryosuke; Nakamura, Tomomi; Murayama, Yumi; Fukami, Kiyoko

    2014-01-01

    Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in CRC predict the ineffectiveness of EGF receptor-targeted therapy. Previous transcriptional microarray analysis suggests the association between phospholipase Cδ1 (PLCδ1) expression and KRAS mutation status in CRC. However, both the roles and the regulatory mechanisms of PLCδ1 in CRC are not known. Here, we found that the expression of PLCδ1, one of the most basal PLCs, is down-regulated in CRC specimens compared with normal colon epithelium by immunohistochemistry. Furthermore, we examined the roles of PLCδ1 in CRC cell lines that harbor an activating KRAS mutation. Ectopic expression of PLCδ1 in CRC cells induced the expression of E-cadherin, whereas knockdown of PLCδ1 repressed the expression of E-cadherin. Moreover, the overexpression of PLCδ1 suppressed the expression of several mesenchymal genes and reduced cell motility, invasiveness, and in vivo tumorigenicity of SW620 CRC cells. We also showed that PLCδ1 expression is repressed by the KRAS/mitogen-activated protein kinase kinase (MEK) pathway. Furthermore, PLCδ1 suppressed the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 through E-cadherin induction in CRC cells, suggesting the presence of a negative regulatory loop between KRAS/MEK/ERK signaling and PLCδ1. These data indicate that PLCδ1 has tumor-suppressive functions in CRC through E-cadherin induction and KRAS/MEK/ERK signal attenuation. PMID:25197077

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

    PubMed Central

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

    2015-01-01

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

  9. 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. PMID:18621911

  10. Group X Secretory Phospholipase A2 Regulates Insulin Secretion through a Cyclooxygenase-2-dependent Mechanism*

    PubMed Central

    Shridas, Preetha; Zahoor, Lubna; Forrest, Kathy J.; Layne, Joseph D.; Webb, Nancy R.

    2014-01-01

    Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA2 is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA2-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA2 overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA2 (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA2 on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA2 plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production. PMID:25122761

  11. Development of a Cell-Based Bioassay for Phospholipase A2-Triggered Liposomal Drug Release

    PubMed Central

    Arouri, Ahmad; Trojnar, Jakub; Schmidt, Steffen; Hansen, Anders H.; Mollenhauer, Jan; Mouritsen, Ole G.

    2015-01-01

    The feasibility of exploiting secretory phospholipase A2 (sPLA2) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies. PMID:25945937

  12. Purification, sequencing and characterization of phospholipase D from Indian mustard seeds.

    PubMed

    Khatoon, Hafeeza; Mansfeld, Johanna; Schierhorn, Angelika; Ulbrich-Hofmann, Renate

    2015-09-01

    Phospholipase D (PLD; E.C. 3.1.4.4) is widespread in plants where it fulfills diverse functions in growth and in the response to stresses. The enzyme occurs in multiple forms that differ in their biochemical properties. In the present paper PLD from medicinally relevant Indian mustard seeds was purified by Ca(2+)-mediated hydrophobic interaction and anion exchange chromatography to electrophoretic homogeneity. Based on mass-spectrometric sequence analysis of tryptic protein fragments, oligonucleotide primers for cloning genomic DNA fragments that encoded the enzyme were designed and used to derive the complete amino acid sequence of this PLD. The sequence data, as well as the molecular properties (molecular mass of 92.0 kDa, pI 5.39, maximum activity at pH 5.5-6.0 and Ca(2+) ion concentrations ⩾60 mM), allowed the assignment of this enzyme to the class of α-type PLDs. The apparent kinetic parameters Vmax and Km, determined for the hydrolysis of phosphatidylcholine (PC) in an aqueous mixed-micellar system were 356±15 μmol min(-1) mg(-1) and 1.84±0.17 mM, respectively. Phosphate analogs such as NaAlF4 and Na3VO4 displayed strong inhibition of the enzyme. Phosphatidylinositol 4,5-bisphosphate had a strong activating effect at 2-10 mM CaCl2. PLD was inactivated at temperatures >45 °C. The enzyme exhibited the highest activity toward PC followed by phosphatidylethanolamine and phosphatidylglycerol. PCs with short-chain fatty acids were better substrates than PCs with long fatty acid chains. Lyso-PC was not accepted as substrate. PMID:26057230

  13. Phospholipase cgamma1 is required for activation of store-operated channels in human keratinocytes.

    PubMed

    Tu, Chia-Ling; Chang, Wenhan; Bikle, Daniel D

    2005-01-01

    Store-operated calcium entry depicts the movement of extracellular Ca2+ into cells through plasma membrane Ca2+ channels activated by depletion of intracellular Ca2+ stores. The members of the canonical subfamily of transient receptor potential channels (TRPC) have been implicated as the molecular bases for store-operated channels (SOC). Here we investigate the role of phospholipase C (PLC) in regulation of native SOC and the expression of endogenous TRPC in human epidermal keratinocytes. Calcium entry in response to store depletion with thapsigargin was reversibly blocked by 2-aminoethoxydiphenyl borane, an effective SOC inhibitor, and suppressed by the diacylglycerol analoge, 1-oleoyl-2-acetyl-sn-glycerol. Inhibition of PLC with U73122 or transfection of a PLCgamma1 antisense cDNA construct completely blocked SOC activity, indicating a requirement for PLC, especially PLCgamma1, in the activation of SOC. RT-PCR and immunoblotting analyses showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are expressed in keratinocytes. Knockdown of the level of endogenous TRPC1 or TRPC4 inhibited store-operated calcium entry, indicating they are part of the native SOC. Co-immunoprecipitation studies demonstrated that TRPC1, but not TRPC4, interacts with PLCgamma1 and the inositol 1,4,5-trisphosphate receptor (IP3R). The association of TRPC1 with PLCgamma1 and IP3R decreased in keratinocytes with higher intracellular Ca2+, coinciding with a downregulation in SOC activity. Our results indicate that the activation of SOC in keratinocytes depends, at least partly, on the interaction of TRPC with PLCgamma1 and IP3R. PMID:15654973

  14. Nonspecific Binding Domains in Lipid Membranes Induced by Phospholipase A2.

    PubMed

    Hong, Chia Yee; Han, Chung-Ta; Chao, Ling

    2016-07-12

    Phospholipase A2 (PLA2) is a peripheral membrane protein that can hydrolyze phospholipids to produce lysolipids and fatty acids. It has been found to play crucial roles in various cellular processes and is thought as a potential candidate for triggering drug release from liposomes for medical treatment. Here, we directly observed that PLA2 hydrolysis reaction can induce the formation of PLA2-binding domains at lipid bilayer interface and found that the formation was significantly influenced by the fluidity of the lipid bilayer. We prepared supported lipid bilayers (SLBs) with various molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to adjust the reactivity and fluidity of the lipid bilayers. A significant amount of the PLA2-induced domains was observed in mixtures of DPPC and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) but not in either pure DPPC or pure DOPC bilayer, which might be the reason that previous studies rarely observed these domains in lipid bilayer systems. The fluorescently labeled PLA2 experiment showed that newly formed domains acted as binding templates for PLA2. The AFM result showed that the induced domain has stepwise plateau structure, suggesting that PLA2 hydrolysis products may align as bilayers and accumulate layer by layer on the support, and the hydrophobic acyl chains at the side of the layer structure may be exposed to the outside aqueous environment. The introduced hydrophobic region could have hydrophobic interactions with proteins and therefore can attract the binding of not only PLA2 but also other types of proteins such as proteoglycans and streptavidin. The results suggest that the formation of PLA2-induced domains may convert part of a zwitterionic nonsticky lipid membrane to a site where biomolecules can nonspecifically bind. PMID:27218880

  15. Cytosolic phospholipaseA2 inhibition with PLA-695 radiosensitizes tumors in lung cancer animal models.

    PubMed

    Thotala, Dinesh; Craft, Jeffrey M; Ferraro, Daniel J; Kotipatruni, Rama P; Bhave, Sandeep R; Jaboin, Jerry J; Hallahan, Dennis E

    2013-01-01

    Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted. PMID:23894523

  16. Cytosolic PhospholipaseA2 Inhibition with PLA-695 Radiosensitizes Tumors in Lung Cancer Animal Models

    PubMed Central

    Ferraro, Daniel J.; Kotipatruni, Rama P.; Bhave, Sandeep R.; Jaboin, Jerry J.; Hallahan, Dennis E.

