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Sample records for stimulates phospholipase a2

  1. 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

  2. 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

  3. 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

  4. 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

  5. Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

    SciTech Connect

    Okayasu, T.; Hasegawa, K.; Ishibashi, T.

    1987-07-01

    Addition of platelet-activating factor (PAF) to cells doubly labeled with (/sup 14/C)glycerol plus (/sup 3/H)arachidonic acid resulted in a transient decrease of (/sup 14/C)glycerol-labeled phosphatidylinositol (PI) and a transient increase of (/sup 14/C)glycerol-labeled lysophosphatidylinositol (LPI). (/sup 3/H)Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The /sup 3/H//sup 14/C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of (/sup 14/C)glycerol-labeled DG paralleled the loss of triacyl (/sup 14/C)glycerol and the /sup 3/H//sup 14/C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- (/sup 3/H)inositol-prelabeled cells, PAF induced a transient decrease of (/sup 3/H)phosphatidylinositol-4,5-bis-phosphate (TPI) and (/sup 3/H)phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with /sup 32/Pi induced a transient decrease of (/sup 32/P)polyphosphoinositides at 20 sec to 1 min. (/sup 32/P)LPI appeared within 10 sec after stimulation and paralleled the loss of (/sup 32/P)PI. (/sup 3/H)Inositol triphosphate, (/sup 3/H)inositol diphosphate, and (/sup 3/H)inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.

  6. Inhibition of PAF synthesis by stimulated human polymorphonuclear leucocytes with cloricromene, an inhibitor of phospholipase A2 activation.

    PubMed Central

    Ribaldi, E.; Mezzasoma, A. M.; Francescangeli, E.; Prosdocimi, M.; Nenci, G. G.; Goracci, G.; Gresele, P.

    1996-01-01

    1. A phospholipase A2 (PLA2) represents the key enzyme in the remodelling pathway of platelet-activating factor (PAF) synthesis in human polymorphonuclear (PMN) leucocytes. 2. PLA2 activation is also the rate-limiting step for the release of the arachidonic acid utilized for the synthesis of leukotrienes in stimulated leucocytes; however, it is unknown whether the PLA2s involved in the two biosynthetic pathways are identical. 3. Cloricromene (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy- carbonylmethoxy coumarin) is an antithrombotic coumarin derivative which inhibits platelet and leucocyte function and suppresses arachidonic acid liberation by interfering with PLA2 activation. 4. The aim of the present study was to assess whether chloricromene inhibits PAF synthesis by stimulated human polymorphonuclear leucocytes (PMNs). 5. Cloricromene (50-500 microM) inhibited in a concentration-dependent manner the release of PAF, as measured by h.p.l.c. bioassay, from A23187-stimulated PMNs. Significant inhibition (45%) of PAF-release was obtained with 50 microM cloricromene and the IC50 was 85 microM. Mepacrine (500 microM), a non-specific PLA2 inhibitor, strikingly reduced PAF release. 6. The incorporation of [3H]-acetate into [3H]-PAF induced by serum-treated zymosan in human PMNs was also inhibited concentration-dependently by cloricromene, with an IC50 of 105 microM. Mepacrine also suppressed [3H]-acetate incorporation into [3H]-PAF. 7. Cloricromene did not affect the activities of the enzymes involved in PAF-synthesis acetyltransferase or phosphocholine transferase. 8. Our data demonstrate that cloricromene, an inhibitor of PLA2-activation in human leucocytes, reduces the synthesis of PAF by stimulated PMNs. This finding has a twofold implication: the PLA2s (or the mechanisms that regulate their activation) involved in PAF synthesis and arachidonate release in human leucocytes are either identical or else indistinguishable by their sensitivity to cloricromene

  7. Serotonin stimulates phospholipase A2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis.

    PubMed Central

    Felder, C C; Kanterman, R Y; Ma, A L; Axelrod, J

    1990-01-01

    Serotonin (5-HT) stimulated the release of arachidonic acid in hippocampal neurons cocultured with glial cells but not in glial cultures alone. Similar results were observed for the 5-HT-stimulated release of inositol phosphates. These results suggest a neural but not glial origin of both responses. Pharmacological studies suggested that release of arachidonic acid and inositol phosphates was mediated by a type 2 5-HT (5-HT2) receptor. 5-HT-stimulated release of arachidonic acid was also detected in cortical neurons, which contain high levels of 5-HT2 receptors, but not striatum, spinal cord, or cerebellar granule cells, which have very low levels or are devoid of 5-HT2 receptors. The phorbol ester phorbol 12-myristate 13-acetate augmented the 5-HT-stimulated release of arachidonic acid but inhibited the 5-HT-stimulated release of inositol phosphates. 5-HT-stimulated release of arachidonic acid, but not inositol phosphates, was dependent on extracellular calcium. 5-HT stimulated the release of [3H]lysophosphatidylcholine from [3H]choline-labeled cells with no increase in the release of [3H]choline or phospho[3H]choline. These data suggest that 5-HT stimulated the release of arachidonic acid in hippocampal neurons through the activation of phospholipase A2, independent of the activation of phospholipase C. PMID:2315313

  8. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. )

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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...

  20. 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

  1. 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

  2. Bisgma Stimulates Prostaglandin E2 Production in Macrophages via Cyclooxygenase-2, Cytosolic Phospholipase A2, and Mitogen-Activated Protein Kinases Family

    PubMed Central

    Lee, Shiuan-Shinn; Li, Yi-Ching; Chang, Yu-Chao

    2013-01-01

    Background Bisphenol A-glycidyl-methacrylate (BisGMA) employs as a monomer in dental resins. The leakage of BisGMA from composite resins into the peripheral environment can result in inflammation via macrophage activation. Prostaglandin E2 (PGE2) is a key regulator of immunopathology in inflammatory reactions. Little is known about the mechanisms of BisGMA-induced PGE2 expression in macrophage. The aim of this study was to evaluate the signal transduction pathways of BisGMA-induced PGE2 production in murine RAW264.7 macrophages. Methodology/Principal Findings Herein, we demonstrate that BisGMA can exhibit cytotoxicity to RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). In addition, PGE2 production, COX-2 expression, and cPLA2 phosphorylation were induced by BisGMA on RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). Moreover, BisGMA could induce the phosphorylation of ERK1/2 pathway (MEK1/2, ERK1/2, and Elk), p38 pathway (MEK3/6, p38, and MAPKAPK2), and JNK pathway (MEK4, JNK, and c-Jun) in a dose- and time-dependent manner (p<0.05). Pretreatment with AACOCF3, U0126, SB203580, and SP600125 significantly diminished the phosphorylation of cPLA2, ERK1/2, p38, and JNK stimulated by BisGMA, respectively (p<0.05). BisGMA-induced cytotoxicity, cPLA2 phosphorylation, PGE2 generation, and caspases activation were reduced by AACOCF3, U0126, SB203580, and SP600125, respectively (p<0.05). Conclusions These results suggest that BisGMA induced-PGE2 production may be via COX-2 expression, cPLA2 phosphorylation, and the phosphorylation of MAPK family. Cytotoxicity mediated by BisGMA may be due to caspases activation through the phosphorylation of cPLA2 and MAPKs family. PMID:24376609

  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. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. [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

  5. 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

  6. 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

  7. 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

  8. 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

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

    PubMed

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

    2002-10-01

    An acidic phospholipase A(2) (OH APLA(2)-II) with an isoelectric point (pI) of 4.0 was recently isolated from Ophiophagus hannah (king cobra) from Guangxi province, China. Comparison of this enzyme to a previously reported homologous phospholipase A(2) from the same venom shows that it lacks toxicity and exhibits a greater phospholipase activity. OH APLA(2)-II has been crystallized by the hanging-drop vapour-diffusion method using 1,6-hexanediol and magnesium chloride as precipitants. The crystal belongs to space group P6(3), with unit-cell parameters a = b = 98.06, c = 132.39 A. The diffraction data were collected under cryoconditions (100 K) and reduced to 2.1 A resolution. A molecular-replacement solution has been determined and shows that there are six molecules in one asymmetric unit. PMID:12351830

  10. 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

  11. 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...

  12. 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

  13. 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

  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. 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. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. Sequential release of TNFα and phospholipase A2 in a rat model of LPS-induced pleurisy

    PubMed Central

    Bucci, M.; D′Acquisto, F.; Parente, L.; Cirino, G.

    1997-01-01

    The levels of extracellular phospholipase A2 (sPLA2) and TNFα, and cell accumulation were measured in the pleural washings obtained at different times following the induction of Escherichia coli lipopolysaccharide (LPS, 100 μg/cavity) pleurisy in rats. TNFα peaked at 2 hours (3036 ± 160.3 units/ml) and decreased thereafter. Conversely, levels of sPLA2 peaked at 48 hours (1.97 ± 0.64 ng/ml) and were increased further (14.02 ± 4.16 ng/ml) by pretreatment with anti-TNFα antibody. Cell accumulation was not affected by antibody pretreatment. These data indicate that the sPLA2 enzyme is involved in LPS-induced pleurisy. The enzyme seems not to be stimulated by TNFα which may be involved in the downregulation of sPLA2 in this model of inflammation. PMID:18472822

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

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

    PubMed

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

    2002-06-01

    The crystal structure of an acidic phospholipase A(2) from Ophiophagus hannah (king cobra) has been determined by molecular replacement at 2.6-A resolution to a crystallographic R factor of 20.5% (R(free)=23.3%) with reasonable stereochemistry. The venom enzyme contains an unusual "pancreatic loop." The conformation of the loop is well defined and different from those in pancreas PLA(2), showing its structural variability. This analysis provides the first structure of a PLA(2)-type cardiotoxin. The sites related to the cardiotoxic and myotoxic activities are explored and the oligomer observed in the crystalline state is described. PMID:12217659

  14. 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

  15. 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

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

    PubMed Central

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

    2014-01-01

    The M-type phospholipase A2 receptor (PLA2R) is expressed in podocytes in human glomeruli. Group IB secretory phospholipase A2 (sPLA2 IB), which is one of the ligands of the PLA2R, is more highly expressed in chronic renal failure patients than in controls. However, the roles of the PLA2R and sPLA2 IB in the pathogenesis of glomerular diseases are unknown. In the present study, we found that more podocyte apoptosis occurs in the kidneys of patients with higher PLA2R and serum sPLA2 IB levels. In vitro, we demonstrated that human podocyte cells expressed the PLA2R in the cell membrane. After binding with the PLA2R, sPLA2 IB induced podocyte apoptosis in a time- and concentration-dependent manner. sPLA2 IB-induced podocyte PLA2R upregulation was not only associated with increased ERK1/2 and cPLA2α phosphorylation but also displayed enhanced apoptosis. In contrast, PLA2R-silenced human podocytes displayed attenuated apoptosis. sPLA2 IB enhanced podocyte arachidonic acid (AA) content in a dose-dependent manner. These data indicate that sPLA2 IB has the potential to induce human podocyte apoptosis via binding to the PLA2R. The sPLA2 IB-PLA2R interaction stimulated podocyte apoptosis through activating ERK1/2 and cPLA2α and through increasing the podocyte AA content. PMID:25335547

  17. 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

  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. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Intracellular signaling in the regulation of renal Na-K-ATPase. I. Role of cyclic AMP and phospholipase A2.