    2013-01-01

    Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted. PMID:23894523

  17. Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

    PubMed Central

    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. PMID:26978518

  18. Ovarian steroid regulation of endometrial phospholipase A2 isoforms in horses.

    PubMed

    Ababneh, M M; Troedsson, M H T

    2013-04-01

    Real-time PCR was used to investigate the role of progesterone (P4) and oestradiol (E2) in regulation of endometrial cytosolic, secretory and calcium-independent phospholipase A2 (PLA2G4A, PLA2G2A and PLA2G6, respectively) gene expression. Ovariectomized mares underwent 6 days of E2 pre-treatment followed by 14 days of P4 supplementation. At the start of P4 treatment (Day 1), mares were assigned in a 2 × 2 factorial design to receive either E2 or vehicle starting on Day 11 and endometrial biopsy collection on either Day 14 when P4 concentrations remained high (>4 ng/ml) or Day 16 when P4 concentrations had declined (0.5-2 ng/ml). Additional biopsies were collected from ovariectomized mares on Day 8, which served as control. Blood samples were collected for P4 determination. PLA2G4A expression was higher (p < 0.05) on Day 14 compared with Day 8. In contrast, PLA2G2A did not change significantly (p < 0.12). PLA2G4A and PLA2G2A gene expression increased (p < 0.05), as P4 concentration dropped, on Day 16. In contrast, PLA2G6 gene expression did not show differences between days. Treatment with oestradiol did not increase PLA2 isoforms expression when compared to treatment with the vehicle. PLA2G4A and PLA2G2A were positively correlated with each other and negatively correlated with P4 concentrations. In conclusion, P4 withdrawal upregulated PLA2G4A and PLA2G2A gene expression, and this was not affected by E2. PLA2G4A and PLA2G2A but not PLA2G6 gene expression may be involved in controlling prostaglandin F2 alpha synthesis and luteolysis. PMID:22882596

  19. Filamin and Phospholipase C-ε Are Required for Calcium Signaling in the Caenorhabditis elegans Spermatheca

    PubMed Central

    Kovacevic, Ismar; Orozco, Jose M.; Cram, Erin J.

    2013-01-01

    The Caenorhabditis elegans spermatheca is a myoepithelial tube that stores sperm and undergoes cycles of stretching and constriction as oocytes enter, are fertilized, and exit into the uterus. FLN-1/filamin, a stretch-sensitive structural and signaling scaffold, and PLC-1/phospholipase C-ε, an enzyme that generates the second messenger IP3, are required for embryos to exit normally after fertilization. Using GCaMP, a genetically encoded calcium indicator, we show that entry of an oocyte into the spermatheca initiates a distinctive series of IP3-dependent calcium oscillations that propagate across the tissue via gap junctions and lead to constriction of the spermatheca. PLC-1 is required for the calcium release mechanism triggered by oocyte entry, and FLN-1 is required for timely initiation of the calcium oscillations. INX-12, a gap junction subunit, coordinates propagation of the calcium transients across the spermatheca. Gain-of-function mutations in ITR-1/IP3R, an IP3-dependent calcium channel, and loss-of-function mutations in LFE-2, a negative regulator of IP3 signaling, increase calcium release and suppress the exit defect in filamin-deficient animals. We further demonstrate that a regulatory cassette consisting of MEL-11/myosin phosphatase and NMY-1/non-muscle myosin is required for coordinated contraction of the spermatheca. In summary, this study answers long-standing questions concerning calcium signaling dynamics in the C. elegans spermatheca and suggests FLN-1 is needed in response to oocyte entry to trigger calcium release and coordinated contraction of the spermathecal tissue. PMID:23671426

  20. Epigenetic Regulation of Cytosolic Phospholipase A2 in SH-SY5Y Human Neuroblastoma Cells.

    PubMed

    Tan, Charlene Siew-Hon; Ng, Yee-Kong; Ong, Wei-Yi

    2016-08-01

    Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and eicosanoids. While much is known about regulation of cPLA2 by posttranslational modification such as phosphorylation, little is known about its epigenetic regulation. In this study, treatment with histone deacetylase (HDAC) inhibitors, trichostatin A (TSA), valproic acid, tubacin and the class I HDAC inhibitor, MS-275, were found to increase cPLA2α messenger RNA (mRNA) expression in SH-SY5Y human neuroblastoma cells. Co-treatment of the histone acetyltransferase (HAT) inhibitor, anacardic acid, modulated upregulation of cPLA2α induced by TSA. Specific involvement of class I HDACs and HAT in cPLA2α regulation was further shown, and a Tip60-specific HAT inhibitor, NU9056, modulated the upregulation of cPLA2α induced by MS-275. In addition, co-treatment of with histone methyltransferase (HMT) inhibitor, 5'-deoxy-5'-methylthioadenosine (MTA) suppressed TSA-induced cPLA2α upregulation. The above changes in cPLA2 mRNA expression were reflected at the protein level by Western blots and immunocytochemistry. Chromatin immunoprecipitation (ChIP) showed TSA increased binding of trimethylated H3K4 to the proximal promoter region of the cPLA2α gene. Cell injury after TSA treatment as indicated by lactate dehydrogenase (LDH) release was modulated by anacardic acid, and a role of cPLA2 in mediating TSA-induced injury shown, after co-incubation with the cPLA2 selective inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3). Together, results indicate epigenetic regulation of cPLA2 and the potential of such regulation for treatment of chronic inflammation. PMID:26162318

  1. Heterogeneous distribution of PIP/sub 2/-specific phospholipase C in rat liver

    SciTech Connect

    Lutz, R.J.; Fukui, T.; Lowenstein, J.M.

    1987-05-01

    Phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PIP/sub 2/-PLC) activity was found in both a plasma membrane-enriched fraction prepared by Percoll gradient centrifugation and cytosol isolated from rat liver. PIP/sub 2/-PLC was assayed in the presence of 50 mM Tris-HCl pH 7.4, 100 ..mu..M free Ca/sup 2 +/, 150 mM KCl, 0.1% deoxycholate, 50 ..mu..M (/sup 3/H-inositol)PIP/sub 2/. No PIP/sub 2/-PLC activity was released from the membrane fraction whe washed with 50 mM Tris-HCl, pH 7.4, under which conditions residual lactate dehydrogenase was completely removed. Approximately 70% of the membrane-associated activity was solubilized by 0.5 M KCl in 50 mM Tris-HCl pH 7.4 (S-PLC). 30% of the membrane-associated activity was especially resistant to solubilization (M-PLC); it remained in the particulate fraction following three successive washes with 0.5 M KCl and an extraction with 1% Triton X-100. Cytosolic PIP/sub 2/-PLC, S-PLC, and M-PLC hydrolyze PIP/sub 2/ at rates 4, 20, and 15-times greater than phosphatidylinositol and at rates 20, 430, and 60-times greater than phosphatidylcholine respectively. The three PIP/sub 2/-PLC activities are activated by calcium and enhanced by deoxycholate and monovalent salts.

  2. INHIBITION OF CALCIUM INDEPENDENT PHOSPHOLIPASE A2 PREVENTS INFLAMMATORY MEDIATOR PRODUCTION IN PULMONARY MICROVASCULAR ENDOTHELIUM

    PubMed Central

    Rastogi, Prerna; McHowat, Jane

    2010-01-01

    Inhalation of allergens can result in mast cell degranulation and release of granule contents, including tryptase, in the lung. Injury to human pulmonary microvascular endothelial cells (HMVEC-L) can also result in activation of the coagulation cascade and thrombin generation. We hypothesize that these proteases activate calcium-independent phospholipase A2 (iPLA2), in HMVEC-L, leading to the production of membrane phospholipids-derived inflammatory mediators. Both thrombin and tryptase stimulation of HMVEC-L increased iPLA2 activity that was inhibited by pretreatment with the iPLA2 selective inhibitor bromoenol lactone (BEL). Arachidonic acid and prostaglandin I2 (PGI2) release were also increased in tryptase and thrombin stimulated cells and inhibited by BEL pretreatment. Pretreating the endothelial cells with AACOCF3 a cytosolic PLA2 inhibitor did not inhibit tryptase or thrombin induced arachidonic acid and PGI2 release. In addition thrombin and tryptase also increased HMVEC-L platelet activating factor (PAF) production that significantly contributes to the recruitment and initial adherence of polymorphonuclear neutrophils (PMN) to the endothelium. Tryptase or thrombin stimulated increase in PMN adherence to the endothelium was inhibited by pretreatment of HMVEC-L with BEL or pretreatment of PMN with CV3988, a PAF receptor specific antagonist. Collectively, these data support our hypothesis that iPLA2 activity is responsible for membrane phospholipid hydrolysis in response to tryptase or thrombin stimulation in HMVEC-L. Therefore selective inhibition of iPLA2 may be a pharmacological target to inhibit the early inflammation in pulmonary vasculature that occurs as a consequence of mast cell degranulation or acute lung injury. PMID:19059366

  3. Structures and binding studies of the complexes of phospholipase A2 with five inhibitors.

    PubMed

    Shukla, Prakash Kumar; Gautam, Lovely; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P

    2015-04-01

    Phospholipase A2 (PLA2) catalyzes the hydrolysis of phospholipids into arachidonic acid and lysophospholipids. Arachidonic acid is used as a substrate in the next step of the multistep pathway leading to the production of eicosanoids. The eicosanoids, in extremely low concentrations, are required in a number of physiological processes. However, the increase in their concentrations above the essential physiological requirements leads to various inflammatory conditions. In order to prevent the unwanted rise in the concentrations of eicosanoids, the actions of PLA2 and other enzymes of the pathway need to be blocked. We report here the structures of five complexes of group IIA PLA2 from Daboia russelli pulchella with tightly binding inhibitors, (i) p-coumaric acid, (ii) resveratrol, (iii) spermidine, (iv) corticosterone and (v) gramine derivative. The binding studies using fluorescence spectroscopy and surface plasmon resonance techniques for the interactions of PLA2 with the above five compounds showed high binding affinities with values of dissociation constants (KD) ranging from 3.7×10(-8) M to 2.1×10(-9) M. The structure determinations of the complexes of PLA2 with the above five compounds showed that all the compounds bound to PLA2 in the substrate binding cleft. The protein residues that contributed to the interactions with these compounds included Leu2, Leu3, Phe5, Gly6, Ile9, Ala18, Ile19, Trp22, Ser23, Cys29, Gly30, Cys45, His48, Asp49 and Phe106. The positions of side chains of several residues including Leu2, Leu3, Ile19, Trp31, Lys69, Ser70 and Arg72 got significantly shifted while the positions of active site residues, His48, Asp49, Tyr52 and Asp99 were unperturbed. PMID:25541253

  4. Interaction of surfactant protein A with peroxiredoxin 6 regulates phospholipase A2 activity.

    PubMed

    Wu, Yong-Zheng; Manevich, Yefim; Baldwin, James L; Dodia, Chandra; Yu, Kevin; Feinstein, Sheldon I; Fisher, Aron B

    2006-03-17

    Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA(2) activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA(2) activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA(2) activity) showed K(m)0.35 mm and V(max) 138 nmol/min/mg of protein. SP-A inhibited PLA(2) activity non-competitively with K(i) 10 mug/ml and was Ca(2+) -independent. Activity at pH 7.4 was approximately 50% less, and inhibition by SP-A was partially dependent on Ca(2+). Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni(2) -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca(2+) dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA(2) activity of Prdx6 by SP-A. PMID:16330552

  5. Stimulation of Ca(2+)-regulated olfactory phospholipase C by amino acids.

    PubMed

    Lo, Y H; Bradley, T M; Rhoads, D E

    1993-11-23

    L-Amino acids are potent olfactory stimuli for Atlantic salmon. A plasma membrane fraction, previously shown to be rich in amino acid binding sites, was prepared from olfactory rosettes of Atlantic salmon (Salmo salar) and utilized to investigate the role of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in olfactory signal transduction. A cocktail of L-amino acids (Ser, Glu, Lys, and Gly) stimulated PIP2 hydrolysis by phospholipase C (PLC) in a dose-dependent manner with half-maximal stimulation when all amino acids were present at approximately 1 microM. Stimulation of PIP2 hydrolysis by amino acids required GTP gamma S, which alone had no effect on PLC activity. Unlike GTP gamma S, AlF4- and Ca2+ stimulated PIP2 breakdown. Preincubation with 1 mM GDP beta S eliminated the effect of amino acids and AlF4- on PIP2 hydrolysis, suggesting the involvement of G protein regulation. The lack of stimulation by GTP gamma S alone suggested that there was negligible exchange of GTP gamma S for GDP in the absence of odorant. There were no significant effects of amino acids on either adenylate cyclase or guanylate cyclase activities in the membrane preparation under these conditions. The effect of the amino acid cocktail was maximal at 1-10 nM free Ca2+. At or above 100 nM free Ca2+, no effect of amino acids on PIP2 hydrolysis was found. However, between 100 nM and 100 microM, Ca2+ directly stimulated PLC activity in a dose-dependent manner. This stimulation by Ca2+ appeared to be G protein independent because it did not require GTP gamma S and was not inhibited by GDP beta S.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8241123

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

    PubMed

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

    2015-04-14

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

  7. Negative regulation of cytosolic phospholipase A(2) by melatonin in the rat pineal gland.

    PubMed Central

    Li, B; Zhang, H; Akbar, M; Kim, H Y

    2000-01-01

    In this paper evidence that supports a new role for melatonin as a negative endogenous regulator of cytosolic phospholipase A(2) (cPLA(2)) is presented. When rat pineal glands were incubated in culture, time-dependent release of arachidonic acid (AA) was observed, which was significantly inhibited by a known 85-kDa cPLA(2) inhibitor, methyl arachidonyl fluorophosphonate. Co-incubation with melatonin inhibited the AA release in a concentration-dependent manner, and this decrease was accompanied by a reduction of cPLA(2) protein and mRNA expression. Melatonin-receptor agonists, 2-iodo-N-butanoyl-5-methoxytryptamine and 5-methoxycarbonylamino-N-acetyltryptamine, also decreased AA release and cPLA(2) protein and mRNA levels, while pre-incubation with the melatonin receptor antagonists luzindole and 2-phenylmelatonin abolished the melatonin effect. In vivo, as melatonin production reflected a typical diurnal variation, endogenous non-esterified AA and cPLA(2) mRNA levels in the rat pineal gland showed an off-phase diurnal pattern in relation to melatonin levels. Intravenous administration of isoproterenol, which has been shown to elevate melatonin production, also decreased the levels of non-esterified AA and cPLA(2) mRNA significantly. Direct administration of melatonin to rats by intravenous injection decreased the levels of non-esterified AA, cPLA(2) protein and mRNA in rat pineal glands. In conclusion, melatonin endogenously down-regulates cPLA(2) expression, presumably through melatonin-receptor-mediated processes. PMID:11042126

  8. Splicing of a human endogenous retrovirus to a novel phospholipase A2 related gene.

    PubMed Central

    Feuchter-Murthy, A E; Freeman, J D; Mager, D L

    1993-01-01

    As part of an investigation into the effects of endogenous retroviruses on adjacent genes, we have isolated a cDNA clone derived from the human teratocarcinoma cell line NTera2D1 representing a chimeric transcript in which an endogenous retrovirus-like element, RTVL-H, has been spliced to downstream cellular sequences. The 5' terminus of this clone, termed AF-5, occurs one bp downstream of the predicted transcriptional start site in the RTVL-H long terminal repeat (LTR). AF-5 contains an open reading frame of 689 amino acids beginning within RTVL-H sequences that has two domains of homology with phospholipase A2 (PLA2). These domains, of approximately 120 amino acids each, are 30-38% identical to secreted PLA2s and contain sequence features of both group I and II enzymes. The corresponding AF-5 transcript is 2.5 kb and is derived from a single copy novel gene termed PLA2L. Southern analysis indicates that the RTVL-H element is normally present in human DNA upstream of the PLA2L gene. RTVL-H/PLA2L chimeric transcripts were detected in two independent teratocarcinoma cell lines but not in several other cell lines or primary human tissues. Characterization of additional cDNA clones and PCR analysis indicates that multiple RTVL-H/PLA2L alternatively spliced transcripts are expressed. No evidence has been found for transcription from a non-LTR promoter. These findings strongly suggest that the endogenous LTR promotes expression of the human PLA2L gene in teratocarcinoma cells. Images PMID:8382789

  9. Phospholipase B activity and organophosphorus compound toxicity in cultured neural cells

    SciTech Connect

    Read, David J.; Langford, Lynda; Barbour, Helen R.; Forshaw, Philip J.; Glynn, Paul . E-mail: pg8@le.ac.uk

    2007-03-15

    Organophosphorus compounds (OP) such as phenyl saligenin phosphate (PSP) and mipafox (MPX) which cause delayed neuropathy, inhibit neuropathy target esterase (NTE), while OPs such as paraoxon (PXN) react more readily with acetylcholinesterase. In yeast and mammalian cell lines, NTE has been shown to have phospholipase B (PLB) activity which deacylates intracellular phosphatidylcholine to glycerophosphocholine (GroPCho) and can be detected by metabolic labeling with [{sup 14}C]choline. Here we investigated PLB activity in primary cultures of mouse neural cells. In cortical and cerebellar granule neurons and astrocytes, [{sup 14}C]GroPCho labeling was inhibited by PSP and MPX: phenyl dipentylphosphinate (PDPP), a non-neuropathic NTE inhibitor, was more potent, while PXN, was substantially less so. In all three cell types, conversion of [{sup 14}C]phosphatidylcholine to [{sup 14}C]GroPCho over 24 h was relatively small (2.3-14%). Consequently, even with > 80% inhibition of [{sup 14}C]GroPCho production, increased [{sup 14}C]phosphatidylcholine was not detected. At concentrations of 1-10 {mu}M, only PSP was cytotoxic to cortical and cerebellar granule neurons after 24-h exposure. Moreover, dramatic changes in glial cell morphology were induced by PSP, but not PDPP or MPX, with rapid (2-3 h) rounding up of astrocytes and of Schwann cells in cultures of dissociated mouse dorsal root ganglia. We conclude that PLB activity is present in a variety of cultured mouse neural cell types but that acute loss of this activity is not cytotoxic. Conversely, the rapid toxic effects of PSP in vitro suggest that a serine hydrolase distinct from NTE is required continuously by neurons and glia.