    PubMed Central

    Satoh, T; Cohen, H T; Katz, A I

    1992-01-01

    We have reported that dopamine (DA) inhibits Na-K-ATPase activity in the cortical collecting duct (CCD) by stimulating the DA1 receptor, and the present study was designed to evaluate the mechanism of this effect. Short-term exposure (15-30 min) of microdissected rat CCD to DA, a DA1 agonist (fenoldopam), vasopressin (AVP), forskolin, or dibutyryl cAMP (dBcAMP), which increase cAMP content by different mechanisms, strongly (approximately 60%) inhibited Na-K-ATPase activity. 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, completely blocked Na-K-ATPase inhibition by DA or fenoldopam, and IP20, an inhibitor peptide of cAMP-dependent protein kinase A (PKA), abolished the Na:K pump effect of all the cAMP agonists listed above. To verify whether the mechanism of pump inhibition by agents that increase cell cAMP involves phospholipase A2 (PLA2), we used mepacrine, a PLA2 inhibitor, which also abolished Na-K-ATPase inhibition by DA or fenoldopam, as well as by AVP, forskolin, or dBcAMP. Arachidonic acid (10(-7) - 10(-4) M) inhibited Na-K-ATPase activity in dose-dependent fashion. Corticosterone, which induces lipomodulin, a PLA2 inhibitor protein inactivated by PKA, equally abolished the pump effects of DA, fenoldopam, forskolin, and dBcAMP, suggesting that lipomodulin might act between PKA and PLA2 in cAMP-dependent pump regulation. We conclude that dopamine inhibits Na-K-ATPase activity in the CCD through a DA1 receptor-mediated cAMP-PKA pathway that involves the stimulation of PLA2 and arachidonic acid release, possibly mediated by inactivation of lipomodulin. This pathway is shared by other agonists that increase cell cAMP and thus stimulate PKA activity. PMID:1349027

  13. Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation

    PubMed Central

    Xie, Chanlu; Hua, Sheng; Li, Jianfang; Wang, Tingfeng; Yao, Mu; Vignarajan, Soma; Teng, Ying; Hejazi, Leila; Liu, Bingya; Dong, Qihan

    2014-01-01

    A constitutive activation of protein kinase B (AKT) in a hyper-phosphorylated status at Ser473 is one of the hallmarks of anti-EGFR therapy-resistant colorectal cancer (CRC). The aim of this study was to examine the role of cytosolic phospholipase A2α (cPLA2α) on AKT phosphorylation at Ser473 and cell proliferation in CRC cells with mutation in phosphoinositide 3-kinase (PI3K). AKT phosphorylation at Ser473 was resistant to EGF stimulation in CRC cell lines of DLD-1 (PIK3CAE545K mutation) and HT-29 (PIK3CAP499T mutation). Over-expression of cPLA2α by stable transfection increased basal and EGF-stimulated AKT phosphorylation and proliferation in DLD-1 cells. In contrast, silencing of cPLA2α with siRNA or inhibition with Efipladib decreased basal and EGF-stimulated AKT phosphorylation and proliferation in HT-29. Treating animals transplanted with DLD-1 with Efipladib (10 mg/kg, i.p. daily) over 14 days reduced xenograft growth by >90% with a concomitant decrease in AKT phosphorylation. In human CRC tissue, cPLA2α expression and phosphorylation were increased in 63% (77/120) compared with adjacent normal mucosa determined by immunohistochemistry. We conclude that cPLA2α is required for sustaining AKT phosphorylation at Ser473 and cell proliferation in CRC cells with PI3K mutation, and may serve as a potential therapeutic target for treatment of CRC resistant to anti-EGFR therapy. PMID:25365190

  14. 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

  15. 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

  16. 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

  17. 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

  18. Neuromuscular effects of a toxic phospholipase A2 and its nontoxic homologue from the venom of the sea snake, Laticauda colubrina.

    PubMed

    Rowan, E G; Harvey, A L; Takasaki, C; Tamiya, N

    1989-01-01

    A single chain phospholipase A2 (LcPLA-II) and a homologous protein lacking enzymatic activity (LcPLH-I) isolated from the venom of the Solomon Island sea snake (Laticauda colubrina) were tested for effects on neuromuscular transmission and muscle contractility on chick biventer cervicis and mouse hemidiaphragm preparations. LcPLA-II (7.5 nM-1.5 microM) blocked indirectly elicited muscle contractions of both preparations. Low concentrations of LcPLA-II caused little change in sensitivity to acetylcholine, carbachol and KCl. The homologue LcPLH-I (375 nM-1.5 microM) reduced the responses of the biventer cervicis preparation to indirect stimulation and abolished responses to acetylcholine and carbachol, but it did not block KCl responses. These effects were due to minor contamination by a post-junctional neurotoxin. LcPLH-I (375 nM-750 nM) had no effect on indirectly stimulated hemidiaphragm preparations. It is concluded that LcPLA-II blocks neuromuscular transmission by a prejunctional action, and that the homologue lacking phospholipase A2 activity also lacks neuromuscular activity. PMID:2749757

  19. Design of Group IIA Secreted/Synovial Phospholipase A2 Inhibitors: An Oxadiazolone Derivative Suppresses Chondrocyte Prostaglandin E2 Secretion

    PubMed Central

    Dong, Chang Zhi; Plocki, Stéphanie; Tsagris, Lydia; Rannou, François; Massicot, France; Djimdé, Atimé; El-Hayek, Elissar; Shi, Yiming; Heymans, Françoise; Gresh, Nohad; Chauvet, Caroline

    2010-01-01

    Group IIA secreted/synovial phospholipase A2 (GIIAPLA2) is an enzyme involved in the synthesis of eicosanoids such as prostaglandin E2 (PGE2), the main eicosanoid contributing to pain and inflammation in rheumatic diseases. We designed, by molecular modeling, 7 novel analogs of 3-{4-[5(indol-1-yl)pentoxy]benzyl}-4H-1,2,4-oxadiazol-5-one, denoted C1, an inhibitor of the GIIAPLA2 enzyme. We report the results of molecular dynamics studies of the complexes between these derivatives and GIIAPLA2, along with their chemical synthesis and results from PLA2 inhibition tests. Modeling predicted some derivatives to display greater GIIAPLA2 affinities than did C1, and such predictions were confirmed by in vitro PLA2 enzymatic tests. Compound C8, endowed with the most favorable energy balance, was shown experimentally to be the strongest GIIAPLA2 inhibitor. Moreover, it displayed an anti-inflammatory activity on rabbit articular chondrocytes, as shown by its capacity to inhibit IL-1β-stimulated PGE2 secretion in these cells. Interestingly, it did not modify the COX-1 to COX-2 ratio. C8 is therefore a potential candidate for anti-inflammatory therapy in joints. PMID:20531958

  20. Phospholipase treatment of accessory cells that have been exposed to antigen selectively inhibits antigen-specific Ia-restricted, but not allospecific, stimulation of T lymphocytes.

    PubMed Central

    Falo, L D; Benacerraf, B; Rock, K L

    1986-01-01

    The corecognition of antigen and class II major histocompatibility complex (MHC) molecules (Ia molecules) by the T-cell receptor is a cell surface event. Before antigen is recognized, it must be taken up, processed, and displayed on the surface of an Ia-bearing accessory cell (antigen-presenting cell, APC). The exact nature of antigen processing and the subsequent associations of antigen with the APC plasma membrane, Ia molecules, and/or the T-cell receptor are not well defined. To further analyze these events, we have characterized the processing and presentation of the soluble polypeptide antigen bovine insulin. We found that this antigen requires APC-dependent processing, as evidenced by the inability of metabolically inactivated APCs to present native antigen to antigen plus Ia-specific T-T hybridomas. The ability of the same APCs to present antigen after uptake and processing showed that this antigen subsequently becomes stably associated with the APC plasma membrane. To characterize the basis for this association, we analyzed its sensitivity to enzymatic digestion. APCs exposed to antigen, treated with phospholipase A2, and then immediately fixed lost the ability to stimulate bovine insulin plus I-Ad-specific hybridomas. In contrast, the ability of these same APCs to stimulate I-Ad allospecific hybridomas was unaffected. This effect of phospholipase is not mimicked by the broadly active protease Pronase, nor is there evidence for contaminating proteases in the phospholipase preparation. These results suggest that one consequence of antigen processing may be an antigen-lipid association that contributes to the anchoring of antigen to the APC membrane. The implications of this model are discussed. PMID:3529095

  1. 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

  2. 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

  3. Oxidant-mediated activation of cytosolic phospholipase a(2) in pulmonary endothelium: role of protein kinase C alpha and a pertussis toxin-sensitive protein.

    PubMed

    Chakraborti, Sajal; Das, Sudip; Chakraborti, Tapati

    2005-01-01

    The authors have previously demonstrated that the oxidant t-buOOH stimulates phospholipase A(2) (PLA(2)) activity in bovine pulmonary artery endothelial cells (S. Chakraborti et al. American Journal of Physiology, 257, L430-L437, 1989). Herein, the authors sought to investigate the mechanism by which t-buOOH stimulates PLA(2) activity and the role of protein kinase C (PKC) in this scenario. Treatment of bovine pulmonary artery endothelial cells with t-buOOH stimulated an aprotinin-sensitive protease activity, PKC activity, and PLA(2) activity in the cell membrane. Pretreatment with intracellular Ca(2+) chelator (BAPTA-AM), PKCalpha inhibitor (Go6976), cPLA(2) inhibitor (AACOCF(3)), and pertussis toxin prevented t-buOOH-stimulated PLA(2) activity. Immunoblot studies with aprotinin, cPLA(2), PKCalpha, and Gialpha antibodies revealed their presence in the endothelial membrane. Immunoblot studies of the cell membrane isolated from t-buOOH-stimulated cells with cPLA(2) and PKCalpha antibodies elicited an apparent increase in their immunoreactive protein profiles along with an additional 47-kDa immunoreactive fragment in the membrane. t-buOOH caused Gialpha phosphorylation in the membrane and pretreatment with Go6976 prevented the phosphorylation. Overall, these results suggest that t-buOOH stimulates an aprotinin-sensitive protease activity that proteolytically activates PKCalpha and that subsequently phosphorylates a pertussis toxin-sensitive protein, resulting in the stimulation of cPLA(2) activity in the cell membrane. PMID:16291515

  4. Inhibitory effect of acteoside on melittin-induced catecholamine exocytosis through inhibition of Ca(2+)-dependent phospholipase A2 and extracellular Ca(2+) influx in PC12 cells.

    PubMed

    Song, Ho Sun; Ko, Myung Soo; Jo, Young Soo; Whang, Wan Kyunn; Sim, Sang Soo

    2015-10-01

    To investigate the inhibitory effect of acteoside on the process of exocytosis induced by melittin, we measured Ca(2+) mobilization, arachidonic acid (AA) release and catecholamine exocytosis in PC12 chromaffin cells. Melittin significantly increased the intracellular Ca(2+) mobilization via receptor-operated calcium channel but not the intracellular Ca(2+) release. It caused AA release via activation of Ca(2+)-dependent phospholipase A2 (PLA2) and catecholamine secretion in a dose-dependent manner. Acteoside dose-dependently inhibited the release of AA and intracellular Ca(2+) mobilization induced by melittin. Acteoside reduced the catecholamine release and raised the amount of intracellular chromogranin A which is co-released with catecholamine from melittin-stimulated PC12 cells. Taken together, our results suggest that acteoside could suppress the exocytosis via inhibition of Ca(2+)-dependent PLA2 and extracellular Ca(2+) influx in PC12 cells stimulated by melittin. PMID:25899996

  5. Activation of Cytosolic Phospholipase A2α in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2*

    PubMed Central

    Noor, Shahid; Goldfine, Howard; Tucker, Dawn E.; Suram, Saritha; Lenz, Laurel L.; Akira, Shizuo; Uematsu, Satoshi; Girotti, Milena; Bonventre, Joseph V.; Breuel, Kevin; Williams, David L.; Leslie, Christina C.

    2016-01-01

    Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2α). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (ΔhlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and ΔhlyLM. The attenuated release of arachidonic acid that is observed in TLR2−/− and MyD88−/− macrophages infected with WTLM and ΔhlyLM correlates with diminished MAPK activation. WTLM but not ΔhlyLM increases intracellular calcium, which is implicated in regulation of cPLA2α. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2α+/+ but not cPLA2α−/− macrophages in response to WTLM and ΔhlyLM. Tumor necrosis factor (TNF)-α production is significantly lower in cPLA2α+/+ than in cPLA2α−/− macrophages infected with WTLM and ΔhlyLM. Treatment of infected cPLA2α+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFα production to the level produced by cPLA2α−/− macrophages implicating prostaglandins in TNFα down-regulation. Therefore activation of cPLA2α in macrophages may impact immune responses to L. monocytogenes. PMID:18083708

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Cytosolic Phospholipase A2α and Eicosanoids Regulate Expression of Genes in Macrophages Involved in Host Defense and Inflammation

    PubMed Central

    Suram, Saritha; Silveira, Lori J.; Mahaffey, Spencer; Brown, Gordon D.; Bonventre, Joseph V.; Williams, David L.; Gow, Neil A. R.; Bratton, Donna L.; Murphy, Robert C.; Leslie, Christina C.