  10. Fluctuation of lysosomal phospholipase A2 in experimental autoimmune uveitis in rats.

    PubMed

    Ohkawa, Ei; Hiraoka, Miki; Abe, Akira; Murata, Masaki; Ohguro, Hiroshi

    2016-08-01

    Intraocular inflammation leads to oxidative stress and may generate lipid oxidation products. The present study was conducted to elucidate the pathophysiological roles of the lysosomal phospholipase A2 (LPLA2), a phospholipid-degrading enzyme, and the production of oxidized phospholipids (oxPLs) in autoimmune uveitis using a rat model. Lewis rats were immunized with a bovine interphotoreceptor retinoid-binding protein (bIRBP) peptide with complete Freund's adjuvant (CFA) to induce experimental autoimmune uveitis (EAU). The aqueous humor (AH) and serum were collected every week for 4 weeks from the immunized rats. The LPLA2 activity of the AH and serum was detected using liposomes consisting of 1,2-dioleoylphosphatidylglycerol/N-acetylsphingosine as the substrate under acidic conditions. Immunohistochemical analysis was performed using antibodies against LPLA2 and oxPLs. The ocular inflammation was exacerbated at 2 weeks after immunization. The LPLA2 activity in the rat AH was increased by EAU induction, and was concomitant with the extent of inflammation in the anterior chamber (AC). In contrast, the LPLA2 activity in the rat serum was not influenced by EAU induction. At 2 weeks after immunization, immunoreactivity of LPLA2 was observed in infiltrated macrophages in the AC and vitreous cavity of the EAU rats. Furthermore, immunoreactivity of oxPLs was observed in the infiltrated macrophages of EAU rat eyes. These results demonstrated that the LPLA2 activity of the AH is augmented with the inflammation in the AC. The high expression of LPLA2 and production of oxPLs are found in the infiltrated macrophages in the acute inflammation of EAU rats. The present findings suggest the connection between LPLA2 activity and oxPL metabolism in the inflammation sites in the eye. PMID:27344956

  11. Correlation between secretion and phospholipase D activation in differentiated HL60 cells.

    PubMed Central

    Stutchfield, J; Cockcroft, S

    1993-01-01

    Receptor-directed agonists including N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), C5a, ATP and UTP all activate phospholipase D (PLD), which is accompanied by secretion in differentiated HL60 cells. Interference in the production of phosphatidase (PA) by the PLD pathway by diverting it towards the production of phosphatidylethanol (PEt) in the presence of ethanol leads to near-total inhibition of the secretion evoked by ATP and UTP and a partial inhibition of that evoked by fMetLeuPhe and C5a. In streptolysin-O-permeabilized cells, fMetLeuPhe is able to activate PLD, and this is dependent on the presence of a low concentration of guanosine 5'-[gamma-thio]-triphosphate (GTP[S]). Ca2+ (10 microM) and GTP[S] individually or in combination are also able to activate PLD and secretion. The stimulation of secretion in permeabilized cells stimulated by Ca2+ alone or fMetLeuPhe or GTP[S] is also abrogated when the production of PA is diverted to PEt by the presence of ethanol. Activation of PLD by GTP[S] or fMetLeuPhe is decreased if the cells are permeabilized first and GTP[S] or fMetLeuPhe is added subsequently. This corresponds well with the loss of the secretory response. We conclude that the ability of GTP[S] or fMetLeuPhe to stimulate secretion from permeabilized cells is dependent on a prior activation of the PLD signalling pathway. PA, generated as a consequence of PLD activation, acts as second messenger that can provide an initiating signal for secretion and is not required for exocytosis itself. PMID:8352731

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    Flick, J S; Thorner, J

    1993-01-01

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

  14. Characterization of a novel inhibitor of cytosolic phospholipase A2alpha, pyrrophenone.

    PubMed Central

    Ono, Takashi; Yamada, Katsutoshi; Chikazawa, Yukiko; Ueno, Masahiko; Nakamoto, Shozo; Okuno, Takayuki; Seno, Kaoru

    2002-01-01

    Cytosolic phospholipase A(2)alpha (cPLA(2)alpha), one of the three subtypes of cPLA(2) (alpha, beta and gamma), is thought to be a rate-limiting enzyme in eicosanoid biosynthesis. We developed a novel and potent cPLA(2)alpha inhibitor with an optically active pyrrolidine, termed pyrrophenone, and characterized this compound in detail using enzyme and cellular assay systems. Pyrrophenone, which shows strong inhibition of cPLA(2)alpha activity, is one of the most potent cPLA(2)alpha inhibitors reported to date. Similar inhibitory potencies for cPLA(2)alpha were obtained from three different assays. The inhibitory activity of pyrrophenone is two or three orders of magnitude more potent than arachidonyl trifluoromethyl ketone (AACOCF(3)) under the same assay conditions. Pyrrophenone shows reversible inhibition of cPLA(2)alpha and displays no characteristics of the slow-binding inhibition observed for AACOCF(3). Pyrrophenone also inhibited the esterase and lysophospholipase activities of cPLA(2)alpha. However, the inhibition by pyrrophenone of 14 kDa secretory PLA(2)s, types IB and IIA, was over two orders of magnitude less potent than that for cPLA(2)alpha. Pyrrophenone strongly inhibited arachidonic acid release in calcium ionophore (A23187)-stimulated human monocytic cells (THP-1 cells) in a dose-dependent manner with an IC(50) value of 0.024 microM, followed by suppression of eicosanoid synthesis, and also showed dose-dependent inhibition for interleukin-1-induced prostaglandin E(2) synthesis in human renal mesangial cells with an IC(50) value of 0.0081 microM. The mechanism of inhibition of eicosanoid synthesis in these cell-based assays was due to inhibition of only one step of arachidonic acid release without any effect on cyclo-oxygenase or lipoxygenase pathways. These results suggest that pyrrophenone could be a potential therapeutic agent for inflammatory diseases. PMID:11964173

  15. Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction.

    PubMed

    Supinski, Gerald S; Alimov, Alexander P; Wang, Lin; Song, Xiao-Hong; Callahan, Leigh A

    2016-05-15

    Calpain activation contributes to the development of infection-induced diaphragm weakness, but the mechanisms by which infections activate calpain are poorly understood. We postulated that skeletal muscle calcium-dependent phospholipase A2 (cPLA2) is activated by cytokines and has downstream effects that induce calpain activation and muscle weakness. We determined whether cPLA2 activation mediates cytokine-induced calpain activation in isolated skeletal muscle (C2C12) cells and infection-induced diaphragm weakness in mice. C2C12 cells were treated with the following: 1) vehicle; 2) cytomix (TNF-α 20 ng/ml, IL-1β 50 U/ml, IFN-γ 100 U/ml, LPS 10 μg/ml); 3) cytomix + AACOCF3, a cPLA2 inhibitor (10 μM); or 4) AACOCF3 alone. At 24 h, we assessed cell cPLA2 activity, mitochondrial superoxide generation, calpain activity, and calpastatin activity. We also determined if SS31 (10 μg/ml), a mitochondrial superoxide scavenger, reduced cytomix-mediated calpain activation. Finally, we determined if CDIBA (10 μM), a cPLA2 inhibitor, reduced diaphragm dysfunction due to cecal ligation puncture in mice. Cytomix increased C2C12 cell cPLA2 activity (P < 0.001) and superoxide generation; AACOCF3 and SS31 blocked increases in superoxide generation (P < 0.001). Cytomix also activated calpain (P < 0.001) and inactivated calpastatin (P < 0.01); both AACOCF3 and SS31 prevented these changes. Cecal ligation puncture reduced diaphragm force in mice, and CDIBA prevented this reduction (P < 0.001). cPLA2 modulates cytokine-induced calpain activation in cells and infection-induced diaphragm weakness in animals. We speculate that therapies that inhibit cPLA2 may prevent diaphragm weakness in infected, critically ill patients. PMID:26968769

  16. 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.

  17. Toxoplasma gondii inhibits mast cell degranulation by suppressing phospholipase Cγ-mediated Ca2+ mobilization

    PubMed Central

    Smith, Norah L.; Abi Abdallah, Delbert S.; Butcher, Barbara A.; Denkers, Eric Y.; Baird, Barbara; Holowka, David

    2013-01-01

    Toxoplasma gondii is well-known to subvert normal immune responses, however, mechanisms are incompletely understood. In particular, its capacity to alter receptor-activated Ca2+-mediated signaling processes has not been well-characterized. In initial experiments, we found evidence that T. gondii infection inhibits Ca2+ responses to fMetLeuPhe in murine macrophages. To further characterize the mechanism of inhibition of Ca2+ mobilization by T. gondii, we used the well-studied RBL mast cell model to probe the capacity of T. gondii to modulate IgE receptor-activated signaling within the first hour of infection. Ca2+ mobilization that occurs via IgE/FcεRI signaling leads to granule exocytosis in mast cells. We found that T. gondii inhibits antigen-stimulated degranulation in infected cells in a strain-independent manner. Under these conditions, we found that cytoplasmic Ca2+ mobilization, particularly antigen-mediated Ca2+ release from intracellular stores, is significantly reduced. Furthermore, stimulation-dependent activation of Syk kinase leading to tyrosine phosphorylation and activation of phospholipase Cγ is inhibited by infection. Therefore, we conclude that inhibitory effects of infection are likely due to parasite-mediated inhibition of the tyrosine kinase signaling cascade that results in reduced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Interestingly, inhibition of IgE/FcεRI signaling persists when tachyzoite invasion is arrested via cytochalasin D treatment, suggesting inhibition is mediated by a parasite-derived factor secreted into the cells during the invasion process. Our study provides direct evidence that immune subversion by T. gondii is initiated concurrently with invasion. PMID:23847603

  18. Targeted Inhibition of Phospholipase C γ2 Adaptor Function Blocks Osteoclastogenesis and Protects from Pathological Osteolysis*