    2013-01-01

    The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α+/+ than cPLA2α-/- macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α+/+ than cPLA2α-/- macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α+/+ and cPLA2α-/- macrophages (3 h) was compared by microarray. cPLA2α+/+ macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α-/- macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α+/+ macrophages. Representative genes expressed lower in cPLA2α+/+ macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation. PMID:23950842

  3. Amyloid beta peptide and NMDA induce ROS from NADPH oxidase and AA release from cytosolic phospholipase A2 in cortical neurons.

    PubMed

    Shelat, Phullara B; Chalimoniuk, Malgorzata; Wang, Jing-Hung; Strosznajder, Joanna B; Lee, James C; Sun, Albert Y; Simonyi, Agnes; Sun, Grace Y

    2008-07-01

    Increase in oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurodegenerative diseases including Alzheimer's disease. There is evidence for involvement of amyloid-beta peptide (Abeta) in mediating the oxidative damage to neurons. Despite yet unknown mechanism, Abeta appears to exert action on the ionotropic glutamate receptors, especially the N-methyl-D-aspartic acid (NMDA) receptor subtypes. In this study, we showed that NMDA and oligomeric Abeta(1-42) could induce reactive oxygen species (ROS) production from cortical neurons through activation of NADPH oxidase. ROS derived from NADPH oxidase led to activation of extracellular signal-regulated kinase 1/2, phosphorylation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha), and arachidonic acid (AA) release. In addition, Abeta(1-42)-induced AA release was inhibited by d(-)-2-amino-5-phosphonopentanoic acid and memantine, two different NMDA receptor antagonists, suggesting action of Abeta through the NMDA receptor. Besides serving as a precursor for eicosanoids, AA is also regarded as a retrograde messenger and plays a role in modulating synaptic plasticity. Other phospholipase A(2) products such as lysophospholipids can perturb membrane phospholipids. These results suggest an oxidative-degradative mechanism for oligomeric Abeta(1-42) to induce ROS production and stimulate AA release through the NMDA receptors. This novel mechanism may contribute to the oxidative stress hypothesis and synaptic failure that underline the pathogenesis of Alzheimer's disease. PMID:18346200

  4. 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

  5. Interleukin-22-Induced Antimicrobial Phospholipase A2 Group IIA Mediates Protective Innate Immunity of Nonhematopoietic Cells against Listeria monocytogenes

    PubMed Central

    Okita, Yamato; Shiono, Takeru; Yahagi, Ayano; Hamada, Satoru; Umemura, Masayuki

    2015-01-01

    Listeria monocytogenes is a bacterial pathogen which establishes intracellular parasitism in various cells, including macrophages and nonhematopoietic cells, such as hepatocytes. It has been reported that several proinflammatory cytokines have pivotal roles in innate protection against L. monocytogenes infection. We found that a proinflammatory cytokine, interleukin 22 (IL-22), was expressed by CD3+ CD4+ T cells at an early stage of L. monocytogenes infection in mice. To assess the influence of IL-22 on L. monocytogenes infection in hepatocytes, cells of a human hepatocellular carcinoma line, HepG2, were treated with IL-22 before L. monocytogenes infection in vitro. Gene expression analysis of the IL-22-treated HepG2 cells identified phospholipase A2 group IIA (PLA2G2A) as an upregulated antimicrobial molecule. Addition of recombinant PLA2G2A to the HepG2 culture significantly suppressed L. monocytogenes infection. Culture supernatant of the IL-22-treated HepG2 cells contained bactericidal activity against L. monocytogenes, and the activity was abrogated by a specific PLA2G2A inhibitor, demonstrating that HepG2 cells secreted PLA2G2A, which killed extracellular L. monocytogenes. Furthermore, colocalization of PLA2G2A and L. monocytogenes was detected in the IL-22-treated infected HepG2 cells, which suggests involvement of PLA2G2A in the mechanism of intracellular killing of L. monocytogenes by HepG2 cells. These results suggest that IL-22 induced at an early stage of L. monocytogenes infection enhances innate immunity against L. monocytogenes in the liver by stimulating hepatocytes to produce an antimicrobial molecule, PLA2G2A. PMID:26644377

  6. Interleukin-22-Induced Antimicrobial Phospholipase A2 Group IIA Mediates Protective Innate Immunity of Nonhematopoietic Cells against Listeria monocytogenes.

    PubMed

    Okita, Yamato; Shiono, Takeru; Yahagi, Ayano; Hamada, Satoru; Umemura, Masayuki; Matsuzaki, Goro

    2016-02-01

    Listeria monocytogenes is a bacterial pathogen which establishes intracellular parasitism in various cells, including macrophages and nonhematopoietic cells, such as hepatocytes. It has been reported that several proinflammatory cytokines have pivotal roles in innate protection against L. monocytogenes infection. We found that a proinflammatory cytokine, interleukin 22 (IL-22), was expressed by CD3(+) CD4(+) T cells at an early stage of L. monocytogenes infection in mice. To assess the influence of IL-22 on L. monocytogenes infection in hepatocytes, cells of a human hepatocellular carcinoma line, HepG2, were treated with IL-22 before L. monocytogenes infection in vitro. Gene expression analysis of the IL-22-treated HepG2 cells identified phospholipase A2 group IIA (PLA2G2A) as an upregulated antimicrobial molecule. Addition of recombinant PLA2G2A to the HepG2 culture significantly suppressed L. monocytogenes infection. Culture supernatant of the IL-22-treated HepG2 cells contained bactericidal activity against L. monocytogenes, and the activity was abrogated by a specific PLA2G2A inhibitor, demonstrating that HepG2 cells secreted PLA2G2A, which killed extracellular L. monocytogenes. Furthermore, colocalization of PLA2G2A and L. monocytogenes was detected in the IL-22-treated infected HepG2 cells, which suggests involvement of PLA2G2A in the mechanism of intracellular killing of L. monocytogenes by HepG2 cells. These results suggest that IL-22 induced at an early stage of L. monocytogenes infection enhances innate immunity against L. monocytogenes in the liver by stimulating hepatocytes to produce an antimicrobial molecule, PLA2G2A. PMID:26644377

  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. 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

  9. 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

  10. Leptin signalling and leptin-mediated activation of human platelets: importance of JAK2 and the phospholipases Cgamma2 and A2.

    PubMed

    Dellas, Claudia; Schäfer, Katrin; Rohm, Ilonka K; Lankeit, Mareike; Leifheit, Maren; Loskutoff, David J; Hasenfuss, Gerd; Konstantinides, Stavros V

    2007-11-01

    Leptin enhances agonist-induced platelet aggregation, and human platelets have been reported to express the leptin receptor. However, the pathways and mediators lying downstream of leptin binding to platelets remain, with few exceptions, unknown. In the present study, we sought to gain further insight into the possible role of leptin as a platelet agonist. Stimulation of platelets with leptin promoted thromboxane generation and activation of alpha(IIb)beta(3), as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p<0.001) and induced cytoskeletal rearrangement of both platelets and Meg01 cells. Leptin time- and dose-dependently phosphorylated the intracellular signalling molecules JAK2 and STAT3, although the importance of STAT3 for leptin-induced platelet activation remains to be determined. Important intracellular mediators and pathways activated by leptin downstream of JAK2 were found to include phosphatidylinositol-3 kinase, phospholipase Cgamma2 and protein kinase C, as well as the p38 MAP kinase-phospholipase A(2) axis. Accordingly, incubation with the specific inhibitors AG490, Ly294002, U73122, and SB203580 prevented leptin-mediated platelet activation. These results help delineate biologically relevant leptin signalling pathways in platelets and may improve our understanding of the mechanisms linking hyperleptinaemia to the increased thrombosis risk in human obesity. PMID:18000612

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Lipoprotein-Associated Phospholipase A2 Activity Predicts Progression of Subclinical Coronary Atherosclerosis

    PubMed Central

    Kinney, Gregory L.; Snell-Bergeon, Janet K.; Maahs, David M.; Eckel, Robert H.; Ehrlich, James; Rewers, Marian

    2011-01-01

    Abstract Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a lipoprotein-associated enzyme that cleaves oxidized phosphatidylcholines, generating pro-atherosclerotic lysophosphatidylcholine and oxidized free fatty acids. Lp-PLA2 is independently associated with cardiovascular disease (CVD) in a variety of populations. Coronary calcium is a measure of subclinical CVD, and progression of coronary calcification predicts future CVD events. In type 1 diabetes there is an increase in coronary calcium and CVD despite a favorable lipid profile. Levels of Lp-PLA2 in type 1 diabetes are not known, nor is the relationship between Lp-PLA2 and progression of coronary calcification. Methods The Coronary Artery Calcification in Type 1 Diabetes study measured coronary calcium by electron-beam computed tomography twice over a 2.6 ± 0.3-year interval. Lp-PLA2 mass and activity were measured at baseline (n = 1,097 subjects, 506 with and 591 without type 1 diabetes). Results In type 1 diabetes Lp-PLA2 mass was marginally higher (285 ± 79 vs. 278 ± 78 ng/mL, P = 0.1), and Lp-PLA2 activity was significantly lower (137 ± 30 vs. 146 ± 36 nmol/min/mL, P < 0.0001) than in those without diabetes. There was a greater proportion of those with progression of coronary calcification in type 1 diabetes compared with those without diabetes (24% vs. 10%, P < 0.0001). Lp-PLA2 activity was independently associated with progression of coronary calcification in multivariate analysis (4th quartile verses bottom three quartiles, odds ratio = 1.77 [1.08–2.91], P = 0.02). LpPLA2 mass was not significantly associated with progression of coronary calcification in this cohort (P = 0.09). Conclusions Lp-PLA2 activity predicts progression of subclinical atherosclerosis in individuals with and without type 1 diabetes. PMID:21291330

  12. Darapladib, a Lipoprotein-Associated Phospholipase A2 Inhibitor, Reduces Rho Kinase Activity in Atherosclerosis

    PubMed Central

    Xu, Dong-Ling; Liu, Xiao-Bo; Bi, Shao-jie; Zhao, Tong; Sui, Shu-Jian; Ji, Xiao-Ping

    2016-01-01

    Purpose Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and Rho kinase activity may be associated with atherosclerosis. The principal aim of this study was to examine whether darapladib (a selective Lp-PLA2 inhibitor) could reduce the elevated Lp-PLA2 and Rho kinase activity in atherosclerosis. Materials and Methods Studies were performed in male Sprague-Dawley rats. The atherosclerosis rats were prepared by feeding them with a high-cholesterol diet for 10 weeks. Low-dose darapladib (25 mg·kg-1·d-1) and high-dose darapladib (50 mg·kg-1·d-1) interventions were then administered over the course of 2 weeks. Results The serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP), and Lp-PLA2, significantly increased in atherosclerosis model groups, as did Rho kinase activity and cardiomyocyte apoptosis (p<0.05 vs. sham group), whereas nitric oxide (NO) production was reduced. Levels of TC, LDL-C, CRP, Lp-PLA2, and Rho kinase activity were respectively reduced in darapladib groups, whereas NO production was enhanced. When compared to the low-dose darapladib group, the reduction of the levels of TC, LDL-C, CRP, and Lp-PLA2 was more prominent in the high-dose darapladib group (p<0.05), and the increase of NO production was more prominent (p<0.05). Cardiomyocyte apoptosis of the high-dose darapladib group was also significantly reduced compared to the low-dose darapladib group (p<0.05). However, there was no significant difference in Rho kinase activity between the low-dose darapladib group and the high-dose darapladib group (p>0.05). Conclusion Darapladib, a Lp-PLA2 inhibitor, leads to cardiovascular protection that might be mediated by its inhibition of both Rho kinase and Lp-PLA2 in atherosclerosis. PMID:26847282