    PubMed Central

    Decker, Corinne; Hesker, Pamela; Zhang, Kaihua; Faccio, Roberta

    2013-01-01

    Phospholipase C γ2 (PLCγ2) is a critical regulator of innate immune cells and osteoclasts (OCs) during inflammatory arthritis. Both the catalytic domain and the adaptor motifs of PLCγ2 are required for OC formation and function. Due to the high homology between the catalytic domains of PLCγ2 and the ubiquitously expressed PLCγ1, molecules encompassing the adaptor motifs of PLCγ2 were designed to test the hypothesis that uncoupling the adaptor and catalytic functions of PLCγ2 could specifically inhibit osteoclastogenesis and bone erosion. Wild-type (WT) bone marrow macrophages (BMM) that overexpress the tandem Src homology 2 (SH2) domains of PLCγ2 (SH2(N+C)) failed to form mature OCs and resorb bone in vitro. Activation of the receptor activator of NF-κB (RANK) signaling pathway, which is critical for OC development, was impaired in cells expressing SH2(N+C). Arrest in OC differentiation was evidenced by a reduction of p38 and Iκ-Bα phosphorylation as well as decreased NFATc1 and c-Fos/c-Jun levels. Consistent with our hypothesis, SH2(N+C) abrogated formation of the RANK-Gab2 complex, which mediates NF-κB and AP-1 activation following RANK ligand (RANKL) stimulation. Furthermore, the ability of SH2(N+C) to prevent inflammatory osteolysis was examined in vivo following RANKL or LPS injections over the calvaria. Both models induced osteolysis in the control group, whereas the SH2(N+C)-treated cohort was largely protected from bone erosion. Collectively, these data indicate that inflammatory osteolysis can be abrogated by treatment with a molecule composed of the tandem SH2 domains of PLCγ2. PMID:24081142

  19. Phospholipase A2 Receptor Autoantibodies and Clinical Outcome in Patients with Primary Membranous Nephropathy

    PubMed Central

    Hoxha, Elion; Thiele, Ina; Zahner, Gunther; Panzer, Ulf; Harendza, Sigrid

    2014-01-01

    Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, with an uncertain clinical outcome. The characterization of the phospholipase A2 receptor (PLA2R) as the major target antigen in primary MN and the detection of circulating autoantibodies in these patients is a major advance in understanding this disease. To test whether PLA2R antibody levels reflect disease activity or clinical outcome, we performed a prospective multicenter study of 133 adult patients with primary MN and detectable serum PLA2R antibodies who had not received immunosuppressive therapy. Patients were followed ≤24 months. PLA2R antibody levels associated with clinical disease activity (proteinuria) in patients with immunosuppressive therapy (n=101) or supportive care (n=32). Within 3 months, immunosuppressive therapy led to a sustained 81% reduction in PLA2R antibody levels paralleled by a 39% reduction in proteinuria. Patients who experienced remission of proteinuria after 12 months had significantly lower PLA2R antibody levels at the time of study inclusion compared with patients with no remission. Patients with high PLA2R antibody levels achieved remission of proteinuria significantly later than patients with low PLA2R antibody levels. PLA2R antibody levels fell over time in patients with spontaneous remission but remained elevated in patients who did not show a reduction in proteinuria. Multivariable Cox regression analysis confirmed PLA2R antibody level as an independent risk factor for not achieving remission of proteinuria. We conclude that a decrease in PLA2R antibody level is associated with a decrease of proteinuria in patients with primary MN. PMID:24610926

  20. Translational studies of lipoprotein-associated phospholipase A2 in inflammation and atherosclerosis

    PubMed Central

    Ferguson, Jane F; Hinkle, Christine C; Mehta, Nehal N; Bagheri, Roshanak; DerOhannessian, Stephanie L; Shah, Rhia; Mucksavage, Megan I; Bradfield, Jonathan P; Hakonarson, Hakon; Wang, Xuexia; Master, Stephen R; Rader, Daniel J; Li, Mingyao; Reilly, Muredach P

    2012-01-01

    Objectives To examine the role of lipoprotein-associated phospholipase A2 (Lp-PLA2/PLA2G7) in human inflammation and coronary atherosclerosis. Background Lp-PLA2 has emerged as a potential therapeutic target in coronary heart disease (CHD). Data supporting Lp-PLA2 are indirect and confounded by species differences; whether Lp-PLA2 is causal in CHD remains in question. Methods We examined inflammatory regulation of Lp-PLA2 during experimental endotoxemia in human, probed the source of Lp-PLA2 in human leukocytes under inflammatory conditions, and assessed the relationship of variation in PLA2G7, the gene encoding Lp-PLA2, with coronary artery calcification (CAC). Results In contrast to circulating TNFα and CRP, blood and monocyte Lp-PLA2 mRNA decreased transiently, and plasma Lp-PLA2 mass declined modestly during endotoxemia. In vitro, Lp-PLA2 expression increased dramatically during human monocyte to macrophage differentiation and further in inflammatory macrophages and foam like-cells. Despite only a marginal association of SNPs in PLA2G7 with Lp-PLA2 activity or mass, numerous PLA2G7 SNPs were associated with CAC. In contrast, several SNPs in CRP were significantly associated with plasma CRP levels but had no relation with CAC. Conclusions Circulating Lp-PLA2 did not increase during acute phase response in human, while inflammatory macrophages and foam cells, but not circulating monocytes, are major leukocyte sources of Lp-PLA2. Common genetic variation in PLA2G7 is associated with sub-clinical coronary atherosclerosis. These data link Lp-PLA2 to atherosclerosis in human while highlighting the challenge in using circulating Lp-PLA2 as a biomarker of Lp-PLA2 actions in the vasculature. PMID:22340269

  1. Kinetics of phospholipase A2, arachidonic acid, and eicosanoid appearance in mouse zymosan peritonitis.

    PubMed

    Lundy, S R; Dowling, R L; Stevens, T M; Kerr, J S; Mackin, W M; Gans, K R

    1990-04-01

    Intraperitoneal injection of zymosan into mice induces a peritonitis characterized by cellular influx, plasma leakage and the appearance of arachidonic acid (AA) metabolites. We report that zymosan injection also stimulates the accumulation of AA, docosahexaenoic acid, linoleic acid, and phospholipase A2 (PLA2) activity. The amount of the unsaturated fatty acids (UnFA) varies both with the zymosan dose and time. Significantly increased levels of UnFA were first detected 15 min after zymosan injection. Maximal levels of the UnFA were reached 1 to 2 h post zymosan injection (AA: 725 +/- 29 ng/mouse, docosahexaenoic acid: 296 +/- 23 ng/mouse, linoleic acid: 4489 +/- 179 ng/mouse) and declined to saline control levels by 8 h. PLA2 activity was significantly increased 5 to 15 min after zymosan injection. Maximal levels of PLA2 activity occurred 15 to 30 min after zymosan injection (31.8 +/- 9.1 nmol phospholipid/mg protein/h) and then decreased by 30% through 24 h. Neither the appearance of UnFA nor PLA2 activity correlated with cellular influx, but both were coincident with plasma exudation at 5 to 15 min after zymosan. However, maximal exudation occurred 1 to 2 h post zymosan injection similar to that seen with the UnFA but not PLA2. These latter results suggest that a significant portion of the UnFA found in the peritoneal cavity of zymosan-injected mice originates from the plasma. PLA2 activity at the early time points (5 to 15 min) may also contribute to the levels of UnFA via hydrolysis of tissue and/or cellular phospholipids. PMID:2108209

  2. Implication of phospholipase D in response of Hordeum vulgare root to short-term potassium deprivation.

    PubMed

    Hafsi, Chokri; Russo, Marco A; Sgherri, Cristina; Izzo, Riccardo; Navari-Izzo, Flavia; Abdelly, Chedly

    2009-03-15

    To verify the possible involvement of lipids and several other compounds including hydrogen peroxide (H(2)O(2)) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in the response of Hordeum vulgare to early potassium deprivation, plants were grown in hydroponic conditions for 30d with a modified Hewitt nutrient solution containing 3mM K(+). They were then incubated for increasing periods of time ranging from 2 to 36h in the same medium deprived of K(+). In contrast to leaves, root K(+) concentration showed its greatest decrease after 6h of treatment. The main lipids of the control barley roots were phospholipids (PL), representing more than 50% of the total lipids. PL did not change with treatment, whereas free sterols (FS) decreased following K(+) deprivation, showing a reduction of approximately 17% after 36h. With respect to the individual PL, 30h K(+) deprivation led to a reduction in phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI) levels, whereas phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidic acid (PA) levels increased. The maximum PA accumulation and the highest phospholipase D (PLD) activation, estimated by an accumulation of phosphatidylbutanol (PtBut), were observed after 24h of K(+) starvation. At the root level, H(2)O(2) showed the maximum value after 6h of incubation in -K solution. In parallel, G3PDH activity reached its minimum. On the basis of a concomitant stimulation of PLD activity and, consequently, PA accumulation, enhancement of H(2)O(2) production, and inhibition of G3PDH activity, we suggest a possible involvement of these three compounds in an early response to K(+) deprivation. PMID:18814934

  3. Phosphatidylcholine-Specific Phospholipase C and Sphingomyelinase Activities in Bacteria of the Bacillus cereus Group

    PubMed Central

    Pomerantsev, A. P.; Kalnin, K. V.; Osorio, M.; Leppla, S. H.