  13. Heparin-enhanced plasma phospholipase A2 activity and prostacyclin synthesis in patients undergoing cardiac surgery.

    PubMed Central

    Nakamura, H; Kim, D K; Philbin, D M; Peterson, M B; Debros, F; Koski, G; Bonventre, J V

    1995-01-01

    Although eicosanoid production contributes to physiological and pathophysiological consequences of cardiopulmonary bypass (CPB), the mechanisms accounting for the enhanced eicosanoid production have not been defined. Plasma phospholipase A2 (PLA2) activity, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TXB2) levels were measured at various times during cardiac surgery. Plasma PLA2 activity increased after systemic heparinization, before CPB. This was highly correlated with concurrent increases in plasma 6-keto-PGF1 alpha, TXB2 concentrations did not increase with heparin administration but did increase significantly after initiation of CPB. High plasma PLA2 activity, 6-keto-PGF1 alpha, and TXB2 concentrations were measured throughout the CPB period. Protamine, administered to neutralize the heparin, caused an acute reduction of both plasma PLA2 activity and plasma 6-keto-PGF1 alpha, but no change in plasma TXB2 concentrations. Thus the ratio of TXB2 to 6-keto-PGF1 alpha increased significantly after protamine administration. Enhanced plasma PLA2 activity was also measured in patients with lower doses of heparin used clinically for nonsurgical applications. Human plasma PLA2 was identified as group II PLA2 by its sensitivity to deoxycholate and dithiothreitol, its substrate specificity, and its elution characteristics on heparin affinity chromatography. Heparin addition to PMNs in vitro resulted in dose-dependent increases in cellular PLA2 activity and release of PLA2. The PLA2 released from the PMN had characteristics similar to those of post-heparin plasma PLA2. In conclusion, plasma PLA2 activity and 6-keto-PGF1 alpha concentrations are markedly enhanced with systemic heparinization. Part of the anticoagulant and vasodilating effects of heparin may be due to increased plasma prostacyclin (PGI2) levels. In addition the pulmonary vasoconstriction sometimes associated with protamine infusion during cardiac surgery might be due to decreased

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

    PubMed Central

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

    2015-01-01

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

  15. Platelet phospholipase A(2) activity in Alzheimer's disease and mild cognitive impairment.

    PubMed

    Gattaz, W F; Forlenza, O V; Talib, L L; Barbosa, N R; Bottino, C M C

    2004-05-01

    Phospholipase A(2) (PLA(2)) controls the metabolism of phospholipids in cell membranes. In the brain, PLA(2) influences the processing of the amyloid precursor protein (APP) and thus the production of the amyloid-beta peptides (Abeta), which are the major components of the senile plaques in Alzheimer's disease (AD). Reduced PLA(2) activity has been reported in brain and in platelets of AD patients. In the present study we investigated PLA(2) activity in platelets from 21 AD patients as compared to 17 healthy elderly controls and 11 individuals with mild cognitive impairment (MCI). Subjects were cognitively assessed by the Mini-Mental State Examination (MMSE) and the CAMDEX schedule. Platelet PLA(2) activity was determined by radio-enzymatic assay, which mainly detected a calcium-independent form of the enzyme present also in the brain (iPLA(2)). PLA(2) activity was significantly lower in AD than in controls (p < 0.001). Mean PLA(2) activity in MCI individuals was between the values of AD patients and controls, with a subgroup showing PLA as low as the lowest AD patients, but the differences from MCI were not significant from AD and control groups. Lower PLA(2) activity was significantly correlated with a worse cognitive performance both at the MMSE (p = 0.001) and the cognitive sub-scale of the CAMDEX inventory (p = 0.002). Our data replicate previous findings of reduced platelet PLA(2) activity in AD. Both reduced PLA(2) activity and the correlation with impaired cognition were also reported in brain tissue of AD patients, suggesting thus that the present determinations in platelets may be related to a reduction in the brain. In the brain the inhibition of PLA(2) inhibits the physiological secretion of the APP, a mechanism that increases Abeta formation. Further longitudinal studies should investigate whether those MCI individuals with the lowest PLA(2) values in platelets would be at a higher risk to develop AD during a longitudinal follow up. PMID:15088152

  16. Ex Vivo Effect of Varespladib on Secretory Phospholipase A2 Alveolar Activity in Infants with ARDS

    PubMed Central

    De Luca, Daniele; Minucci, Angelo; Piastra, Marco; Cogo, Paola E.; Vendittelli, Francesca; Marzano, Laura; Gentile, Leonarda; Giardina, Bruno; Conti, Giorgio; Capoluongo, Ettore D.

    2012-01-01

    Background Secretory phospholipase A2 (sPLA2) plays a pivotal role in acute respiratory distress syndrome (ARDS). This enzyme seems an interesting target to reduce surfactant catabolism and lung tissue inflammation. Varespladib is a specifically designed indolic sPLA2 inhibitor, which has shown promising results in animals and adults. No specific data in pediatric ARDS patients are yet available. Methods We studied varespladib in broncho-alveolar lavage (BAL) fluids obtained ex vivo from pediatric ARDS patients. Clinical data and worst gas exchange values during the ARDS course were recorded. Samples were treated with saline or 10–40–100 µM varespladib and incubated at 37°C. Total sPLA2 activity was measured by non-radioactive method. BAL samples were subjected to western blotting to identify the main sPLA isotypes with different sensitivity to varespladib. Results was corrected for lavage dilution using the serum-to-BAL urea ratio and for varespladib absorbance. Results Varespladib reduces sPLA2 activity (p<0.0001) at 10,40 and 100 µM; both sPLA2 activity reduction and its ratio to total proteins significantly raise with increasing varespladib concentrations (p<0.001). IC50 was 80 µM. Western blotting revealed the presence of sPLA2-IIA and –IB isotypes in BAL samples. Significant correlations exist between the sPLA2 activity reduction/proteins ratio and PaO2 (rho = 0.63;p<0.001), PaO2/FiO2 (rho = 0.7; p<0.001), oxygenation (rho = −0.6; p<0.001) and ventilation (rho = −0.4;p = 0.038) indexes. Conclusions Varespladib significantly inhibits sPLA2 in BAL of infants affected by post-neonatal ARDS. Inhibition seems to be inversely related to the severity of gas exchange impairment. PMID:23071714

  17. Epigenetic control of group V phospholipase A2 expression in human malignant cells.

    PubMed

    Menschikowski, Mario; Hagelgans, Albert; Nacke, Brit; Jandeck, Carsten; Mareninova, Olga A; Asatryan, Liana; Siegert, Gabriele

    2016-06-01

    Secreted phospholipases A2 (sPLA2) are suggested to play an important role in inflammation and tumorigenesis. Different mechanisms of epigenetic regulation are involved in the control of group IIA, III and X sPLA2s expression in cancer cells, but group V sPLA2 (GV-PLA2) in this respect has not been studied. Here, we demonstrate the role of epigenetic mechanisms in regulation of GV-PLA2 expression in different cell lines originating from leukaemia and solid cancers. In blood leukocytes from leukaemic patients, levels of GV-PLA2 transcripts were significantly lower in comparison to those from healthy individuals. Similarly, in DU-145 and PC-3 prostate and CAL-51 and MCF-7 mammary cancer cell lines, levels of GV-PLA2 transcripts were significantly lower in relation to those found in normal epithelial cells of prostate or mammary. By sequencing and methylation-specific high-resolution melting (MS-HRM) analyses of bisulphite-modified DNA, distinct CpG sites in the GV-PLA2 promoter region were identified that were differentially methylated in cancer cells in comparison to normal epithelial and endothelial cells. Spearman rank order analysis revealed a significant negative correlation between the methylation degree and the cellular expression of GV-PLA2 (r = -0.697; p = 0.01). The effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on GV-PLA2 transcription in the analysed cells confirmed the importance of DNA methylation and histone modification in the regulation of the GV-PLA2 gene expression in leukaemic, prostate and mammary cancer cell lines. The exposure of tumour cells to human recombinant GV-PLA2 resulted in a reduced colony forming activity of MCF-7, HepG2 and PC-3 cells, but not of DU-145 cells suggesting a cell-type-dependent effect of GV-PLA2 on cell growth. In conclusion, our results suggest that epigenetic mechanisms such as DNA methylation and histone modification play an important role in

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

    PubMed

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

    2015-04-01

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

  19. Rapamycin-insensitive up-regulation of adipocyte phospholipase A2 in tuberous sclerosis and lymphangioleiomyomatosis.

    PubMed

    Li, Chenggang; Zhang, Erik; Sun, Yang; Lee, Po-Shun; Zhan, Yongzhong; Guo, Yanan; Osorio, Juan C; Rosas, Ivan O; Xu, Kai-Feng; Kwiatkowski, David J; Yu, Jane J

    2014-01-01

    Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/- mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2-/-MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived "mesenchymal" cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role

  20. Control of the chemical step by leucine-31 of pancreatic phospholipase A2.

    PubMed

    Yu, B Z; Janssen, M J; Verheij, H M; Jain, M K

    2000-05-16

    A well-defined region of pancreatic and other secreted phospholipase A2 (PLA2), which we call the i-face, makes a molecular contact with the interface to facilitate and control the events and processivity of the interfacial catalytic turnover cycles. The structural features of the i-face and its allosteric relationship to the active site remain to be identified. As a part of the calcium binding (26-34) loop, Leu-31 is located on the surface near the substrate binding slot of PLA2. Analysis of the primary rate and equilibrium parameters of the Leu-31 substitution mutants of the pig pancreatic PLA2 shows that the only significant effect of the substitution is to impair the chemical step at the zwitterionic interface in the presence of added NaCl, and only a modest effect is seen on kcat at the anionic interface. Leu-31 substitutions have little effect on the binding of the enzyme to the interface; the affinity for certain substrate mimics is modestly influenced in W3F, L31W double mutant. The fluorescence emission results with the double mutant show that the microenvironment of Trp-31 is qualitatively different at the zwitterionic versus anionic interfaces. At both of the interfaces Trp-31 is not shielded from the bulk aqueous environment as it remains readily accessible to acrylamide and water. The NaCl-induced change in the Trp-31 emission spectrum of the double mutant on the zwitterionic interface is similar to that seen on the binding to the anionic interface. Together, the kinetic and spectroscopic results show that the form of PLA2 at the zwitterionic interface (Ez) is distinguishably different from the catalytically more efficient form at the anionic interface (Ea). This finding provides a structural basis for the two-state model for kcat activation by the anionic interface. In conjunction with earlier results we suggest that neutralization of certain cationic residues of PLA2 exerts a control on the calcium loop through residue 31. PMID:10801320

  1. 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

  2. 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

  3. 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

  4. 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 and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2μg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2. PMID:27282409

  5. Influence of product phase separation on phospholipase A(2) hydrolysis of supported phospholipid bilayers studied by force microscopy.

    PubMed Central

    Nielsen, Lars K; Balashev, Konstatin; Callisen, Thomas H; Bjørnholm, Thomas

    2002-01-01

    In situ atomic force microscopy studies reveal a marked influence of the initial presence of hydrolysis products on the hydrolysis of supported phospholipid bilayers by phospholipase A(2). By analysis of the nano-scale topography of a number of supported bilayers with different initial product concentrations, made by Langmuir-Blodgett deposition, we show that small depressions enriched in products are efficiently promoting enzyme degradation of the bilayer. These small depressions, which are indicative of phase separation, are initially present in samples with 75% products. The kinetics of phospholipase A(2) exhibit under certain conditions an initial phase of slow hydrolysis, termed the latency phase, followed by a marked increase in the hydrolysis rate. The appearance of the phase-separated bilayer is strikingly similar to that of bilayers at the end of the latency phase. By analysis of individual nano-scale defects we illustrate a quantitative difference in the growth rates of defects caused by product aggregation and other structural defects. This difference shows for the first time how the enzyme prefers one type of defect to another. PMID:12414695

  6. Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

    PubMed Central

    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

    2015-01-01

    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 Ca2+. 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 Ca2+ and regulation of Ca2+-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 Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

  7. Biological characterization of the Amazon coral Micrurus spixii snake venom: Isolation of a new neurotoxic phospholipase A2.

    PubMed

    Terra, Angelo L C; Moreira-Dill, Leandro S; Simões-Silva, Rodrigo; Monteiro, José Roniele N; Cavalcante, Walter L G; Gallacci, Márcia; Barros, Neuza B; Nicolete, Roberto; Teles, Carolina B G; Medeiros, Patrícia S M; Zanchi, Fernando B; Zuliani, Juliana P; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M

    2015-09-01

    The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A2 called MsPLA2-I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC50 1.24 μg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum, which are unprecedented for Micrurus venoms. MsPLA2-I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii. The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA2 from Micrurus altirostris. This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA2-I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA2-I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential. PMID:26095535

  8. Higher Levels of Lipoprotein Associated Phospholipase A2 is associated with Increased Prevalence of Cognitive Impairment: the APAC Study

    PubMed Central

    Jiang, Ruixuan; Chen, Shengyun; Shen, Yuan; Wu, Jianwei; Chen, Shuohua; Wang, Anxin; Wu, Shouling; Zhao, Xingquan

    2016-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a unique circulating phospholipase with inflammatory and oxidative activities and the limited data regarding the relationship between Lp-PLA2 and cognitive impairment are conflicted. We conducted a cross-sectional study including 1,374 Chinese adults recruited from 2010 to 2011, aiming to evaluate the relationship between Lp-PLA2 levels and the prevalence of cognitive impairment in a Chinese community-based population. Participants underwent standardized evaluation. Serum Lp-PLA2 mass was measured by ELISA. Cognition status was evaluated via the Mini-Mental Status Exam (MMSE) and cognitive impairment was identified as MMSE <24. Multivariable logistic regression models were used to assess the associations of Lp-PLA2 mass with cognitive impairment. Lp-PLA2 mass was significantly associated with the prevalence of cognitive impairment after adjusting for other potential confounding factors (compared with the first quartile, adjusted ORs of the second, third, and fourth quartile were 2.058 (95% CI, 0.876–4.835), 2.834 (95% CI, 1.255–6.398), and 4.882 (95% CI, 2.212–10.777), p < 0.0001). In conclusion, elevated level of Lp-PLA2 mass was independently associated with the prevalence of cognitive impairment in Chinese adults. PMID:27609335

  9. Quantitative Proteomic Analysis of Venoms from Russian Vipers of Pelias Group: Phospholipases A2 are the Main Venom Components

    PubMed Central

    Kovalchuk, Sergey I.; Ziganshin, Rustam H.; Starkov, Vladislav G.; Tsetlin, Victor I.; Utkin, Yuri N.

    2016-01-01

    Venoms of most Russian viper species are poorly characterized. Here, by quantitative chromato-mass-spectrometry, we analyzed protein and peptide compositions of venoms from four Vipera species (V. kaznakovi, V. renardi, V. orlovi and V. nikolskii) inhabiting different regions of Russia. In all these species, the main components were phospholipases A2, their content ranging from 24% in V. orlovi to 65% in V. nikolskii. Altogether, enzyme content in venom of V. nikolskii reached ~85%. Among the non-enzymatic proteins, the most abundant were disintegrins (14%) in the V. renardi venom, C-type lectin like (12.5%) in V. kaznakovi, cysteine-rich venom proteins (12%) in V. orlovi and venom endothelial growth factors (8%) in V. nikolskii. In total, 210 proteins and 512 endogenous peptides were identified in the four viper venoms. They represented 14 snake venom protein families, most of which were found in the venoms of Vipera snakes previously. However, phospholipase B and nucleotide degrading enzymes were reported here for the first time. Compositions of V. kaznakovi and V. orlovi venoms were described for the first time and showed the greatest similarity among the four venoms studied, which probably reflected close relationship between these species within the “kaznakovi” complex. PMID:27077884

  10. Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation.

    PubMed

    Boudreau, Luc H; Duchez, Anne-Claire; Cloutier, Nathalie; Soulet, Denis; Martin, Nicolas; Bollinger, James; Paré, Alexandre; Rousseau, Matthieu; Naika, Gajendra S; Lévesque, Tania; Laflamme, Cynthia; Marcoux, Geneviève; Lambeau, Gérard; Farndale, Richard W; Pouliot, Marc; Hamzeh-Cognasse, Hind; Cognasse, Fabrice; Garraud, Olivier; Nigrovic, Peter A; Guderley, Helga; Lacroix, Steve; Thibault, Louis; Semple, John W; Gelb, Michael H; Boilard, Eric

    2014-10-01

    Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses. PMID:25082876

  11. Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation

    PubMed Central

    Boudreau, Luc H.; Duchez, Anne-Claire; Cloutier, Nathalie; Soulet, Denis; Martin, Nicolas; Bollinger, James; Paré, Alexandre; Rousseau, Matthieu; Naika, Gajendra S.; Lévesque, Tania; Laflamme, Cynthia; Marcoux, Geneviève; Lambeau, Gérard; Farndale, Richard W.; Pouliot, Marc; Hamzeh-Cognasse, Hind; Cognasse, Fabrice; Garraud, Olivier; Nigrovic, Peter A.; Guderley, Helga; Lacroix, Steve; Thibault, Louis; Semple, John W.; Gelb, Michael H.

    2014-01-01

    Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses. PMID:25082876

  12. Higher Levels of Lipoprotein Associated Phospholipase A2 is associated with Increased Prevalence of Cognitive Impairment: the APAC Study.

    PubMed

    Jiang, Ruixuan; Chen, Shengyun; Shen, Yuan; Wu, Jianwei; Chen, Shuohua; Wang, Anxin; Wu, Shouling; Zhao, Xingquan

    2016-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a unique circulating phospholipase with inflammatory and oxidative activities and the limited data regarding the relationship between Lp-PLA2 and cognitive impairment are conflicted. We conducted a cross-sectional study including 1,374 Chinese adults recruited from 2010 to 2011, aiming to evaluate the relationship between Lp-PLA2 levels and the prevalence of cognitive impairment in a Chinese community-based population. Participants underwent standardized evaluation. Serum Lp-PLA2 mass was measured by ELISA. Cognition status was evaluated via the Mini-Mental Status Exam (MMSE) and cognitive impairment was identified as MMSE <24. Multivariable logistic regression models were used to assess the associations of Lp-PLA2 mass with cognitive impairment. Lp-PLA2 mass was significantly associated with the prevalence of cognitive impairment after adjusting for other potential confounding factors (compared with the first quartile, adjusted ORs of the second, third, and fourth quartile were 2.058 (95% CI, 0.876-4.835), 2.834 (95% CI, 1.255-6.398), and 4.882 (95% CI, 2.212-10.777), p < 0.0001). In conclusion, elevated level of Lp-PLA2 mass was independently associated with the prevalence of cognitive impairment in Chinese adults. PMID:27609335

  13. Serotonin stimulates phospholipase A sub 2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis

    SciTech Connect

    Felder, C.C.; Ma, A.L.; Axelrod, J.; Kanterman, R.Y. )

    1990-03-01

    Serotonin (5-HT) stimulated the release of arachidonic acid in hippocampal neurons cocultured with glial cells but not in glial cultures alone. Similar results were observed for the 5-HT-stimulated release of inositol phosphates. These results suggest a neural but not glial origin of both responses. Pharmacological studies suggested that release of arachidonic acid and inositol phosphates was mediated by a type 2 5-HTT (5-HT{sub 2}) receptor. 5-HT-stimulated release of arachidonic acid was also detected in cortical neurons, which contain high levels of 5-HT{sub 2} receptors, but not striatum, spinal cord, or cerebellar granule cells, which have very low levels or are devoid of 5-HT{sub 2} receptors. The phorbol ester phorbol 12-myristate 13-acetate augmented the 5-HT-stimulated release of arachidonic acid but inhibited the 5-HT-stimulated release of inositol phosphates. 5-HT-stimulated release of arachidonic acid, but not inositol phosphates, was dependent on extracellular calcium. 5-HT stimulated the release of ({sup 3}H)lysophosphatidylcholine from ({sup 3}H)choline-labeled cells with no increase in the release of ({sup 3}H)choline or phospho({sup 3}H)choline. These data suggest that 5-HT stimulated the release of arachidonic acid in hippocampal neurons through the activation of phospholipase A{sub 2}, independent of the activation of phospholipase C.

  14. 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

  15. A group IIA-secreted phospholipase A2 from snake venom induces lipid body formation in macrophages: the roles of intracellular phospholipases A2 and distinct signaling pathways.

    PubMed

    Leiguez, Elbio; Zuliani, Juliana Pavan; Cianciarullo, Aurora Marques; Fernandes, Cristina Maria; Gutiérrez, José Maria; Teixeira, Catarina

    2011-07-01

    We investigated the ability of the sPLA(2), known as MT-III, isolated from the viperid snake Bothrops asper, to induce LB formation in macrophages and the major cellular signaling pathways involved in this process. The effects of MT-III on ADRP localization and expression and macrophage ultrastructure were assessed. Our results showed that this sPLA(2) induced a marked increase in LB numbers in macrophages, induced the recruitment of ADRP in macrophages, and up-regulated ADRP expression. Ultrastructural analysis showed the presence of weakly and strongly osmiophilic LBs in sPLA(2)-stimulated cells. Enlargement of the ER and Golgi cisterns was also observed. Pretreatment of cells with H7 or staurosporine (PKC inhibitors), LY294002 or wortmannin (PI3K inhibitors), SB202190 or PD98059 (p38(MAPK) and ERK1/2 inhibitors, respectively), or Pyr-2 or Bel (cPLA(2) and iPLA(2) inhibitors, respectively) significantly reduced sPLA(2)-induced LB formation. Herbimycin (a PTK inhibitor) and indomethacin or etoricoxib (COX inhibitors) failed to alter sPLA(2)-induced effects. In conclusion, our results show for the first time the ability of a venom sPLA(2) to induce the formation of LBs and the expression of ADRP in macrophages. Venom PLA(2)-induced LB formation is dependent on PKC, PI3K, p38(MAPK), ERK1/2, cPLA(2), and iPLA(2) signaling pathways but not on PTK, COX-1, or COX-2 pathways. Activation of the ER and Golgi complex may play an important role in the formation of LBs induced by this sPLA(2) in macrophages. PMID:21478270

  16. Prevalence of serum anti M-type phospholipase A2 receptor antibody in primary membranous nephropathy: A single center experience

    PubMed Central

    Gopalakrishnan, N.; Abeesh, P.; Dineshkumar, T.; Murugananth, S.; Sakthirajan, R.; Raman, G. Srinivasa; Dhanapriya, J.; Balasubramaniyan, T.; Haris, Md.

    2016-01-01

    We conducted a prospective study to assess utility of detection of antibodies to phospholipase A2receptor (PLA2R) in the serum of patients with membranous nephropathy. Seventy five patients with biopsy proven membranous nephropathy admitted between January 2011 and September 2014 were studied. Serum anti- PLA2R was tested by indirect immunofluorescence. The test was positive in 45 out of 60 patients with primary membranous nephropathy (PMN) and in none of the 15 patients with secondary membranous nephropathy, with a sensitivity of 75% and specificity of 100% for PMN. Anti PLA2R positivity also showed a significant correlation with quantum of proteinuria and negative correlation with serum albumin. This study has validated detection of serum anti PLA2R in PMN as a non invasive diagnostic tool in Indian patients. PMID:27512297

  17. A novel phospholipase A(2) from the venom glands of Bungarus candidus: cloning and sequence-comparison.

    PubMed

    Tsai, Inn-Ho; Hsu, Hwa-Yao; Wang, Ying-Ming

    2002-09-01

    The presence of phospholipase A(2) (PLA(2)) in the venom of Malayan krait (Bungarus candidus) and its structure were studied. The PLA(2) cDNAs from the venom gland of B. candidus (Indonesia origin) were amplified by the polymerase chain reactions (PCR) and cloned. The primers used were based on the cDNA sequences of several homologous B. multicinctus venom PLA(2)s. In addition to the A-chains of beta-bungarotoxins, a novel B. candidus PLA(2) was cloned and its full amino acid sequence deduced. Having totally 125 amino acid residues, the PLA(2) contains a pancreatic loop and is 61% identical to the acidic PLA(2) of king cobra venom. However, the enzyme was not detected from the venom sample. Its structural relationships to other elapid venom PLA(2)s were analyzed with a phylogenetic tree and discussed. PMID:12220723

  18. Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A2 and Humicola lanuginosa lipase.

    PubMed

    Balashev, Konstantin; John DiNardo, N; Callisen, Thomas H; Svendsen, Allan; Bjørnholm, Thomas

    2007-01-01

    An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A(2) (PLA(2)) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates. PMID:17084807

  19. Plasma membrane phospholipase A2 controls hepatocellular fatty acid uptake and is responsive to pharmacological modulation: implications for nonalcoholic steatohepatitis.

    PubMed

    Stremmel, Wolfgang; Staffer, Simone; Wannhoff, Andreas; Pathil, Anita; Chamulitrat, Walee

    2014-07-01

    Excess hepatic fat accumulation leads to nonalcoholic steatohepatitis (NASH), a serious threat to health for which no effective treatment is available. However, the mechanism responsible for fatty acid uptake by hepatocytes remains unclear. Using the human hepatocyte-derived tumor cell line HepG2, we found that fatty acid influx is mediated by a heterotetrameric plasma membrane protein complex consisting of plasma membrane fatty acid-binding protein, caveolin-1, CD36, and calcium-independent membrane phospholipase A2 (iPLA2β). Blocking iPLA2β with the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) caused the dissociation of the complex, thereby inhibiting fatty acid influx (IC50 47 μM), and suppressed the synthesis of all subunits through a reduction in lysophosphatidylcholine from 8.0 to 3.5 μmol/mg of protein and corresponding depletion of phosphorylated c-Jun N-terminal kinase. These findings were substantiated by an observed 56.5% decrease in fatty acid influx in isolated hepatocytes derived from iPLA2β-knockout mice. Moreover, steatosis and inflammation were abrogated by UDCA-LPE treatment in a cellular model of NASH. Thus, iPLA2β acts as an upstream checkpoint for mechanisms that regulate fatty acid uptake, and its inhibition by UDCA-LPE qualifies this nontoxic compound as a therapeutic candidate for the treatment of NASH.-Stremmel, W., Staffer, S., Wannhoff, A., Pathil, A., Chamulitrat, W. Plasma membrane phospholipase A2 controls hepatocellular fatty acid uptake and is responsive to pharmacological modulation: implications for nonalcoholic steatohepatitis. PMID:24719358

  20. Effect of azithromycin on the LPS-induced production and secretion of phospholipase A2 in lung cells.

    PubMed

    Kitsiouli, Eirini; Antoniou, Georgia; Gotzou, Helen; Karagiannopoulos, Michalis; Basagiannis, Dimitris; Christoforidis, Savvas; Nakos, George; Lekka, Marilena E

    2015-07-01

    Azithromycin is a member of macrolides, utilized in the treatment of infections. Independently, these antibiotics also possess anti-inflammatory and immunomodulatory properties. Phospholipase A2 isotypes, which are implicated in the pathophysiology of inflammatory lung disorders, are produced by alveolar macrophages and other lung cells during inflammatory response and can promote lung injury by destructing lung surfactant. The aim of the study was to investigate whether in lung cells azithromycin can inhibit secretory and cytosolic phospholipases A2, (sPLA2) and (cPLA2), respectively, which are induced by an inflammatory trigger. In this respect, we studied the lipopolysaccharide (LPS)-mediated production or secretion of sPLA2 and cPLA2 from A549 cells, a cancer bronchial epithelial cell line, and alveolar macrophages, isolated from bronchoalveolar lavage fluid of ARDS and control patients without cardiopulmonary disease or sepsis. Pre-treatment of cells with azithromycin caused a dose-dependent decrease in the LPS-induced sPLA2-IIA levels in A549 cells. This inhibition was rather due to reduced PLA2G2A mRNA expression and secretion of sPLA2-IIA protein levels, as observed by western blotting and indirect immunofluorescence by confocal microscopy, respectively, than to the inhibition of the enzymic activity per se. On the contrary, azithromycin had no effect on the LPS-induced production or secretion of sPLA2-IIA from alveolar macrophages. The levels of LPS-induced c-PLA2 were not significantly affected by azithromycin in either cell type. We conclude that azithromycin exerts anti-inflammatory properties on lung epithelial cells through the inhibition of both the expression and secretion of LPS-induced sPLA2-IIA, while it does not affect alveolar macrophages. PMID:25791017

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

    PubMed

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

    2016-03-25

    Within the secreted phospholipase A2(sPLA2) family, group X sPLA2(sPLA2-X) has the highest capacity to hydrolyze cellular membranes and has long been thought to promote inflammation by releasing arachidonic acid, a precursor of pro-inflammatory eicosanoids. Unexpectedly, we found that transgenic mice globally overexpressing human sPLA2-X (PLA2G10-Tg) displayed striking immunosuppressive and lean phenotypes with lymphopenia and increased M2-like macrophages, accompanied by marked elevation of free ω3 polyunsaturated fatty acids (PUFAs) and their metabolites. Studies usingPla2g10-deficient mice revealed that endogenous sPLA2-X, which is highly expressed in the colon epithelium and spermatozoa, mobilized ω3 PUFAs or their metabolites to protect against dextran sulfate-induced colitis and to promote fertilization, respectively. In colitis, sPLA2-X deficiency increased colorectal expression of Th17 cytokines, and ω3 PUFAs attenuated their production by lamina propria cells partly through the fatty acid receptor GPR120. In comparison, cytosolic phospholipase A2(cPLA2α) protects from colitis by mobilizing ω6 arachidonic acid metabolites, including prostaglandin E2 Thus, our results underscore a previously unrecognized role of sPLA2-X as an ω3 PUFA mobilizerin vivo, segregated mobilization of ω3 and ω6 PUFA metabolites by sPLA2-X and cPLA2α, respectively, in protection against colitis, and the novel role of a particular sPLA2-X-driven PUFA in fertilization. PMID:26828067

  2. Structure/Function Relationships of Adipose Phospholipase A2 Containing a Cys-His-His Catalytic Triad*

    PubMed Central

    Pang, Xiao-Yan; Cao, Jian; Addington, Linsee; Lovell, Scott; Battaile, Kevin P.; Zhang, Na; Rao, J. L. Uma Maheswar; Dennis, Edward A.; Moise, Alexander R.

    2012-01-01

    Adipose phospholipase A2 (AdPLA or Group XVI PLA2) plays an important role in the onset of obesity by suppressing adipose tissue lipolysis. As a consequence, AdPLA-deficient mice are resistant to obesity induced by a high fat diet or leptin deficiency. It has been proposed that AdPLA mediates its antilipolytic effects by catalyzing the release of arachidonic acid. Based on sequence homology, AdPLA is part of a small family of acyltransferases and phospholipases related to lecithin:retinol acyltransferase (LRAT). To better understand the enzymatic mechanism of AdPLA and LRAT-related proteins, we solved the crystal structure of AdPLA. Our model indicates that AdPLA bears structural similarity to proteins from the NlpC/P60 family of cysteine proteases, having its secondary structure elements configured in a circular permutation of the classic papain fold. Using both structural and biochemical evidence, we demonstrate that the enzymatic activity of AdPLA is mediated by a distinctive Cys-His-His catalytic triad and that the C-terminal transmembrane domain of AdPLA is required for the interfacial catalysis. Analysis of the enzymatic activity of AdPLA toward synthetic and natural substrates indicates that AdPLA displays PLA1 in addition to PLA2 activity. Thus, our results provide insight into the enzymatic mechanism and biochemical properties of AdPLA and LRAT-related proteins and lead us to propose an alternate mechanism for AdPLA in promoting adipose tissue lipolysis that is not contingent on the release of arachidonic acid and that is compatible with its combined PLA1/A2 activity. PMID:22923616

  3. 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

  4. 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

  5. Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1.

    PubMed

    Huang, Ping; Altshuller, Yelena M; Hou, June Chunqiu; Pessin, Jeffrey E; Frohman, Michael A

    2005-06-01

    Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion. PMID:15772157

  6. Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid.

    PubMed

    Mangum, Lee C; Borazjani, Abdolsamad; Stokes, John V; Matthews, Anberitha T; Lee, Jung Hwa; Chambers, Janice E; Ross, Matthew K

    2015-04-20

    ) levels and enhanced p47(phox) membrane localization compared to that in vehicle-treated cells. p47(phox) is a cytosolic regulatory subunit of Nox, and its phosphorylation and translocation to the NOX2 catalytic subunit in membranes is a requisite step for Nox assembly and activation. Dieldrin and trans-nonachlor treatments of monocytes also resulted in marked increases in arachidonic acid (AA) and eicosanoid production, which could be abrogated by the phospholipase A2 (PLA2) inhibitor arachidonoyltrifluoromethyl ketone (ATK) but not by calcium-independent PLA2 inhibitor bromoenol lactone. This suggested that cytosolic PLA2 plays a crucial role in the induction of Nox activity by increasing the intracellular pool of AA that activates protein kinase C, which phosphorylates p47(phox). In addition, ATK also blocked OC-induced p47(phox) serine phosphorylation and attenuated ROS levels, which further supports the notion that the AA pool liberated by cytosolic PLA2 is responsible for Nox activation. Together, the results suggest that trans-nonachlor and dieldrin are capable of increasing intracellular superoxide levels via a Nox-dependent mechanism that relies on elevated intracellular AA levels. These findings are significant because chronic activation of monocytes by environmental toxicants might contribute to pathogenic oxidative stress and inflammation. PMID:25633958

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

    PubMed Central

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

    2014-01-01

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

  8. Interplay between ABA and phospholipases A(2) and D in the response of citrus fruit to postharvest dehydration.

    PubMed

    Romero, Paco; Gandía, Mónica; Alférez, Fernando

    2013-09-01

    The interplay between abscisic acid (ABA) and phospholipases A2 and D (PLA2 and PLD) in the response of citrus fruit to water stress was investigated during postharvest by using an ABA-deficient mutant from 'Navelate' orange named 'Pinalate'. Fruit from both varieties harvested at two different maturation stages (mature-green and full-mature) were subjected to prolonged water loss inducing stem-end rind breakdown (SERB) in full-mature fruit. Treatment with PLA2 inhibitor aristolochic acid (AT) and PLD inhibitor lysophosphatidylethanolamine (LPE) reduced the disorder in both varieties, suggesting that phospholipid metabolism is involved in citrus peel quality. Expression of CsPLDα and CsPLDβ, and CssPLA2α and CssPLA2β was studied by real-time RT-PCR during water stress and in response to ABA. CsPLDα expression increased in mature-green fruit from 'Navelate' but not in 'Pinalate' and ABA did not counteract this effect. ABA enhanced repression of CsPLDα in full-mature fruit. CsPLDβ gene expression decreased in mature-green 'Pinalate', remained unchanged in 'Navelate' and was induced in full-mature fruit from both varieties. CssPLA2α expression increased in mature-green fruit from both varieties whereas in full-mature fruit only increased in 'Navelate'. CssPLA2β expression increased in mature-green flavedo from both varieties, but in full-mature fruit remained steady in 'Navelate' and barely increased in 'Pinalate' fruit. ABA reduced expression in both after prolonged storage. Responsiveness to ABA increased with maturation. Our results show interplay between PLA2 and PLD and suggest that ABA action is upstream phospholipase activation. Response to ABA during water stress in citrus is regulated during fruit maturation and involves membrane phospholipid degradation. PMID:23800664

  9. Platelet-derived growth factor stimulates protein kinase D through the activation of phospholipase Cgamma and protein kinase C.

    PubMed

    Van Lint, J; Ni, Y; Valius, M; Merlevede, W; Vandenheede, J R

    1998-03-20

    Platelet-derived growth factor (PDGF) stimulates protein kinase D (PKD) in a time- and dose-dependent manner. We have used a series of PDGF receptor mutants that display a selective impairment of the binding of SH2-containing proteins (GTPase-activating protein, SHP-2, phospholipase Cgamma (PLCgamma), or phosphatidylinositol 3'-kinase (PI3K)) to show that Tyr-1021, the PLCgamma-binding site, is essential for PKD stimulation by PDGF in A431 cells. We next investigated whether any one of these four binding sites could mediate PKD activation in the absence of the other three sites. F5, a receptor mutant that lacks all four binding sites for GTPase-activating protein, PLCgamma, PI3K, and SHP-2, fails to activate PKD. A panel of single add-back mutants was used to investigate if any one of these four sites could restore signaling to PKD. Of the four sites, only the PLCgamma+ single add-back receptor restored PDGF-mediated activation of PKD, and only this add-back receptor produced diacylglycerol (DAG) in a PDGF-dependent manner. 1,2-Dioctanoyl-sn-glycerol, a membrane-permeant DAG analog, was found to be sufficient for activation of PKD. Taken together, these data indicate that PLCgamma activation is not only necessary, but also sufficient to mediate PDGF-induced PKD activation. Although the presence of a pleckstrin homology domain makes PKD a potential PI3K target, PKD was not stimulated by selective PI3K activation, and wortmannin, an inhibitor of PI3K, did not inhibit PDGF signaling to PKD. The activation of PKD by DAG or by the wild-type and PLCgamma+ add-back PDGF receptors was inhibited by GF109203X, suggesting a role for protein kinase C in the stimulation of PKD by PDGF. PDGF induced a time-dependent phosphorylation of PKD that closely correlated with activation. The PDGF-induced activation and phosphorylation of PKD were reversed by in vitro incubation of PKD with protein phosphatase 1 or 2A, indicating that PDGF signaling to PKD involves the Ser

  10. Effects of phospholipase A2 and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets[S

    PubMed Central

    Jayaraman, Shobini; Gantz, Donald L.; Gursky, Olga

    2011-01-01

    Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A2 (PLA2), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial β-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA. PMID:21220788

  11. Interleukin-1beta-induced type IIA secreted phospholipase A2 gene expression and extracellular activity in rat vascular endothelial cells.

    PubMed

    Schwemmer, M; Aho, H; Michel, J B

    2001-06-01

    Two phospholipase A2 (PLA2) isoforms, secretory and cytosolic, have been implicated in inflammation. Secretory type IIA PLA2 (sPLA2-IIA), which hydrolyzes fatty acids bound at the sn-2 position of glycerophospholipids, has been detected universally in a variety of mammalian tissues and cells. The expression of the sPLA2-IIA gene and its extracellular activity were shown to be regulated by different factors such as hypoxia, cytokines and phorbol esters. In the present study, we examined the effects of interleukin-1beta (IL-1beta) on the expression of the 14kDa sPLA2-IIA, determined using reverse transcription polymerase chain reaction and radiometric Escherichia coli enzyme assay in primary cultures of rat endothelial cells and in two different rat endothelial cell lines (SVAREC and RBE4). These experiments revealed that IL-1beta induces sPLA2-IIa gene expression and secretion of the enzyme in endothelial cells in a dose- and time-dependent manner. The cAMP-elevator forskolin did not augment the cytokine-induced elevation of sPLA2-IIa enzyme activity but significantly increased the IL-1beta-stimulated sPLA2-IIa mRNA contents in endothelial cells. PMID:11469536

  12. Mapping the Human Platelet Lipidome Reveals Cytosolic Phospholipase A2 as a Regulator of Mitochondrial Bioenergetics during Activation.

    PubMed

    Slatter, David A; Aldrovandi, Maceler; O'Connor, Anne; Allen, Stuart M; Brasher, Christopher J; Murphy, Robert C; Mecklemann, Sven; Ravi, Saranya; Darley-Usmar, Victor; O'Donnell, Valerie B

    2016-05-10

    Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via β-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation. Phospholipase activity is linked to energy metabolism, revealing cPLA2 as a central regulator of both lipidomics and energy flux. Using a lipidomic approach (LipidArrays), we also estimated the total number of lipids in resting, thrombin-activated, and aspirinized platelets. Significant diversity between genetically unrelated individuals and a wealth of species was revealed. Resting platelets demonstrated ∼5,600 unique species, with only ∼50% being putatively identified. Thrombin elevated ∼900 lipids >2-fold with 86% newly appearing and 45% inhibited by aspirin supplementation, indicating COX-1 is required for major activation-dependent lipidomic fluxes. Many lipids were structurally identified. With ∼50% of the lipids being absent from databases, a major opportunity for mining lipids relevant to human health and disease is presented. PMID:27133131

  13. Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production.

    PubMed

    Ronco, A M; Moraga, P F; Llanos, M N

    2002-01-01

    We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose- and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A(2) (PLA(2)) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [(14)C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180+/-12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA(2) inhibitors, decreased the hormone-stimulated AA release to 85+/-9 and 70+/-24% respectively. Conversely, melittin, a PLA(2) stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G(i) family was involved in the hCG-dependent release of AA. Cells treated with PLA(2) inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of

  14. Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

    PubMed

    Arastoo, Mohammed; Hacker, Christian; Popovics, Petra; Lucocq, John M; Stewart, Alan J

    2016-02-01

    Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis. PMID:26671787

  15. Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1.

    PubMed Central

    Hernández, M; Bayón, Y; Sánchez Crespo, M; Nieto, M L

    1997-01-01

    The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways. PMID:9359863

  16. 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

  17. Secreted Phospholipases A2 of Snake Venoms: Effects on the Peripheral Neuromuscular System with Comments on the Role of Phospholipases A2 in Disorders of the CNS and Their Uses in Industry

    PubMed Central

    Harris, John B.; Scott-Davey, Tracey

    2013-01-01

    Neuro- and myotoxicological signs and symptoms are significant clinical features of envenoming snakebites in many parts of the world. The toxins primarily responsible for the neuro and myotoxicity fall into one of two categories—those that bind to and block the post-synaptic acetylcholine receptors (AChR) at the neuromuscular junction and neurotoxic phospholipases A2 (PLAs) that bind to and hydrolyse membrane phospholipids of the motor nerve terminal (and, in most cases, the plasma membrane of skeletal muscle) to cause degeneration of the nerve terminal and skeletal muscle. This review provides an introduction to the biochemical properties of secreted sPLA2s in the venoms of many dangerous snakes and a detailed discussion of their role in the initiation of the neurologically important consequences of snakebite. The rationale behind the experimental studies on the pharmacology and toxicology of the venoms and isolated PLAs in the venoms is discussed, with particular reference to the way these studies allow one to understand the biological basis of the clinical syndrome. The review also introduces the involvement of PLAs in inflammatory and degenerative disorders of the central nervous system (CNS) and their commercial use in the food industry. It concludes with an introduction to the problems associated with the use of antivenoms in the treatment of neuro-myotoxic snakebite and the search for alternative treatments. PMID:24351716

  18. Secreted phospholipases A2 of snake venoms: effects on the peripheral neuromuscular system with comments on the role of phospholipases A2 in disorders of the CNS and their uses in industry.

    PubMed

    Harris, John B; Scott-Davey, Tracey

    2013-12-01

    Neuro- and myotoxicological signs and symptoms are significant clinical features of envenoming snakebites in many parts of the world. The toxins primarily responsible for the neuro and myotoxicity fall into one of two categories--those that bind to and block the post-synaptic acetylcholine receptors (AChR) at the neuromuscular junction and neurotoxic phospholipases A2 (PLAs) that bind to and hydrolyse membrane phospholipids of the motor nerve terminal (and, in most cases, the plasma membrane of skeletal muscle) to cause degeneration of the nerve terminal and skeletal muscle. This review provides an introduction to the biochemical properties of secreted sPLA2s in the venoms of many dangerous snakes and a detailed discussion of their role in the initiation of the neurologically important consequences of snakebite. The rationale behind the experimental studies on the pharmacology and toxicology of the venoms and isolated PLAs in the venoms is discussed, with particular reference to the way these studies allow one to understand the biological basis of the clinical syndrome. The review also introduces the involvement of PLAs in inflammatory and degenerative disorders of the central nervous system (CNS) and their commercial use in the food industry. It concludes with an introduction to the problems associated with the use of antivenoms in the treatment of neuro-myotoxic snakebite and the search for alternative treatments. PMID:24351716

  19. Progesterone-induced Acrosome Exocytosis Requires Sequential Involvement of Calcium-independent Phospholipase A2β (iPLA2β) and Group X Secreted Phospholipase A2 (sPLA2).

    PubMed

    Abi Nahed, Roland; Martinez, Guillaume; Escoffier, Jessica; Yassine, Sandra; Karaouzène, Thomas; Hograindleur, Jean-Pascal; Turk, John; Kokotos, George; Ray, Pierre F; Bottari, Serge; Lambeau, Gérard; Hennebicq, Sylviane; Arnoult, Christophe

    2016-02-01

    Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2β and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2β is critical for spontaneous AR, whereas both iPLA2β and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 μm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2β, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 μm) but not at higher P4 concentrations (~10 μm). PMID:26655718

  20. Role of an aprotinin-sensitive protease in protein kinase Calpha-mediated activation of cytosolic phospholipase A2 by calcium ionophore (A23187) in pulmonary endothelium.

    PubMed

    Chakraborti, Sajal; Michael, John R; Chakraborti, Tapati

    2004-06-01

    Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 microM)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 microM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 microM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the

  1. Phospholipase A2, oxidative stress, and neurodegeneration in binge ethanol-treated organotypic slice cultures of developing rat brain

    PubMed Central

    Moon, Kwan-Hoon; Tajuddin, Nuzhath; Brown, James; Neafsey, Edward J.; Kim, Hee-Yong; Collins, Michael A.

    2013-01-01

    Background Brain neurodamage from chronic binge ethanol exposure is linked to neuroinflammation and associated oxidative stress. Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures of developing brain age, we reported that binge ethanol promotes release of a neuroinflammatory instigator, arachidonic acid (AA), concomitant with neurodegeneration, and that mepacrine, a global inhibitor of phospholipase A2 (PLA2) enzymes mobilizing AA from phospholipids, is neuroprotective. Here we sought with binge ethanol-treated HEC cultures to establish that PLA2 activity is responsible in part for significant oxidative stress, and to ascertain the PLA2 families responsible for AA release and neurodegeneration. Methods HEC slices, prepared from one wk-old rats and cultured 2–2½ wks, were exposed to 100 mM ethanol over 6 successive days, with 4 daytime “withdrawals” (no ethanol). Brain 3-nitrotyrosinated (3-NT) and 4-hydroxynonenal (4-HNE)-adducted proteins, oxidative stress footprints, were immunoassayed on days 3 through 6, and mepacrine’s effect was determined on day 6. The effects of specific PLA2 inhibitors on neurodegeneration (propidium iodide staining) and AA release (ELISA levels in media) in the cultures were then determined. Also, the effect of JZL184, an inhibitor of monoacylglycerol lipase (MAGL) which is reported to mobilize AA from endocannabinoids during neuroinflammatory insults, was examined. Results 3-NT- and 4-HNE-adducted proteins were significantly increased by the binge ethanol exposure, consistent with oxidative stress, and mepacrine prevented the increases. The PLA2 inhibitor results implicated secretory PLA2 (GII sPLA2) and to some extent Ca+2-independent PLA2 (GVI iPLA2) in binge ethanol-induced neurotoxicity and in AA release, but surprisingly, Ca+2-dependent PLA2 (GIV cPLA2) did not appear important. Furthermore, unlike PLA2 inhibition, MAGL inhibition failed to prevent the neurodegeneration. Conclusions In these

  2. 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

  3. Studies of synthetic chalcone derivatives as potential inhibitors of secretory phospholipase A2, cyclooxygenases, lipoxygenase and pro-inflammatory cytokines

    PubMed Central

    Jantan, Ibrahim; Bukhari, Syed Nasir Abbas; Adekoya, Olayiwola A; Sylte, Ingebrigt

    2014-01-01

    Arachidonic acid metabolism leads to the generation of key lipid mediators which play a fundamental role during inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as a synergistic anti-inflammatory effect with enhanced spectrum of activity. A series of 1,3-diphenyl-2-propen-1-one derivatives were investigated for anti-inflammatory related activities involving inhibition of secretory phospholipase A2, cyclooxygenases, soybean lipoxygenase, and lipopolysaccharides-induced secretion of interleukin-6 and tumor necrosis factor-alpha in mouse RAW264.7 macrophages. The results from the above mentioned assays exhibited that the synthesized compounds were effective inhibitors of pro-inflammatory enzymes and cytokines. The results also revealed that the chalcone derivatives with 4-methlyamino ethanol substitution seem to be significant for inhibition of enzymes and cytokines. Molecular docking experiments were carried out to elucidate the molecular aspects of the observed inhibitory activities of the investigated compounds. Present findings increase the possibility that these chalcone derivatives might serve as a beneficial starting point for the design and development of improved anti-inflammatory agents. PMID:25258510

  4. A novel phospholipase A2 (D49) from the venom of the Crotalus oreganus abyssus (North American Grand canyon rattlesnake).

    PubMed

    Martins, W; Baldasso, P A; Honório, K M; Maltarollo, V G; Ribeiro, R I M A; Carvalho, B M A; Soares, A M; Calderon, L A; Stábeli, R G; Caballol, M A O; Acosta, G; Oliveira, E; Marangoni, S; Albericio, F; Da Silva, S L

    2014-01-01

    Currently, Crotalus viridis was divided into two species: Crotalus viridis and Crotalus oreganus. The current classification divides "the old" Crotalus viridis into two new and independent species: Crotalus viridis (subspecies: viridis and nuntius) and Crotalus oreganus (subspecies: abyssus, lutosus, concolor, oreganus, helleri, cerberus, and caliginis). The analysis of a product from cDNA (E6d), derived from the gland of a specie Crotalus viridis viridis, was found to produce an acid phospholipase A2. In this study we isolated and characterized a PLA2 (D49) from Crotalus oreganus abyssus venom. Our studies show that the PLA2 produced from the cDNA of Crotalus viridis viridis (named E6d) is exactly the same PLA2 primary sequence of amino acids isolated from the venom of Crotalus oreganus abyssus. Thus, the PLA2 from E6d cDNA is actually the same PLA2 presented in the venom of Crotalus oreganus abyssus and does not correspond to the venom from Crotalus viridis viridis. These facts highlight the importance of performing more studies on subspecies of Crotalus oreganus and Crotalus viridis, since the old classification may have led to mixed results or mistaken data. PMID:24707493

  5. Multivalent nanoparticle networks enable point-of-care detection of human phospholipase-A2 in serum.

    PubMed

    Chapman, Robert; Lin, Yiyang; Burnapp, Mark; Bentham, Andrew; Hillier, David; Zabron, Abigail; Khan, Shahid; Tyreman, Matthew; Stevens, Molly M

    2015-03-24

    A rapid and highly sensitive point-of-care (PoC) lateral flow assay for phospholipase A2 (PLA2) is demonstrated in serum through the enzyme-triggered release of a new class of biotinylated multiarmed polymers from a liposome substrate. Signal from the enzyme activity is generated by the adhesion of polystreptavidin-coated gold nanoparticle networks to the lateral flow device, which leads to the appearance of a red test line due to the localized surface plasmon resonance effect of the gold. The use of a liposome as the enzyme substrate and multivalent linkers to link the nanoparticles leads to amplification of the signal, as the cleavage of a small amount of lipids is able to release a large amount of polymer linker and adhesion of an even larger amount of gold nanoparticles. By optimizing the molecular weight and multivalency of these biotinylated polymer linkers, the sensitivity of the device can be tuned to enable naked-eye detection of 1 nM human PLA2 in serum within 10 min. This high sensitivity enabled the correct diagnosis of pancreatitis in diseased clinical samples against a set of healthy controls using PLA2 activity in a point-of-care device for the first time. PMID:25756526

  6. Lack of Group X Secreted Phospholipase A2 Increases Survival Following Pandemic H1N1 Influenza Infection

    PubMed Central

    Kelvin, Alyson A.; Degousee, Norbert; Banner, David; Stefanski, Eva; Leon, Alberto J.; Angoulvant, Denis; Paquette, Stéphane G.; Huang, Stephen S. H.; Danesh, Ali; Robbins, Clinton S.; Noyan, Hossein; Husain, Mansoor; Lambeau, Gerard; Gelb, Michael H.; Kelvin, David J.; Rubin, Barry B.

    2014-01-01

    The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of (Reviewer 2 Minor Comment 2) GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX−/−) model and found that survival after infection was significantly greater in GX−/− mice than in GX+/+ mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX−/− mice BAL fluid. Lung microarray analysis identified an earlier and more robust induction of T and B cell associated genes in GX−/− mice. Based on the central role of sPLA2 enzymes as key initiators of inflammatory processes, we propose that activation of GX-sPLA2 during H1N1pdm infection is an early step of pulmonary inflammation and its (Reviewer 2 Minor Comment 2) inhibition increases adaptive immunity and improves survival. Our findings suggest that GX-sPLA2 may be a potential therapeutic target during influenza. PMID:24725934

  7. Trans-Serosal Leakage of Proinflammatory Mediators during Abdominal Aortic Aneurysm Repair: Role of Phospholipase A2 in Activating Leukocytes

    PubMed Central

    2010-01-01

    Gut barrier failure and the resultant translocation of luminal bacteria and bacterial products into the systemic circulation have been proposed as pathogenic mechanisms of multiorgan dysfunction syndrome (MODS) in open repair of abdominal aortic aneurysm (AAA). Our study aimed to demonstrate the direct release of gut-derived inflammatory mediators via the trans-serosal route in humans. Fifteen patients who underwent elective infrarenal open repair of AAA were randomized into two groups. In Group I patients (n = 10), the small intestine was exteriorized into a bowel bag. In Group II patients (n = 5), the small intestine was packed within the peritoneal cavity using large gauzes. We collected the bowel bag fluid in Group I and the ascites fluid, squeezed out from the gauzes at the end of surgery, in Group II. Leukocytes were collected from patients' blood samples. Incubation with the bowel bag fluid and ascites fluid caused a significant increase in both granulocyte pseudopod formation and CD11b expression compared to that with control fluid (p < 0.01). The addition of phospholipase A2 (PLA2) inhibitor quinacrine abolished leukocyte activation by the bowel bag fluid. Based on these results, we consider that trasns-serosal leakage of gut-derived mediators occurred during the open repair of AAA; further, sPLA2 may be the most potent mediator in the activation of leukocytes among such gut-derived mediators in AAA surgery. PMID:23555400

  8. Association between Ala379Val polymorphism of lipoprotein-associated phospholipase A2 and migraine without aura in Iranian population

    PubMed Central

    Haghdoost, Faraidoon; Gharzi, Mahsa; Faez, Farough; Hosseinzadeh, Elinaz; Tajaddini, Mohamadhasan; Rafiei, Laleh; Asgari, Fatemeh; Banihashemi, Mahboobeh; Masjedi, Samaneh Sadat; Zandifar, Alireza; Haghjooy-Javanmard, Shaghayegh

    2016-01-01

    Background: Migraine is a common neurovascular disorder with multifactorial and polygenic inheritance. The aim of this study was to investigate the association of a migraine without aura and Ala379Val polymorphism of lipoprotein-associated phospholipase A2 (Lp-PLA2) gene in the Iranian population. Methods: In this study, 103 migraine patients and 100 healthy controls were enrolled. DNA samples were extracted and the Ala379Val polymorphism of Lp-PLA2 gene was investigated. To assess severity of a headache, patients filled out the headache impact test (HIT-6) and migraine severity (MIGSEV) questionnaires. Results: Allele V had significantly lower frequency in the case group than control subjects [P = 0.001, odds ratio (OR) = 0.25, confidence interval (CI): 0.15-0.40]. The frequency of migraine patients that were a carrier of V allele (V/V and A/V) was statistically significant lower than the control group (P = 0.003, OR = 2.39, CI: 1.35-4.23). There was no significant difference of alleles frequency between three grades of MIGSEV (P = 0.316). Furthermore, total HIT-6 score was not significantly different between different genotypes (P = 0.466). Conclusion: Our results showed that Ala379Val gene polymorphism of LP-PLA2 is associated with lower risk of migraine but not with severity of headaches in an Iranian population. PMID:27326362

  9. Active site mutants of human secreted Group IIA Phospholipase A2 lacking hydrolytic activity retain their bactericidal effect.

    PubMed

    Chioato, Lucimara; Aragão, Elisangela Aparecida; Ferreira, Tatiana Lopes; Ward, Richard J

    2012-01-01

    The Human Secreted Group IIA Phospholipase A(2) (hsPLA2GIIA) presents potent bactericidal activity, and is considered to contribute to the acute-phase immune response. Hydrolysis of inner membrane phospholipids is suggested to underlie the bactericidal activity, and we have evaluated this proposal by comparing catalytic activity with bactericidal and liposome membrane damaging effects of the G30S, H48Q and D49K hsPLA2GIIA mutants. All mutants showed severely impaired hydrolytic activities against mixed DOPC:DOPG liposome membranes, however the bactericidal effect against Micrococcus luteus was less affected, with 50% killing at concentrations of 1, 3, 7 and 9 μg/mL for the wild-type, D49K, H48Q and G30S mutants respectively. Furthermore, all proteins showed Ca(2+)-independent damaging activity against liposome membranes demonstrating that in addition to the hydrolysis-dependent membrane damage, the hsPLA2GIIA presents a mechanism for permeabilization of phospholipid bilayers that is independent of catalytic activity, which may play a role in the bactericidal function of the protein. PMID:21986368

  10. Is Lipoprotein-Associated Phospholipase A2 a Link between Inflammation and Subclinical Atherosclerosis in Rheumatoid Arthritis?

    PubMed Central

    Södergren, Anna; Karp, Kjell; Bengtsson, Christine; Möller, Bozena; Rantapää-Dahlqvist, Solbritt; Wållberg-Jonsson, Solveig

    2015-01-01

    Objective. Lipoprotein-associated phospholipase A2 (Lp-PLA2), a marker of vascular inflammation, is associated with cardiovascular disease. This prospective study of an inception cohort aimed to investigate whether the level of Lp-PLA2 is associated with subclinical atherosclerosis in patients with rheumatoid arthritis (RA). Methods. Patients from northern Sweden diagnosed with early RA were consecutively recruited into an ongoing prospective study. From these, all patients ≤60 years (n = 71) were included for measurements of subclinical atherosclerosis at inclusion (T0) and five years later (T5). Forty age- and sex-matched controls were included. The patients were clinically assessed, SCORE, Reynolds Risk Score, and Larsen score were calculated, and blood samples were drawn from all individuals at T0 and T5. Results. There was no significant difference in the level of Lp-PLA2 between patients with RA and controls (p > 0.05). In simple linear regression models among patients with RA, Lp-PLA2 at T0 was significantly associated with intima media thickness (IMT) at T0 and T5, flow mediated dilation (FMD) at T0 and T5, ever smoking, male sex, HDL-cholesterol (inversely), non-HDL-cholesterol, SCORE, Reynolds Risk Score, and Larsen score (p < 0.05). Conclusion. In this cohort of patients with early RA, the concentration of Lp-PLA2 was associated with both subclinical atherosclerosis and disease severity. PMID:26504820