    2003-01-01

    Bacillus anthracis is nonhemolytic, even though it is closely related to the highly hemolytic Bacillus cereus. Hemolysis by B. cereus results largely from the action of phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelinase (SPH), encoded by the plc and sph genes, respectively. In B. cereus, these genes are organized in an operon regulated by the global regulator PlcR. B. anthracis contains a highly similar cereolysin operon, but it is transcriptionally silent because the B. anthracis PlcR is truncated at the C terminus. Here we report the cloning, expression, purification, and enzymatic characterization of PC-PLC and SPH from B. cereus and B. anthracis. We also investigated the effects of expressing PlcR on the expression of plc and sph. In B. cereus, PlcR was found to be a positive regulator of plc but a negative regulator of sph. Replacement of the B. cereus plcR gene by its truncated orthologue from B. anthracis eliminated the activities of both PC-PLC and SPH, whereas introduction into B. anthracis of the B. cereus plcR gene with its own promoter did not activate cereolysin expression. Hemolytic activity was detected in B. anthracis strains containing the B. cereus plcR gene on a multicopy plasmid under control of the strong B. anthracis protective antigen gene promoter or in a strain carrying a multicopy plasmid containing the entire B. cereus plc-sph operon. Slight hemolysis and PC-PLC activation were found when PlcR-producing B. anthracis strains were grown under anaerobic-plus-CO2 or especially under aerobic-plus-CO2 conditions. Unmodified parental B. anthracis strains did not demonstrate obvious hemolysis under the same conditions. PMID:14573681

  4. Phospholipase A2 in Experimental Allergic Bronchitis: A Lesson from Mouse and Rat Models

    PubMed Central

    Mruwat, Rufayda; Yedgar, Saul; Lavon, Iris; Ariel, Amiram; Krimsky, Miron; Shoseyov, David

    2013-01-01

    Background Phospholipases A2 (PLA2) hydrolyzes phospholipids, initiating the production of inflammatory lipid mediators. We have previously shown that in rats, sPLA2 and cPLA2 play opposing roles in the pathophysiology of ovalbumin (OVA)-induced experimental allergic bronchitis (OVA-EAB), an asthma model: Upon disease induction sPLA2 expression and production of the broncho-constricting CysLTs are elevated, whereas cPLA2 expression and the broncho-dilating PGE2 production are suppressed. These were reversed upon disease amelioration by treatment with an sPLA2 inhibitor. However, studies in mice reported the involvement of both sPLA2 and cPLA2 in EAB induction. Objectives To examine the relevance of mouse and rat models to understanding asthma pathophysiology. Methods OVA-EAB was induced in mice using the same methodology applied in rats. Disease and biochemical markers in mice were compared with those in rats. Results As in rats, EAB in mice was associated with increased mRNA of sPLA2, specifically sPLA2gX, in the lungs, and production of the broncho-constricting eicosanoids CysLTs, PGD2 and TBX2 in bronchoalveolar lavage (BAL). In contrast, EAB in mice was associated also with elevated cPLA2 mRNA and PGE2 production. Yet, treatment with an sPLA2 inhibitor ameliorated the EAB concomitantly with reverting the expression of both cPLA2 and sPLA2, and eicosanoid production. Conclusions In both mice and rats sPLA2 is pivotal in OVA-induced EAB. Yet, amelioration of asthma markers in mouse models, and human tissues, was observed also upon cPLA2 inhibition. It is plausible that airway conditions, involving multiple cell types and organs, require the combined action of more than one, essential, PLA2s. PMID:24204651

  5. Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor

    PubMed Central

    Aurass, Philipp; Oates, Clare V.; Tate, Edward W.; Hartland, Elizabeth L.; Flieger, Antje

    2015-01-01

    Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo. PMID:26216420

  6. Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells

    PubMed Central

    Marchini-Alves, Claudia Maria Meirelles; Barbosa Lorenzi, Valeria Cintra; da Silva, Elaine Zayas Marcelino; Mazucato, Vivian Marino; Jamur, Maria Celia; Oliver, Constance

    2015-01-01

    Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. It has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type. In mast cells it plays an important role in signal transduction. The aim of the present study was to clarify the role of PLD2 in the secretory pathway. RBL-2H3 cells, a mast cell line, transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2 were used. Previous observations showed that the Golgi complex was well organized in CA cells, but was disorganized and dispersed in CI cells. Furthermore, in CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized. These previous observations demonstrated that PLD2 is important for maintaining the morphology and organization of the Golgi complex. To further understand the role of PLD2 in secretory and vesicular trafficking, the role of PLD2 in the secretory process was investigated. Incorporation of sialic acid was used to follow the synthesis and transport of glycoconjugates in the cell lines. The modified sialic acid was subsequently detected by labeling with a fluorophore or biotin to visualize the localization of the molecule after a pulse-chase for various times. Glycoconjugate trafficking was slower in the CI cells and labeled glycans took longer to reach the plasma membrane. Furthermore, in CI cells sialic acid glycans remained at the plasma membrane for longer periods of time compared to RBL-2H3 cells. These results suggest that PLD2 activity plays an important role in regulating glycoconjugate trafficking in mast cells. PMID:26492088

  7. Membrane activity of the phospholipase C-δ1 pleckstrin homology (PH) domain

    PubMed Central

    2005-01-01

    PH-PLCδ1 [the PH domain (pleckstrin homology domain) of PLCδ1 (phospholipase C-δ1)] is among the best-characterized phosphoinositide-binding domains. PH-PLCδ1 binds with high specificity to the headgroup of PtdIns(4,5)P2, but little is known about its interfacial properties. In the present study, we show that PH-PLCδ1 is also membrane-active and can insert significantly into PtdIns(4,5)P2-containing monolayers at physiological (bilayer-equivalent) surface pressures. However, this membrane activity appears to involve interactions distinct from those that target PH-PLCδ1 to the PtdIns(4,5)P2 headgroup. Whereas the majority of PtdIns(4,5)P2-bound PH-PLCδ1 can be displaced by adding excess of soluble headgroup [Ins(1,4,5)P3], membrane activity of PH-PLCδ1 cannot. PH-PLCδ1 differs from other phosphoinositide-binding domains in that its membrane insertion does not require that the phosphoinositide-binding site be occupied. Significant monolayer insertion remains when the phosphoinositide-binding site is mutated, and PH-PLCδ1 can insert into monolayers that contain no PtdIns(4,5)P2 at all. Our results suggest a model in which reversible membrane binding of PH-PLCδ1, mediated by PtdIns(4,5)P2 or other acidic phospholipids, occurs without membrane insertion. Accumulation of the PH domain at the membrane surface enhances the efficiency of insertion, but does not significantly affect its extent, whereas the presence of phosphatidylethanolamine and cholesterol in the lipid mixture promotes the extent of insertion. This is the first report of membrane activity in an isolated PH domain and has implications for understanding the membrane targeting by this common type of domain. PMID:15755258

  8. Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages.

    PubMed

    Shridas, Preetha; Bailey, William M; Talbott, Kayla R; Oslund, Rob C; Gelb, Michael H; Webb, Nancy R

    2011-07-01

    Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content. PMID:21622863

  9. Ambient Air Pollution and Lipoprotein-Associated Phospholipase A2 in Survivors of Myocardial Infarction

    PubMed Central

    Hampel, Regina; Baumgärtner, Zita; Rückerl, Regina; Greven, Sonja; Koenig, Wolfgang; Peters, Annette; Schneider, Alexandra

    2011-01-01

    Background: Increasing evidence suggests a proatherogenic role for lipoprotein-associated phospholipase A2 (Lp-PLA2). A meta-analysis of published cohorts has shown that Lp-PLA2 is an independent predictor of coronary heart disease events and stroke. Objective: In this study, we investigated whether the association between air pollution and cardiovascular disease might be partly explained by increased Lp-PLA2 mass in response to exposure. Methods: A prospective longitudinal study of 200 patients who had had a myocardial infarction was performed in Augsburg, Germany. Up to six repeated clinical examinations were scheduled every 4–6 weeks between May 2003 and March 2004. Supplementary to the multicenter AIRGENE protocol, we assessed repeated plasma Lp-PLA2 concentrations. Air pollution data from a fixed monitoring site representing urban background concentrations were collected. We measured hourly means of particle mass [particulate matter (PM) < 10 µm (PM10) and PM < 2.5 µm (PM2.5) in aerodynamic diameter] and particle number concentrations (PNCs), as well as the gaseous air pollutants carbon monoxide (CO), sulfur dioxide (SO2), ozone (O3), nitric oxide (NO), and nitrogen dioxide (NO2). Data were analyzed using mixed models with random patient effects. Results: Lp-PLA2 showed a positive association with PM10, PM2.5, and PNCs, as well as with CO, NO2, NO, and SO2 4–5 days before blood withdrawal (lag 4–5). A positive association with O3 was much more immediate (lag 0). However, inverse associations with some pollutants were evident at shorter time lags. Conclusion: These preliminary findings should be replicated in other study populations because they suggest that the accumulation of acute and subacute effects or the chronic exposure to ambient particulate and gaseous air pollution may result in the promotion of atherosclerosis, mediated, at least in part, by increased levels of Lp-PLA2. PMID:21356620

  10. Cell-specific regulation of type II phospholipase A2 expression in rat mesangial cells.

    PubMed Central

    Konieczkowski, M; Sedor, J R

    1993-01-01

    IL-1 stimulates mesangial cells to synthesize specific proteins, including a non-pancreatic (Type II) phospholipase A2 (PLA2). We have studied the regulation of PLA2 by proinflammatory mediators, implicated in the pathogenesis of glomerulonephritis, and have assessed whether the activation of second messenger systems modulates or mimics PLA2 gene expression by cytokines. IL-1 alpha and beta, TNF alpha, and LPS, but not serum, IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme expression. IL-1-stimulated mesangial cells express a 1.0 kB PLA2 mRNA transcript that is induced in a dose- and time-dependent manner. IL-1-stimulated increases in steady-state PLA2 mRNA abundance result from a moderate increase in PLA2 transcription rate that is amplified by the prolonged persistence of the transcript. Forskolin and dibutyryl cAMP potentiate IL-1-induced PLA2 mRNA and enzyme expression, but have no effect in the absence of cytokine. 12-tetradecanoyl phorbol 13-acetate, sn-1, 2-dioctanoyl glycerol or 1-oleoyl-2-acetyl-sn-glycerol fail to induce PLA2 expression or to alter the effect of IL-1 when coincubated with the cytokine. In contrast, serum deprivation for 24 h specifically enhances IL-1-stimulated PLA2. Genistein potentiates PLA2 mRNA expression in cells exposed to both IL-1 and serum. The inhibitory effect of serum on IL-1-induced PLA2 mRNA abundance is reproduced by PDGF but not dexamethasone. These data demonstrate that the signaling pathways directly engaged by IL-1 to induce PLA2 expression in mesangial cells interact with several second messenger systems in a cell-specific manner. We speculate that IL-1 induces specialized changes in mesangial cell structure and function through direct activation of a transcription factor(s), that result in induction of a specific gene set. Images PMID:8227365

  11. Botanical Polyphenols Mitigate Microglial Activation and Microglia-Induced Neurotoxicity: Role of Cytosolic Phospholipase A2.

    PubMed

    Chuang, Dennis Y; Simonyi, Agnes; Cui, Jiankun; Lubahn, Dennis B; Gu, Zezong; Sun, Grace Y

    2016-09-01

    Microglia play a significant role in the generation and propagation of oxidative/nitrosative stress, and are the basis of neuroinflammatory responses in the central nervous system. Upon stimulation by endotoxins such as lipopolysaccharides (LPS), these cells release pro-inflammatory factors which can exert harmful effects on surrounding neurons, leading to secondary neuronal damage and cell death. Our previous studies demonstrated the effects of botanical polyphenols to mitigate inflammatory responses induced by LPS, and highlighted an important role for cytosolic phospholipase A2 (cPLA2) upstream of the pro-inflammatory pathways (Chuang et al. in J Neuroinflammation 12(1):199, 2015. doi: 10.1186/s12974-015-0419-0 ). In this study, we investigate the action of botanical compounds and assess whether suppression of cPLA2 in microglia is involved in the neurotoxic effects on neurons. Differentiated SH-SY5Y neuroblastoma cells were used to test the neurotoxicity of conditioned medium from stimulated microglial cells, and WST-1 assay was used to assess for the cell viability of SH-SY5Y cells. Botanicals such as quercetin and honokiol (but not cyanidin-3-O-glucoside, 3CG) were effective in inhibiting LPS-induced nitric oxide (NO) production and phosphorylation of cPLA2. Conditioned medium from BV-2 cells stimulated with LPS or IFNγ caused neurotoxicity to SH-SY5Y cells. Decrease in cell viability could be ameliorated by pharmacological inhibitors for cPLA2 as well as by down-regulating cPLA2 with siRNA. Botanicals effective in inhibition of LPS-induced NO and cPLA2 phosphorylation were also effective in ameliorating microglial-induced neurotoxicity. Results demonstrated cytotoxic factors from activated microglial cells to cause damaging effects to neurons and potential use of botanical polyphenols to ameliorate the neurotoxic effects. PMID:27339657

  12. The diversity of algal phospholipase D homologs revealed by biocomputational analysis.

    PubMed

    Beligni, María Verónica; Bagnato, Carolina; Prados, María Belén; Bondino, Hernán; Laxalt, Ana María; Munnik, Teun; Ten Have, Arjen

    2015-10-01

    Phospholipase D (PLD) participates in the formation of phosphatidic acid, a precursor in glycerolipid biosynthesis and a second messenger. PLDs are part of a superfamily of proteins that hydrolyze phosphodiesters and share a catalytic motif, HxKxxxxD, and hence a mechanism of action. Although HKD-PLDs have been thoroughly characterized in plants, animals and bacteria, very little is known about these enzymes in algae. To fill this gap in knowledge, we performed a biocomputational analysis by means of HMMER iterative profiling, using most eukaryotic algae genomes available. Phylogenetic analysis revealed that algae exhibit very few eukaryotic-type PLDs but possess, instead, many bacteria-like PLDs. Among algae eukaryotic-type PLDs, we identified C2-PLDs and PXPH-like PLDs. In addition, the dinoflagellate Alexandrium tamarense features several proteins phylogenetically related to oomycete PLDs. Our phylogenetic analysis also showed that algae bacteria-like PLDs (proteins with putative PLD activity) fall into five clades, three of which are novel lineages in eukaryotes, composed almost entirely of algae. Specifically, Clade II is almost exclusive to diatoms, whereas Clade I and IV are mainly represented by proteins from prasinophytes. The other two clades are composed of mitochondrial PLDs (Clade V or Mito-PLDs), previously found in mammals, and a subfamily of potentially secreted proteins (Clade III or SP-PLDs), which includes a homolog formerly characterized in rice. In addition, our phylogenetic analysis shows that algae have non-PLD members within the bacteria-like HKD superfamily with putative cardiolipin synthase and phosphatidylserine/phosphatidylglycerophosphate synthase activities. Altogether, our results show that eukaryotic algae possess a moderate number of PLDs that belong to very diverse phylogenetic groups. PMID:26986890

  13. Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.

    PubMed

    Cheng, H F; Jiang, M J; Chen, C L; Liu, S M; Wong, L P; Lomasney, J W; King, K

    1995-03-10

    In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC. PMID:7890667

  14. Modulation of human type II secretory phospholipase A2 by sphingomyelin and annexin VI.

    PubMed

    Koumanov, K; Wolf, C; Béreziat, G

    1997-08-15

    Conjectural results have been reported on the capacity of inflammatory secreted phospholipase A2 (sPLA2) to hydrolyse mammalian membrane phospholipids. Development of an assay based on the release of non-esterified fatty acids by the enzyme acting on the organized phospholipid mixture constituting the membrane matrix has led to the identification of two prominent effectors, sphingomyelin (SPH) and annexin. Recombinant human type II sPLA2 hydrolyses red-cell membrane phospholipids with a marked preference for the inner leaflet. This preference is apparently related to the high content of SPH in the outer leaflet, which inhibits sPLA2. This inhibition by SPH is specific for sPLA2. Cholesterol counteracts the inhibition of sPLA2 by SPH, suggesting that the SPH-to-cholesterol ratio accounts in vivo for the variable susceptibility of cell membranes to sPLA2. Different effects were observed of the presence of the non-hydrolysable D-alpha-dipalmitoyl phosphatidylcholine (D-DPPC), which renders the membranes rigid but does not inhibit sPLA2. Annexin VI was shown, along with other annexins, to inhibit sPLA2 activity by sequestering the phospholipid substrate. The present study has provided the first evidence that annexin VI, in concentrations that inhibit hydrolysis of purified phospholipid substrates, stimulated the hydrolysis of membrane phospholipids by sPLA2. The activation requires the presence of membrane proteins. The effect is specific for type II sPLA2 and is not reproducible with type I PLA2. The activation by annexin VI of sPLA2 acting on red cell membranes results in the preferential release of polyunsaturated fatty acids. It suggests that type II sPLA2, in conjunction with annexin VI, might be involved in the final step of endocytosis and/or exocytosis providing the free polyunsaturated fatty acids acting synergistically to cause membrane fusion. PMID:9337873

  15. Phospholipase A(2)-susceptible liposomes of anticancer double lipid-prodrugs.

    PubMed

    Arouri, Ahmad; Mouritsen, Ole G

    2012-03-12

    A novel approach to anticancer drug delivery is presented based on lipid-like liposome-forming anticancer prodrugs that are susceptible to secretory phospholipase A(2) (sPLA(2)) that is overexpressed in several cancer types. The approach provides a selective unloading of anticancer drugs at the target tissues, as well as circumvents the necessity for "conventional" drug loading. In our attempts to improve the performance of the liposomes in vivo, several PEGylated and non-PEGylated liposomal formulations composed of a retinoid prodrug premixed with the sPLA(2)-hydrolyzable DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared. Besides favorably modifying the physicochemical properties of the liposomes, the incorporation of DPPC and PEG-lipids in the liposomes should substantially enhance the enzymatic activity, as concluded from literature. In addition, one can reap benefits from the presumed permeability enhancing effect of the liberated fatty acids and lysolipids. The size distribution of the prepared liposomes as well as their phase behavior, enzymatic hydrolysis, and cytotoxicity, in the presence and absence of sPLA(2), were determined. The liposomes were around 100nm in diameter and in the gel/fluid coexistence region at 37°C. The enzymatic hydrolysis of the prodrug was pronouncedly accelerated upon the premixing with DPPC, and the hydrolysis was further enhanced by PEGylation. Interestingly, the faster hydrolysis of the prodrug and the released fatty acids and lysolipids from DPPC did not improve the cytotoxicity of the mixture; the effect of combining the prodrug with DPPC was additive and not synergistic. The data presented here question the significance of the permeability enhancing effects claimed for fatty acids and lysolipids at the target cell membrane, and whether these effects can be achieved using physiologically achievable concentrations of fatty acids and lysolipids. PMID:21946258

  16. Phase Variation in the Helicobacter pylori Phospholipase A Gene and Its Role in Acid Adaptation

    PubMed Central

    Tannæs, Tone; Dekker, Niek; Bukholm, Geir; Bijlsma, Jetta J. E.; Appelmelk, Ben J.

    2001-01-01

    Previously, we have shown that Helicobacter pylori can spontaneously and reversibly change its membrane lipid composition, producing variants with low or high content of lysophospholipids. The “lyso” variant contains a high percentage of lysophospholipids, adheres better to epithelial cells, and releases more proteins such as urease and VacA, compared to the “normal” variant, which has a low content of lysophospholipids. Prolonged growth of the normal variant at pH 3.5, but not under neutral conditions, leads to enrichment of lyso variant colonies, suggesting that the colony switch is relevant to acid adaptation. In this study we show that the change in membrane lipid composition is due to phase variation in the pldA gene. A change in the (C) tract length of this gene results in reversible frameshifts, translation of a full-length or truncated pldA, and the production of active or inactive outer membrane phospholipase A (OMPLA). The role of OMPLA in determining the colony morphology was confirmed by the construction of an OMPLA-negative mutant. Furthermore, variants with an active OMPLA were able to survive acidic conditions better than variants with the inactive form. This explains why the lyso variant is selected at low pH. Our studies demonstrate that phase variation in the pldA gene, resulting in an active form of OMPLA, is important for survival under acidic conditions. We also demonstrated the active OMPLA genotype in fresh isolates of H. pylori from patients referred to gastroscopy for dyspepsia. PMID:11705905

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

    PubMed Central

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

    2002-01-01

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

  18. Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin*

    PubMed Central

    Yamaga, Masaki; Kielar-Grevstad, D. Michelle; Martin, Thomas F. J.

    2015-01-01

    PI(4,5)P2 localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca2+-triggered vesicle exocytosis, but the impact of local PI(4,5)P2 hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca2+-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P2 hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (also known as CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca2+-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 or 95 mm KCl buffers increased peak Ca2+ levels to ∼400 or ∼800 nm, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mm K+ preferentially elicited the exocytosis of plasma membrane-resident vesicles, whereas 95 mm K+ preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mm K+ but not with 56 mm K+ activated PLCη2 to catalyze PI(4,5)P2 hydrolysis. The decrease in PI(4,5)P2 promoted F-actin disassembly, which increased exocytosis of newly arriving vesicles. Consistent with its role as a Ca2+-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P2 for coordinating cytoskeletal dynamics with vesicle exocytosis and reveal a new role for PLCη2 as a Ca2+-dependent regulator of F-actin dynamics and vesicle trafficking. PMID:26432644

  19. Phospholipase A2-interacting weak neurotoxins from venom of monocled cobra Naja kaouthia display cell-specific cytotoxicity.

    PubMed

    Mukherjee, Ashis K

    2008-06-15

    The molecular weights of two phospholipase A(2) (PLA(2))-interacting polypeptides (kaouthiotoxins (KTXs)-KTX-A and KTX-B) purified from the venom of monocled cobra Naja kaouthia, were estimated by mass spectrometry as 7722 and 7627Da, respectively. Binary sequence alignment showed that both KTXs share substantial sequence homology with weak neurotoxins from cobra venom and they were devoid of any enzymatic activity. Their pI was determined at pH 8.1 showing basic nature of these proteins. KTXs displayed cell-specific cytotoxicity on mammalian and insect cells. PMID:18456298

  20. Tubular Coupling

    NASA Technical Reports Server (NTRS)

    Rosenbaum, Bernard J. (Inventor)

    2000-01-01

    A system for coupling a vascular overflow graft or cannula to a heart pump. A pump pipe outlet is provided with an external tapered surface which receives the end of a compressible connula. An annular compression ring with a tapered internal bore surface is arranged about the cannula with the tapered internal surface in a facing relationship to the external tapered surface. The angle of inclination of the tapered surfaces is converging such that the spacing between the tapered surfaces decreases from one end of the external tapered surface to the other end thereby providing a clamping action of the tapered surface on a cannula which increases as a function of the length of cannula segment between the tapered surfaces. The annular compression ring is disposed within a tubular locking nut which threadedly couples to the pump and provides a compression force for urging the annular ring onto the cannula between the tapered surfaces. The nut has a threaded connection to the pump body. The threaded coupling to the pump body provides a compression force for the annular ring. The annular ring has an annular enclosure space in which excess cannula material from the compression between the tapered surfaces to "bunch up" in the space and serve as an enlarged annular ring segment to assist holding the cannula in place. The clamped cannula provides a seamless joint connection to the pump pipe outlet where the clamping force is uniformly applied to the cannula because of self alignment of the tapered surfaces. The nut can be easily disconnected to replace the pump if necessary.

  1. Dark coupling

    SciTech Connect

    Gavela, M.B.; Hernández, D.; Honorez, L. Lopez; Mena, O.; Rigolin, S. E-mail: d.hernandez@uam.es E-mail: omena@ific.uv.es

    2009-07-01

    The two dark sectors of the universe—dark matter and dark energy—may interact with each other. Background and linear density perturbation evolution equations are developed for a generic coupling. We then establish the general conditions necessary to obtain models free from non-adiabatic instabilities. As an application, we consider a viable universe in which the interaction strength is proportional to the dark energy density. The scenario does not exhibit ''phantom crossing'' and is free from instabilities, including early ones. A sizeable interaction strength is compatible with combined WMAP, HST, SN, LSS and H(z) data. Neutrino mass and/or cosmic curvature are allowed to be larger than in non-interacting models. Our analysis sheds light as well on unstable scenarios previously proposed.

  2. Thermoacoustic couple

    DOEpatents

    Wheatley, J.C.; Swift, G.W.; Migliori, A.

    1983-10-04

    An apparatus and method for determining acoustic power density level and its direction in a fluid using a single sensor are disclosed. The preferred embodiment of the apparatus, which is termed a thermoacoustic couple, consists of a stack of thin, spaced apart polymeric plates, selected ones of which include multiple bimetallic thermocouple junctions positioned along opposite end edges thereof. The thermocouple junctions are connected in series in the nature of a thermopile, and are arranged so as to be responsive to small temperature differences between the opposite edges of the plates. The magnitude of the temperature difference, as represented by the magnitude of the electrical potential difference generated by the thermopile, is found to be directly related to the level of acoustic power density in the gas.

  3. Changes of phospholipase A₂ and C activities during dry-cured duck processing and their relationship with intramuscular phospholipid degradation.

    PubMed

    Wang, Daoying; Zhang, Muhan; Bian, Huan; Xu, Weimin; Xu, Xinglian; Zhu, Yongzhi; Liu, Fang; Geng, Zhiming; Zhou, Guanghong

    2014-02-15

    Phospholipid hydrolysis, as the main stage and cause of phopholipid degradation, is generally attributed to phospholipases. In this study, the changes of phospholipase A₂ (PLA₂) and C (PLC) activities, neutral lipid, free fatty acids and phospholipids in dry-cured duck leg muscles during processing, were examined. The composition of free fatty acids and neutral lipids increased significantly (P<0.05) with extension of processing time while the phospholipids composition decreased. The PLA₂ and PLC activity decreased in the final product, but retained 83.70% and 86.78% of their initial activities, respectively. The relative activities of both PLA₂ and PLC highly correlated with the decline of phospholipids and the increase of free fatty acids. High correlations were also obtained between the relative activities of PLC and the increase of neutral lipid (P<0.01). All these results suggest that PLA₂ and PLC contribute to the degradation of intramuscular phospholipids during the processing of dry-cured duck. PMID:24128575

  4. Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death

    PubMed Central

    Gonçalves, A. Pedro; Cordeiro, J. Miguel; Monteiro, João; Muñoz, Alberto; Correia-de-Sá, Paulo; Read, Nick D.; Videira, Arnaldo

    2014-01-01

    ABSTRACT The model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca2+ ([Ca2+]c) dynamics and a distinct Ca2+ signature that involves Ca2+ influx from the external medium and internal Ca2+ stores. We investigated the molecular basis of this Ca2+ response by using [Ca2+]c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca2+ signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca2+ entry does not occur through the hitherto described high- and low-affinity Ca2+ uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca2+ release from internal stores following IP3 formation combines with the extracellular Ca2+ influx. PMID:25037570

  5. Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity.

    PubMed

    Buerth, Christoph; Kovacic, Filip; Stock, Janpeter; Terfrüchte, Marius; Wilhelm, Susanne; Jaeger, Karl-Erich; Feldbrügge, Michael; Schipper, Kerstin; Ernst, Joachim F; Tielker, Denis

    2014-06-01

    CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities. PMID:24469105

  6. Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2.

    PubMed

    Casais-E-Silva, Luciana L; Teixeira, Catarina F P; Lebrun, Ivo; Lomonte, Bruno; Alape-Girón, Alberto; Gutiérrez, José María

    2016-08-22

    The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro