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Sample records for phospholipase a2 correlates

  1. Activation of phospholipase A2 by cannabinoids. Lack of correlation with CNS effects.

    PubMed

    Evans, A T; Formukong, E; Evans, F J

    1987-01-26

    Cannabinoids delta 1-tetrahydrocannabinol, cannabinol, cannabidiol and cannabigerol have been shown to affect directly the activity of phospholipase A2 in a cell-free assay. The compounds produced a biphasic activation of the enzyme, with EC50 values in the range 6.0-20.0 X 10(-6) M and IC50 values in the range 50.0-150.0 X 10(-6) M. These results correlated well with the relative potencies reported for the stimulation of prostaglandin release from human synovial cells in vitro, confirming that activation of phospholipase A2 is the predominant action of cannabinoids on arachidonate metabolism in tissue culture. However, since delta 1-tetrahydrocannabinol is unique among these compounds in possessing cataleptic activity, it is unlikely that phospholipase A2 is the major receptor mediating the psychotropic effects of cannabis.

  2. 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

  3. Group XV phospholipase A2, a lysosomal phospholipase A2

    PubMed Central

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

    2010-01-01

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

  4. Correlation between plasma lipoprotein-associated phospholipase A2 and peripheral arterial disease.

    PubMed

    Li, Shuai-Bing; Yang, Fan; Jing, Li; Ma, Juan; Jia, Ya-Dan; Dong, Shao-Ying; Zheng, Wei-Feng; Zhao, Luo-Sha

    2013-05-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a recently identified and potentially useful plasma biomarker for cardiovascular diseases. However, its role in peripheral arterial disease (PAD) remains unclear. The objective of this study was to assess the independent association of Lp-PLA2 and other inflammatory markers with the reduced ankle-brachial blood pressure index (ABI), a marker of PAD. We performed a cross-sectional study in 982 individuals aged ≥40 years who were recruited from the First Affiliated Hospital of Zhengzhou University. PAD was defined as an ABI <0.9 in at least one leg. The individuals were further divided into two groups, 145 with PAD and 837 without PAD. Following adjustment for traditional cardiovascular risk factors, the odds ratios of PAD when comparing the highest to the lowest quartiles were 3.24 (95% CI, 1.68-3.94) for Lp-PLA2, 2.14 (95% CI, 1.07-3.11) for homocysteine, 1.93 (95% CI, 1.02-4.01) for fibrinogen, 2.26 (95% CI, 1.32-5.74) for apolipoprotein B and 1.3 (95% CI, 0.75-2.49) for high-sensitivity C-reactive protein (hsCRP). When Lp-PLA2 and inflammatory markers were simultaneously included in the full model, the corresponding odds ratios were 1.81 (95% CI, 1.14-3.68) for Lp-PLA2, 1.15 (95% CI, 0.49-2.69) for homocysteine, 1.21 (95% CI, 0.88-5.57) for fibrinogen, 0.98 (95% CI, 0.51-3.85) for apolipoprotein B and 1.23 (95% CI, 1.12-3.51) for hsCRP. Lp-PLA2 levels were significantly and independently associated with PAD following adjustment for other inflammatory markers. These findings reflect the potential role of circulating Lp-PLA2 as a marker of atherosclerosis.

  5. [Correlation of antitumor effect of recombinant sea snake basic phospholipase A2 to its enzymatic activity].

    PubMed

    Liang, Yong-Ju; Yang, Xiao-Ping; Wei, Jian-Wen; Fu, Li-Wu; Jiang, Xiao-Yu; Chen, Shang-Wu; Yang, Wen-Li

    2005-12-01

    Snake venom phospolipase A2 (PLA(2)), a large family of homologous (14 ku) soluble proteins, exerts diverse pharmacologic activities as well as enzymatic activities. So far, the structure and function of terrestrial snake PLA(2), especially the relationship of its enzymatic and pharmacologic activities have been studied extensively, but the investigation of sea snake PLA(2) are limited. This study was to investigate the in vitro and in vivo antitumor effects of recombinant sea snake basic PLA(2) (rSSBPLA(2)) and its mutants rN48 and rK4 from sea snake Lapemis hardwickii venom, and to explore the influence of 2 residues related with the enzymatic activity on the antitumor effects. Site-directed mutagenesis of the 2 conserved residues related with enzymatic activity (His48 mutated to Asn and Asp49 mutated to Lys) was performed. The inhibitory effects of rSSBPLA(2), rN48 and rK49 on proliferation of human myeloid leukemia cell line HL-60, human neuroblastoma cell line SK-N-SH, human gastric cancer cell line MGC-803, and human liver cancer cell line HepG2 were assessed by MTT assay. Their antitumor effects on sarcoma cell line S180 xenograft and EAC ascites cancer model in mice were detected. The relative enzymatic activities of rN48 and rK49 were 0 and 5% of that of rSSBPLA(2). The 50% inhibitory concentration (IC(50)) of rSSBPLA(2) for HL60, SK-N-SH, and MGC-803 cells were (45.28+/-0.09) microg/ml, (57.07+/-0.12) microg/ml, and (69.34+/-0.35) microg/ml, respectively, but it had no inhibitory effect on proliferation of HepG2 cells. rSSBPLA(2) obviously inhibited growth of S180 xenograft in miceû the inhibitory rates were 50.8%, 43.2%, 38.2%, and 55.5%, respectively, under the dose of 2 mg/kg (qd x 10), 2 mg/kg (q2d x 5), 4 mg/kg (qd x 1) and 4 mg/kg (q5d x 2). The inhibitory rate of EAC model was 33.5% under the dose of 4 mg/kg (q5d x 2). The inhibitory rates were significantly higher in test groups than in control groups (P<0.01). rN48 and rK49 had no inhibitory

  6. Phospholipase A(2) activates hemostasis.

    PubMed

    Stief, Thomas W

    2007-01-01

    Phospholipases A(2) (PLA(2)) are aggressive enzymes that can destroy phospholipids of cell membranes. The resulting cell fragments trigger the kallikrein-mediated contact phase of coagulation. The aim of the present study was to expose citrated whole blood to PLA(2) and to quantify thrombin generation in recalcified plasma. Normal citrated blood was exposed to bovine pancreatic or snake PLA(2), lipopolysaccharide (LPS), or zymosan A for 30-45 min (RT). After centrifugation the plasma samples were recalcified (10 + 1) with 250 mM CaCl(2) in the recalcified coagulation activity assay (RECA). After 0-45 min coagulation reaction time (CRT at 37°C) 1.6 M arginine (final test concentration) was added to stop hemostasis activation and to depolymerize non-crosslinked fibrin. The generated thrombin activity was chromogenically determined. 100 ng/ml bovine pancreatic or snake PLA(2) generates about 0.2-0.8 IU/ml thrombin after 15 min CRT. This thrombin generation is similar as that induced by 200 ng/ml LPS or 20 μg/ml zymosan A. Up to 60 ng/ml bovine pancreatic PLA(2) the generated thrombin activity is proportional to the PLA(2) activity used; 1 μg/ml PLA(2) induces much less thrombin, but PLA(2) at 10 μg/ml again results into thrombin generation of 0.1-3 IU/ml at 10-15 min CRT. As control, in pooled normal citrated plasma there is no significant change in thrombin generation when exposed to up to 10 μg/ml bovine pancreatic PLA(2). Elevated plasmatic PLA(2) activities (occurring e.g. in trauma, pancreatitis, or sepsis) activate the blood hemostasis system resulting in pathologic disseminated intravascular coagulation (PDIC). It is suggested to diagnose these life threatening states as early as possible, screening all patients for plasmatic thrombin activity.

  7. Phospholipase A2 Activates Hemostasis

    PubMed Central

    Stief, Thomas W.

    2007-01-01

    Background Phospholipases A2 (PLA2) are aggressive enzymes that can destroy phospholipids of cell membranes. The resulting cell fragments trigger the kallikrein—mediated contact phase of coagulation. The aim of the present study was to expose citrated whole blood to PLA2 and to quantify thrombin generation in recalcified plasma. Methods Normal citrated blood was exposed to bovine pancreatic or snake PLA2, lipopolysaccharide (LPS), or zymosan A for 30–45 min (RT). After centrifugation the plasma samples were recalcified (10 + 1) with 250 mM CaCl2 in the recalcified coagulation activity assay (RECA). After 0–45 min coagulation reaction time (CRT at 37°C) 1.6 M arginine (final test concentration) was added to stop hemostasis activation and to depolymerize non-crosslinked fibrin. The generated thrombin activity was chromogenically determined. Results 100 ng/ml bovine pancreatic or snake PLA2 generates about 0.2–0.8 IU/ml thrombin after 15 min CRT. This thrombin generation is similar as that induced by 200 ng/ml LPS or 20 μg/ml zymosan A. Up to 60 ng/ml bovine pancreatic PLA2 the generated thrombin activity is proportional to the PLA2 activity used; 1 μg/ml PLA2 induces much less thrombin, but PLA2 at 10 μg/ml again results into thrombin generation of 0.1–3 IU/ml at 10–15 min CRT. As control, in pooled normal citrated plasma there is no significant change in thrombin generation when exposed to up to 10 μg/ml bovine pancreatic PLA2. Discussion Elevated plasmatic PLA2 activities (occurring e.g. in trauma, pancreatitis, or sepsis) activate the blood hemostasis system resulting in pathologic disseminated intravascular coagulation (PDIC). It is suggested to diagnose these life threatening states as early as possible, screening all patients for plasmatic thrombin activity. PMID:21901065

  8. Antigen receptors on immature, but not mature, B and T cells are coupled to cytosolic phospholipase A2 activation: expression and activation of cytosolic phospholipase A2 correlate with lymphocyte maturation.

    PubMed

    Gilbert, J J; Stewart, A; Courtney, C A; Fleming, M C; Reid, P; Jackson, C G; Wise, A; Wakelam, M J; Harnett, M M

    1996-03-15

    The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.

  9. Lipoprotein associated phospholipase A2 activity & its correlation with oxidized LDL & glycaemic status in early stages of type-2 diabetes mellitus.

    PubMed

    Garg, Seema; Madhu, S V; Suneja, Shilpa

    2015-01-01

    Lipoprotein associated phospholipase A 2 (Lp-PLA 2 ) is an important risk predictor of coronary artery disease (CAD). This study was aimed to evaluate Lp-PLA 2 activity and oxidized low density lipoprotein (oxLDL) in newly diagnosed patients of type 2 diabetes mellitus and to determine the correlation of Lp-PLA 2 activity with oxLDL and plasma glucose levels. Blood samples were collected in patients with newly diagnosed type 2 diabetes (n=40) before any treatment was started and healthy controls (n=40). These were processed for estimating plasma glucose: fasting and post prandial, ox LDL, and Lp-PLA2 activity. The parameters in the two groups were compared. Correlation between different parameters was calculated by Pearson correlation analysis in both groups. Lp-PLA 2 activity (24.48 ± 4.91 vs 18.63 ± 5.29 nmol/min/ml, P<0.001) and oxLDL levels (52.46 ± 40.19 vs 33.26 ± 12.54 μmol/l, P<0.01) were significantly higher in patients as compared to those in controls. Lp-PLA 2 activity correlated positively with oxLDL in both controls (r=0.414, P<0.01), as well in patients (r=0.542, P<0.01). A positive correlation between Lp-PLA 2 activity and fasting plasma glucose levels was observed only in patients (r=0.348, P<0.05). Result of this study implies that higher risk of CAD in patients with diabetes may be due to increase in Lp-PLA 2 activity during the early course of the disease. A positive correlation between enzyme activity and fasting plasma glucose indicates an association between hyperglycaemia and increased activity of Lp-PLA2. This may explain a higher occurrence of CAD in patients with diabetes. A positive correlation between oxLDL and Lp-PLA2 activity suggests that Lp-PLA2 activity may be affected by oxLDL also.

  10. 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 uses subject to reporting. (1) The chemical substances identified generically as lecithins...

  11. 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 uses subject to reporting. (1) The chemical substances identified generically as lecithins...

  12. Secreted phospholipase A2 and mast cells.

    PubMed

    Murakami, Makoto; Taketomi, Yoshitaka

    2015-01-01

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

  13. Secretory Phospholipase A2 Responsive Liposomes

    PubMed Central

    ZHU, GUODONG; MOCK, JASON N.; ALJUFFALI, IBRAHIM; CUMMINGS, BRIAN S.; ARNOLD, ROBERT D.

    2011-01-01

    Secretory phospholipase A2 (sPLA2) expression is increased in several cancers and has been shown to trigger release from some lipid carriers. This study used electrospray ionization mass spectrometry (ESI-MS) and release of 6-carboxyfluorescein (6-CF) to determine the effects of sPLA2 on various liposome formulations. Different combinations of zwitterionic [1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine, and 1,2- distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE)] and anionic [1,2-distearoyl-sn-glycero-3-phosphatidic acid, 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG), 1,2-distearoyl-sn-glycero-3-phosphatidylserine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–N-poly(ethylene glycol) 2000 (DSPE–PEG)] phospholipids were examined. DSPG and DSPE were most susceptible to sPLA2-mediated degradation compared with other phospholipids. Increased 6-CF release was observed after inclusion of 10 mol % DSPE and anionic lipids into different liposome formulations. Group IIa sPLA2-mediated 6-CF release was less than Group III and relatively insensitive to cholesterol (Chol), whereas Chol reduced sPLA2-mediated release. Inclusion of DSPE–PEG increased sPLA2-mediated 6-CF release, whereas serum reduced lipid degradation and 6-CF release significantly. These data demonstrate that ESI-MS and 6-CF release were useful in determining the selectivity of sPLA2 and release from liposomes, that differences in the activity of different sPLA2 isoforms exist, and that DSPE–PEG enhanced sPLA2-mediated release of liposomal constituents. These findings will aid in the selection of lipids and optimization of the kinetics of drug release for the treatment of cancers and diseases of inflammation in which sPLA2 expression is increased. PMID:21455978

  14. Role of distinct phospholipases A2 and their modulators in meconium aspiration syndrome in human neonates.

    PubMed

    De Luca, Daniele; Minucci, Angelo; Tripodi, Domenico; Piastra, Marco; Pietrini, Domenico; Zuppi, Cecilia; Conti, Giorgio; Carnielli, Virgilio P; Capoluongo, Ettore

    2011-07-01

    Meconium aspiration syndrome (MAS) is a life-threatening neonatal lung injury, whose pathophysiology has been mainly studied in animal models. In such models, pancreatic secretory phospholipase A2 (sPLA2-IB) and proinflammatory cytokines present in meconium challenge the lungs, catabolising surfactant and harming the alveoli. Locally produced phospholipases might perpetuate the injury and influence clinical pictures and therapeutic approaches. Our aim is to verify whether pulmonary phospholipase A2 (sPLA2-IIA) is involved in the damage and to determine if phospholipases and their modulators are associated with MAS clinical pictures. We studied distinct phospholipases A2 and their modulators in broncho-alveolar lavage (BAL) fluids and in meconium of five MAS neonates and in five control neonates ventilated for extrapulmonary reasons. MAS patients have higher amounts of pulmonary phospholipase (sPLA2-IIA; P = 0.016) and Clara cell secretory protein (CCSP; P = 0.032). The local production of such proteins by the lung is confirmed by their very low levels in meconium. sPLA2-IIA contributes to the higher total enzyme activity in MAS patients, as compared to controls (P = 0.008). Cytosolic phospholipase was not detected in meconium or alveolar fluid. sPLA2 activity and sPLA2-IIA concentrations are correlated with the TNFα and with the release of CCSP. sPLA2 total activity, sPLA2-IIA and TNFα concentrations in BAL fluids correlate with the oxygenation impairment and haemorrhagic lung oedema. Pulmonary sPLA2 is locally produced and contributes to the total sPLA2 activity during MAS. CCSP is also produced in trying to lower the inflammation. Both sPLA2 activity and sPLA2-IIA are significantly correlated with oxygenation impairment and haemorrhagic lung oedema.

  15. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., phospholipase A2-hydrolyzed (PMN P-93-333) is subject to reporting under this section for the significant new...) Release to water. Requirements as specified in § 721.90 (a)(4), (b)(4), and (c)(4) (where N = 10 ppb). (b...), (f), (g), (h), (i), and (k) are applicable to manufacturers, importers, and processors of this...

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

    PubMed

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

    2016-01-01

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

  17. Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2.

    PubMed

    Reed, J K

    1981-08-06

    The effects of phospholipase A2 treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [3H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A2 from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by beta-bungarotoxin. (2) Phospholipase A2 from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A2 treatment of [3H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.

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

    PubMed

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

    2007-09-15

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

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

    PubMed

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

    2012-11-01

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

  20. Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2.

    PubMed

    Arcila, Mary Luz; Sánchez, María Dulfary; Ortiz, Blair; Barrera, Luis Fernando; García, Luis F; Rojas, Mauricio

    2007-10-01

    Monocyte/macrophage cell death is an important event during mycobacterial infection. To get insights about the influence of mononuclear phagocyte maturation in this event we compared the response to Mycobacterium tuberculosis (Mtb) infection of fresh isolated monocytes and monocyte-derived macrophages (MDM) from healthy tuberculin positive individuals. Both monocytes and MDM underwent apoptosis, however, there was a higher numbers of apoptotic macrophages with active Caspases 8 and 9. We also compared Mtb-induced cell death in U937 pro-monocytes and PMA-differentiated cells (U937D). In response to Mtb infection, U937D cells underwent apoptosis and promonocytes both apoptosis and necrosis. There were high number of U937D cells producing TNF-alpha and high number of IL-10+ promonocytes. These evidences suggest that U937 could be a valid model to study the mechanisms that rule Mtb-induced cell death. Experiments with the cell line and fresh isolated mononuclear cells with pharmacological inhibitors showed that induction of necrosis involved calcium and cAMP signals resulting in IL-10 production. Necrosis also correlated with Caspase 3, PLA2 activity and bacterial growth. In U937D cells and monocytes from healthy donors there was activation of calcium, TNF-alpha and Caspase 8 activation and decreased bacterial load. Understanding the mechanisms that control the dichotomy events between apoptosis and necrosis/oncosis associated with cell maturity might open new strategies to better control the course of mycobacterial infections.

  1. Phospholipase A2: Potential roles in native membrane fusion.

    PubMed

    Dabral, Deepti; Coorssen, Jens R

    2017-04-01

    Membrane fusion is a fundamental molecular mechanism by which two apposed membrane bilayers coalesce in rapid, transient steps that enable the successive merging of the outer and inner leaflets allowing lipid intermixing and subsequent mixing of the two previously separate compartments. The actual membrane merger mechanism - fusion, by definition - is conceptualized to be protein- or lipid-centric. According to the widely vetted stalk-pore hypothesis, membrane fusion proceeds via high curvature lipid intermediates. By cleaving membrane phospholipids at the sn-2 position, Phospholipase A2 generates metabolites that exert spontaneous curvature stress (both negative and positive) on the membrane, thus influencing local membrane bending by altering the packing and conformation of lipids and proteins, respectively. Such changes could potentially modulate priming and attachment/docking steps that precede fusion, as well as the membrane merger steps per se. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2000-01-01

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

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

    PubMed

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

    1988-04-25

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

  4. Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2

    PubMed Central

    Anagón, Alejandro C.; Molinar, Ricardo R.; Possani, Lourival D.; Fletcher, Paul L.; Cronan, John E.; Julia, Jordi Z.

    1980-01-01

    The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD50=4.3μg/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding two more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes have shown that all three phospholipases belong to type A2. Amino acid analysis has shown that phospholipase A2 (type 1) has 97 residues with a calculated mol.wt. of 10978±11. Phospholipase A2 (type 2) has 96 residues with a mol.wt. of 10959±11. Phospholipase A2 (type 3) has 266 residues with 16 half-cystine residues and a calculated mol.wt of 29042±31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val- Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A2 (type 2). ImagesFig. 1. PMID:7387631

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

    PubMed

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

    2013-12-15

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

  6. 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

  7. Interfacial Catalysis: The Mechanism of Phospholipase A2

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2010-06-01

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

  9. 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

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

    PubMed

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

    2011-09-01

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

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

    NASA Astrophysics Data System (ADS)

    Varshney, Rajeev; Kale, R. K.

    1995-04-01

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

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

    PubMed

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

    1989-02-01

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

  13. Role of phospholipase A2 in activation of isolated cardiomyocyte respiration in postinfarction cardiosclerosis.

    PubMed

    Egorova, M V; Afanas'ev, S A; Popov, S V

    2008-12-01

    The rate of oxygen consumption by isolated cardiomyocytes was studied in rats with experimental postinfarction cardiosclerosis. The increase in oxygen consumption under these condition was comparable to that in melittin- and arachidonic acid-induced activation of phospholipase A2 in cardiomyocytes of intact animals. Bromophenacyl bromide inhibition of phospholipase A2 in cardiomyocytes of rats with postinfarction cardiosclerosis led to reduction of oxygen consumption rate to values characteristic of intact animal cardiomyocytes. The results confirm the hypothesis according to which high oxygen consumption in postinfarction cardiosclerosis is related to increased activity of phospholipase A2.

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

    PubMed Central

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

    1994-01-01

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

  15. Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2.

    PubMed

    Van Themsche, C; Jacob, M; Salesse, C

    2001-01-01

    The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.

  16. 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

  17. Divalent cations increase lipid order in erythrocytes and susceptibility to secretory phospholipase A2.

    PubMed

    Vest, Rebekah S; Gonzales, Laurie J; Permann, Seth A; Spencer, Emily; Hansen, Lee D; Judd, Allan M; Bell, John D

    2004-04-01

    Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree to which ions affected membrane properties correlated with the ionic radius and electronegativity of the ions. Lastly, erythrocytes became more susceptible to enzyme hydrolysis in the presence of elevated intracellular levels of nickel and manganese, but not magnesium. These differences appeared related to the ability of the ions to induce a transition in erythrocyte shape.

  18. Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment.

    PubMed

    Balietti, Marta; Giuli, Cinzia; Fattoretti, Patrizia; Fabbietti, Paolo; Postacchini, Demetrio; Conti, Fiorenzo

    2016-01-01

    We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A.

  19. 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

  20. 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

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

    PubMed

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

    2013-01-18

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

  2. Cytosolic Phospholipase A2 and Lysophospholipids in Tumor Angiogenesis

    PubMed Central

    Linkous, Amanda G.

    2010-01-01

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

  3. Point of care testing of phospholipase A2 group IIA for serological diagnosis of rheumatoid arthritis

    NASA Astrophysics Data System (ADS)

    Liu, Nathan J.; Chapman, Robert; Lin, Yiyang; Mmesi, Jonas; Bentham, Andrew; Tyreman, Matthew; Abraham, Sonya; Stevens, Molly M.

    2016-02-01

    Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care.Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08423g

  4. 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.

  5. New insights into the role of lipoprotein(a)-associated lipoprotein-associated phospholipase A2 in atherosclerosis and cardiovascular disease.

    PubMed

    Tsimikas, Sotirios; Tsironis, Loukas D; Tselepis, Alexandros D

    2007-10-01

    Lipoprotein(a) [Lp(a)] plays an important role in atherosclerosis. The biological effects of Lp(a) have been attributed either to apolipoprotein(a) or to its low-density lipoprotein-like particle. Lp(a) contains platelet-activating factor acetylhydrolase, an enzyme that exhibits a Ca2+-independent phospholipase A2 activity and is complexed to lipoproteins in plasma; thus, it is also referred to as lipoprotein-associated phospholipase A2. Substrates for lipoprotein-associated phospholipase A2 include phospholipids containing oxidatively fragmented residues at the sn-2 position (oxidized phospholipids; OxPLs). OxPLs may play important roles in vascular inflammation and atherosclerosis. Plasma levels of OxPLs present on apolipoprotein B-100 particles (OxPL/apolipoprotein B) are correlated with coronary artery, carotid, and peripheral arterial disease. Furthermore, OxPL/apolipoprotein B levels in plasma are strongly correlated with Lp(a) levels, are preferentially sequestered on Lp(a), and thus are potentially subjected to degradation by the Lp(a)-associated lipoprotein-associated phospholipase A2. The present review article focuses specifically on the characteristics of the lipoprotein-associated phospholipase A2 associated with Lp(a) and discusses the possible role of this enzyme in view of emerging data showing that OxPLs in plasma are preferentially sequestered on Lp(a) and may significantly contribute to the increased atherogenicity of this lipoprotein.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Frei, E; Zahler, P

    1979-02-02

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

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

    PubMed

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

    2002-11-26

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

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed

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

    1983-08-01

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

  11. Serotonin-2A homodimers are needed for signalling via both phospholipase A2 and phospholipase C in transfected CHO cells.

    PubMed

    Iglesias, Alba; Cimadevila, Marta; Cadavid, María Isabel; Loza, María Isabel; Brea, José

    2017-04-05

    Different ligands differentially activate phospholipase A2 (PLA2) and phospholipase C (PLC) signalling pathways that are coupled to the serotonin 2A (5-HT2A) receptor, a class-A G-protein coupled receptor (GPCR). The serotonin 5-HT2A receptor has been shown to be expressed as a homodimer displaying some ligands negative cooperativity between protomers in the PLA2 signalling pathway. We hypothesized that the homodimeric complex is the minimum functional unit required for activation of the PLA2 and PLC pathways by the serotonin 5-HT2A receptor. To investigate this hypothesis, we partially blocked the serotonin 5-HT2A receptors with ritanserin and measured PLA2 and PLC activity simultaneously. We subsequently added the competitive antagonist spiperone to release the inactivator through a crosstalk mechanism and thus allow the dimer to return to a reactive state. Partial inactivation of the homodimer by ritanserin binding decreased the activity of the receptor by 59±13% and 70±4% in the PLA2 and PLC pathways respectively (P<0.001), with no difference in the potency of the serotonin (5-HT) was observed. The subsequent binding of spiperone released ritanserin due to the crosstalk between protomers and recovery of the receptor activity to 74±7% and 72±4%. Negative cooperativity between protomers in the dimer was maintained during arachidonic acid (AA) release after blocking ritanserin, as indicated by the biphasic inhibition curves for clozapine over 1μM serotonin (5-HT) in these conditions. These findings provide evidence that serotonin 5-HT2A receptors must be expressed as homodimers in order to activate both the PLA2 and PLC signalling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Secretory phospholipase A2 is required to produce histologic changes associated with gastroduodenal reflux in a murine model.

    PubMed

    Babu, Ashok; Meng, Xianzhong; Banerjee, Anirban M; Gamboni-Robertson, Fabia; Cleveland, Joseph C; Damle, Sagar; Fullerton, David A; Weyant, Michael J

    2008-06-01

    The earliest response of esophageal mucosa to gastric reflux is the development of oxidative damage and inflammation. These processes contribute to the development of metaplasia known as Barrett's esophagus, as well as the progression to malignancy. Secretory phospholipase A(2) is a mediator of inflammation with levels that are increased in Barrett's metaplasia and carcinoma when compared with levels in normal samples. Our goal is to determine the role of secretory phospholipase A(2) in the development of reflux-associated changes in the esophageal mucosa. Secretory phospholipase A(2)-deficient mice (C57BL/6, n = 5) and mice known to express high levels of secretory phospholipase A(2) (BALB/c, n = 5) underwent side-to-side surgical anastomosis of the first portion of the duodenum and gastroesophageal junction, allowing exposure of esophageal mucosa to duodenal and gastric contents duodeno-gastroesophageal anastomosis. Control animals (n = 5) of each strain underwent laparotomy with esophagotomy and repair. Tissue was frozen in embedding medium. Hematoxylin and eosin staining and Ki67 and secretory phospholipase A(2) immunohistochemistry were used to evaluate esophageal tissue and its response to duodeno-gastroesophageal anastomosis. Immunofluorescent staining confirmed the absence of secretory phospholipase A(2) in C57BL/6 mice and its presence in BALB/c mice. Hematoxylin and eosin staining demonstrated significant thickening of the esophageal mucosa in response to gastroesophageal reflux in the presence of secretory phospholipase A(2). Mice known to express high levels of secretory phospholipase A(2) also demonstrated increased numbers of proliferating cells. Secretory phospholipase A(2)-deficient mice were immune to the early changes induced by mixed reflux. The presence of secretory phospholipase A(2) appears necessary for early histologic changes produced by exposure of the esophagus to gastroduodenal contents. This enzyme is identified as a promising target

  13. 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

  14. Expression of Phospholipases A2 and C in Human Corneal Epithelial Cells

    PubMed Central

    Landreville, Solange; Coulombe, Stéphanie; Carrier, Patrick; Gelb, Michael H.; Guérin, Sylvain L.; Salesse, Christian

    2008-01-01

    Purpose To achieve a better understanding of the involvement of phospholipases in the inflammation and wound-healing processes in human corneal epithelial cells (HCECs), expression of phospholipase A2s (PLA2s) and phospholipase Cs (PLCs) was examined in the human corneal epithelium. Methods Specific primers were designed for RT-PCR amplification of the known secreted (s)PLA2, cytosolic (c)PLA2, and PLC mRNAs. Corresponding PCR products were cloned and the DNA sequenced. Immunofluorescence of flatmounted corneal sections and Western blot analyses were used to detect the PLA2s and PLCs expressed by HCECs. Results The mRNAs for the following phospholipases were detected by RT-PCR in the HCECs: sPLA2GIII, -GX, and -GXIIA; cPLA2α and -γ; PLCβ1, -β2, -β3, -β4, -γ1, -γ2, -δ1, -δ3, -δ4, and -ε. Immunofluorescence analyses conducted on corneal epithelium cryosections and Western blot on freshly isolated HCECs demonstrated the presence of sPLA2GIII, -GX, and -GXIIA; cPLA2α and -γ; and PLCβ2, -β3, -γ1, -γ2, and -δ3. Conclusions Many phospholipase isoforms are expressed by HCECs and may play a major role in signal transduction (PLCs) as well as in the release of precursors of potent mediators of inflammation, such as leukotrienes and prostaglandins (PLA2s). Moreover, the sPLA2s expressed by the corneal epithelium could be involved in the normal antibacterial activity in the tears and in wound healing. PMID:15505048

  15. 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

  16. Secretory phospholipase A2 activity in blood serum: the challenge to sense.

    PubMed

    Alekseeva, A S; Korotaeva, A A; Samoilova, E V; Volynsky, P E; Vodovozova, E L; Boldyrev, I A

    2014-11-07

    Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.

  17. Quantum-dynamical picture of a multistep enzymatic process: reaction catalyzed by phospholipase A(2).

    PubMed Central

    Bała, P; Grochowski, P; Nowiński, K; Lesyng, B; McCammon, J A

    2000-01-01

    A quantum-classical molecular dynamics model (QCMD), applying explicit integration of the time-dependent Schrödinger equation (QD) and Newtonian equations of motion (MD), is presented. The model is capable of describing quantum dynamical processes in complex biomolecular systems. It has been applied in simulations of a multistep catalytic process carried out by phospholipase A(2) in its active site. The process includes quantum-dynamical proton transfer from a water molecule to histidine localized in the active site, followed by a nucleophilic attack of the resulting OH(-) group on a carbonyl carbon atom of a phospholipid substrate, leading to cleavage of an adjacent ester bond. The process has been simulated using a parallel version of the QCMD code. The potential energy function for the active site is computed using an approximate valence bond (AVB) method. The dynamics of the key proton is described either by QD or classical MD. The coupling between the quantum proton and the classical atoms is accomplished via Hellmann-Feynman forces, as well as the time dependence of the potential energy function in the Schrödinger equation (QCMD/AVB model). Analysis of the simulation results with an Advanced Visualization System revealed a correlated rather than a stepwise picture of the enzymatic process. It is shown that an sp(2)--> sp(3) configurational change at the substrate carbonyl carbon is mostly responsible for triggering the activation process. PMID:10968989

  18. Secretory phospholipase A2 in the pathogenesis of acute dengue infection

    PubMed Central

    Jeewandara, Chandima; Gomes, Laksiri; Udari, Sukhitha; Paranavitane, S.A.; Shyamali, N.L.A.; Ogg, Graham S.

    2016-01-01

    Abstract Introduction Platelet activating factor (PAF) is an important mediator of vascular leak in acute dengue. Phospholipase A2s (PLA2) are inflammatory lipid enzymes that generate and regulate PAF and other mediators associated with mast cells. We sought to investigate if mast cell activation and increases in secretory sPLA2s are associated with an increase in PAF and occurrence of dengue haemorrhagic fever (DHF). Methods The changes in the levels of mast cell tryptase, PAF and the activity of sPLA2 were determined throughout the course of illness in 13 adult patients with DHF, and 30 patients with dengue fever (DF). Results We found that sPLA2 activity was significantly higher in patients with DHF when compared to those with DF, during the first 120 h of clinical illness. sPLA2 activity was significantly associated with PAF levels, which were also significantly higher in patients with DHF. Although levels of mast cell tryptase were higher in patients with DHF, the difference was not significant, and the levels were not above the reference ranges. sPLA2 activity significantly correlated with the degree of viraemia in patients with DHF but not in those with DF. Conclusion sPLA2 appears to play an important role in the pathogenesis of dengue. Since its activity is significantly increased during the early phase of infection in patients with DHF, this suggests that understanding the underlying mechanisms may provide opportunities for early intervention. PMID:28250920

  19. Lithium activates brain phospholipase A2 and improves memory in rats: implications for Alzheimer's disease.

    PubMed

    Mury, Fábio B; da Silva, Weber C; Barbosa, Nádia R; Mendes, Camila T; Bonini, Juliana S; Sarkis, Jorge Eduardo Souza; Cammarota, Martin; Izquierdo, Ivan; Gattaz, Wagner F; Dias-Neto, Emmanuel

    2016-10-01

    Phospholipase A2 (Pla2) is required for memory retrieval, and its inhibition in the hippocampus has been reported to impair memory acquisition in rats. Moreover, cognitive decline and memory deficits showed to be reduced in animal models after lithium treatment, prompting us to evaluate possible links between Pla2, lithium and memory. Here, we evaluated the possible modulation of Pla2 activity by a long-term treatment of rats with low doses of lithium and its impact in memory. Wistar rats were trained for the inhibitory avoidance task, treated with lithium for 100 days and tested for perdurability of long-term memory. Hippocampal samples were used for quantifying the expression of 19 brain-expressed Pla2 genes and for evaluating the enzymatic activity of Pla2 using group-specific radio-enzymatic assays. Our data pointed to a significant perdurability of long-term memory, which correlated with increased transcriptional and enzymatic activities of certain members of the Pla2 family (iPla2 and sPla2) after the chronic lithium treatment. Our data suggest new possible targets of lithium, add more information on its pharmacological activity and reinforce the possible use of low doses of lithium for the treatment of neurodegenerative conditions such as the Alzheimer's disease.

  20. [Role of secreted and lipoprotein-associated phospholipase A2 in cardiovascular risk].

    PubMed

    Ferri, Nicola; Corsini, Alberto

    2014-12-01

    Phospholipase A(2) (PLA(2)) are enzymes that hydrolyze the ester bond of glycerophospholipids releasing free fatty acids and lysophospholipids, including the arachidonic acid, the precursor of the eicosanoids and the inflammatory cascades. PLA(2) are present in the atherosclerotic plaques and their direct involvement in the proatherogenic inflammatory response is well documented. Epidemiological and genetic studies have demonstrated the correlation of the PLA(2) mass and enzymatic activity with the incidence of cardiovascular diseases. The potential pro-atherogenic role of PLA(2) led to the development of two small molecules, varespladib, a reversible sPLA(2) inhibitor, and darapladib, a selective Lp-PLA(2) inhibitor. Both molecules have demonstrated antiatherosclerotic properties in animal models, and positive effects on atherosclerotic plaque composition evaluated in phase 2 clinical trials. On these grounds, the results of three phase 3 studies have recently been published: the VISTA-16 study with varespladib in patients with acute coronary syndrome, and the STABILITY and SOLID-TIMI 52 studies with darapladib in patients with stable coronary heart disease and acute coronary syndrome, respectively. Unexpectedly, both studies did not demonstrate an additional protective action of PLA 2 inhibitors over the standard of care treatment with statins, antiplatelet drugs, and coronary revascularization. In the present article, the enzymatic properties and the involvement of sPLA(2) and Lp-PLA(2) in atherogenesis are reviewed, with a focus on the results of experimental studies and clinical studies with both varespladib and darapladib inhibitors.

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

    PubMed

    Bae, Jong-Sup

    2015-08-01

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

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

    PubMed

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

    2017-02-01

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

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

    PubMed

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

    2013-12-01

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

  4. Influence of dipalmitoylphosphatidylcholine (or dioleoylphosphatidylcholine) and phospholipase A2 enzyme on the properties of emulsions.

    PubMed

    Wiącek, Agnieszka Ewa

    2012-05-01

    The properties of n-tetradecane emulsions with dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) in 1M ethanol were investigated at 20 and 37°C. The zwitterionic phospholipids having the same headgroup bound to the apolar tail composed of two saturated or unsaturated chains were used as stabilizing agents. Both phospholipids may self-organize into aggregates, which possess different sizes and surface affinities. Electrokinetic properties of the systems at natural pH or pH 8 were investigated taking into account the effective diameter of the droplets as well as the zeta potentials using the dynamic light scattering technique. The effect of both phospholipids decreases the initially negative zeta potential of the n-tetradecane emulsion and is more evident in the case of DPPC especially at a physiological temperature near its main temperature transition. The change of zeta potential by DOPC is visible at both temperatures probably as an effect of a loose packing of this phospholipid on n-tetradecane droplets, because of the presence of double bonds in its molecule. Also, the role of ethanol dipoles on the stability of oil/phospholipid emulsions is obvious. The other aim of paper was the characterization of the phospholipase A(2) influence on DOPC hydrolysis in the emulsion environment in order to emphasize the importance of such methodology. The present work is the first study that explores the effects of both electrolyte ions and ethanol molecules on DOPC hydrolysis by phospholipase. The effect of enzyme on the n-tetradecane/DOPC emulsions was investigated at pH 8 with Na(+) or Ca(2+) ions, which occur in the physiological fluids. The effective diameters do not always correlate with the zeta potentials. A possible reason of such behavior might a mechanism different from the electrostatic stabilization. The particular role of Ca(2+) ions in the emulsions with phospholipids was confirmed. Those investigations provide insight into the

  5. Close-up view of the modifications of fluid membranes due to phospholipase A(2).

    PubMed

    Jakobsen, Ask F; Mouritsen, Ole G; Weiss, Matthias

    2005-11-30

    Phospholipases are a class of molecular machines that are involved in the active remodelling processes of biological membranes. These lipases are interfacially activated enzymes and in the specific case of phospholipase A(2) (PLA(2)) the enzyme catalyses the hydrolysis of di-acyl phospholipids into products of lysolipids and fatty acids, that dramatically change the physical properties of lipid membrane substrates. Using dissipative particle dynamics simulations on a simple coarse-grained bead-spring model of a fluid lipid bilayer in water, the mechanical and diffusive properties of the bilayer in the pure state and after the action of PLA(2) have been calculated. It is found that, in response to hydrolysis, the lipid membrane becomes mechanically softened and the various in-plane and trans-bilayer diffusional modes become enhanced. The results compare favourably with available experimental data.

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

    PubMed Central

    2010-01-01

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

  7. Association of lipoprotein-associated phospholipase A2 with coronary calcification among American and Japanese men

    PubMed Central

    El-Saed, Aiman; Sekikawa, Akira; Zaky, Riad Wahid; Kadowaki, Takashi; Takamiya, Tomoko; Okamura, Tomonori; Edmundowicz, Daniel; Kita, Yoshikuni; Kuller, Lewis H.; Ueshima, Hirotsugu

    2013-01-01

    BACKGROUND We have previously reported that the prevalence of coronary artery calcification (CAC) was substantially lower among Japanese than American men despite a less favorable profile of many traditional risk factors in Japanese men. OBJECTIVES To determine whether lipoprotein-associated phospholipase A2 (Lp-PLA2) levels are related to the difference in the prevalence of CAC between the two populations. METHODS A total of 200 men aged 40-49 were examined: 100 residents in Allegheny County, Pennsylvania, United States, and 100 residents in Kusatsu City, Shiga, Japan. Coronary calcium score (CCS) was evaluated by electron-beam tomography, Lp-PLA2 levels, nuclear magnetic resonance (NMR) lipoprotein subclasses and other factors were assessed centrally in the United States. RESULTS Lp-PLA2 levels were higher among American than Japanese men (Mean±SD 301.7±82.6 versus 275.9±104.7 ng/mL, respectively, p=0.06). Among all Japanese men and those with low density lipoprotein (LDL) cholesterol ≥130 mg/dL, there was an inverse association of the prevalence of CCS>0 with the tertile groups of Lp-PLA2 levels (p=0.08 and p=0.03, respectively). American men did not have any association between CCS>0 with the tertile groups of Lp-PLA2 (p=0.62). Although Lp-PLA2 among both populations correlated positively with LDL and total cholesterol, American and Japanese men had different correlations with NMR lipoprotein subclasses. Reported high odds ratio for CCS>0 among American compared to Japanese men was not reduced after adjusting for Lp-PLA2 levels. CONCLUSION Lp-PLA2 may have different mechanisms of action among American and Japanese men. Lp-PLA2 levels can not explain the observed CAC differences between the two populations. PMID:18094516

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

    PubMed Central

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

    2002-01-01

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

  9. 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.

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

    PubMed

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

    2015-11-01

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

  11. Effects of biological oxidants on the catalytic activity and structure of group VIA phospholipase A2.

    PubMed

    Song, Haowei; Bao, Shunzhong; Ramanadham, Sasanka; Turk, John

    2006-05-23

    Group VIA phospholipase A(2) (iPLA(2)beta) is expressed in phagocytes, vascular cells, pancreatic islet beta-cells, neurons, and other cells and plays roles in transcriptional regulation, cell proliferation, apoptosis, secretion, and other events. A bromoenol lactone (BEL) suicide substrate used to study iPLA(2)beta functions inactivates iPLA(2)beta by alkylating Cys thiols. Because thiol redox reactions are important in signaling and some cells that express iPLA(2)beta produce biological oxidants, iPLA(2)beta might be subject to redox regulation. We report that biological concentrations of H(2)O(2), NO, and HOCl inactivate iPLA(2)beta, and this can be partially reversed by dithiothreitol (DTT). Oxidant-treated iPLA(2)beta modifications were studied by LC-MS/MS analyses of tryptic digests and included DTT-reversible events, e.g., formation of disulfide bonds and sulfenic acids, and others not so reversed, e.g., formation of sulfonic acids, Trp oxides, and Met sulfoxides. W(460) oxidation could cause irreversible inactivation because it is near the lipase consensus sequence ((463)GTSTG(467)), and site-directed mutagenesis of W(460) yields active mutant enzymes that exhibit no DTT-irreversible oxidative inactivation. Cys651-sulfenic acid formation could be one DTT-reversible inactivation event because Cys651 modification correlates closely with activity loss and its mutagenesis reduces sensitivity to inhibition. Intermolecular disulfide bond formation might also cause reversible inactivation because oxidant-treated iPLA(2)beta contains DTT-reducible oligomers, and oligomerization occurs with time- and temperature-dependent iPLA(2)beta inactivation that is attenuated by DTT or ATP. Subjecting insulinoma cells to oxidative stress induces iPLA(2)beta oligomerization, loss of activity, and subcellular redistribution and reduces the rate of release of arachidonate from phospholipids. These findings raise the possibility that redox reactions affect iPLA(2)beta functions.

  12. Requirement of Cytosolic Phospholipase A2 gamma in Lipid Droplet Formation.

    PubMed

    Su, Xi; Liu, Shuhui; Zhang, Xianwen; Lam, Sin Man; Hu, Xue; Zhou, Yuan; Chen, Jizheng; Wang, Yun; Wu, Chunchen; Shui, Guanghou; Lu, Mengji; Pei, Rongjuan; Chen, Xinwen

    2017-03-20

    Lipid droplet (LD) accumulation in hepatocytes is a typical character of steatosis. Hepatitis C virus (HCV) infection, one of the risk factors related to steatosis, induced LD accumulation in cultured cells. However, the mechanisms of which HCV induce LD formation are not fully revealed. Previously we identified cytosolic phospholipase A2 gamma (PLA2G4C) as a host factor upregulated by HCV infection and involved in HCV replication. Here we further revealed that PLA2G4C plays an important role in LD biogenesis and refined the functional analysis of PLA2G4C in LD biogenesis and HCV assembly. LD formation upon fatty acid and HCV stimulation in PLA2G4C knockdown cells was impaired and could not be restored by complementation with PLA2G4A. PLA2G4C was tightly associated in the membrane with the domain around the amino acid residues 260-292, normally in ER but relocated into LDs upon oleate stimulation. Mutant PLA2G4C without enzymatic activity was not able to restore LD formation in PLA2G4C knockdown cells. Thus, both the membrane attachment and the enzymatic activity of PLA2G4C were required for its function in LD formation. The participation of PLA2G4C in LD formation is correlated with its involvement in HCV assembly. Finally, PLA2G4C overexpression itself led to LD formation in hepatic cells and enhanced LD accumulation in the liver of high-fat diet (HFD)-fed mice, suggesting its potential role in fatty liver disease.

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

    PubMed

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

    2013-12-01

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

  14. 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.

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

    PubMed Central

    Sanchez-Mejia, Rene O.; Mucke, Lennart

    2011-01-01

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

  16. Plasma Lipoprotein-associated Phospholipase A2 in Patients with Metabolic Syndrome and Carotid Atherosclerosis

    PubMed Central

    2011-01-01

    Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a recently identified and potentially useful plasma biomarker for cardiovascular and atherosclerotic diseases. However, the correlation between the Lp-PLA2 activity and carotid atherosclerosis remains poorly investigated in patients with metabolic syndrome (MetS). The present study aimed to evaluate the potential role of Lp-PLA2 as a comprehensive marker of metabolic syndrome in individuals with and without carotid atherosclerosis. Methods We documented 118 consecutive patients with MetS and 70 age- and sex-matched healthy subjects served as controls. The patients were further divided into two groups: 39 with carotid plaques and 79 without carotid plaques to elucidate the influence of Lp-PLA2 on carotid atherosclerosis. The plasma Lp-PLA2 activity was measured by using ELISA method and carotid intimal-media thickness (IMT) was performed by ultrasound in all participants. Results Lp-PLA2 activity was significantly increased in MetS subgroups when compared with controls, and was higher in patients with carotid plaques than those without plaques (P < 0.05). Furthermore, we found that significant difference in Lp-PLA2 was obtained between patients with three and four disorders of metabolic syndrome (P < 0.01). Age (β = 0.183, P = 0.029), LDL-cholesterol (β = 0.401, P = 0.000) and waist-hip ratio (β = 0.410, P = 0.000) emerged as significant and independent determinants of Lp-PLA2 activity. Multiple stepwise regression analysis revealed that LDL-cholesterol (β = 0.309, P = 0.000), systolic blood pressure (β = 0.322, P = 0.002) and age (β = 0.235, P = 0.007) significantly correlated with max IMT, and Lp-PLA2 was not an independent predictor for carotid IMT. Conclusions Lp-PLA2 may be a modulating factor for carotid IMT via age and LDL-cholesterol, not independent predictor in the pathophysiological process of carotid atherosclerosis in patients with MetS. PMID:21247435

  17. Mapping the catalytic pocket of phospholipases A2 and C using a novel set of phosphatidylcholines.

    PubMed Central

    Caramelo, J J; Florín-Christensen, J; Florín-Christensen, M; Delfino, J M

    2000-01-01

    A set of radioiodinatable phosphatidylcholines (PCs) derivatized with the Bolton-Hunter reagent (BHPCs) was synthesized to probe the substrate recognition and activity of phospholipases. A common feature of this series is the presence of a bulky 4-hydroxyphenyl group at the end of the fatty acyl chain attached to position sn-2. The distance between the end group and the glycerol backbone was varied by changing the length of the intervening fatty acyl chain (3-25 atoms). Except for the shortest, this chain includes at least one amide linkage. The usefulness of this series of substrates as a molecular ruler was tested by measuring the hydrolytic activities of Naja naja naja phospholipase A(2) (PLA(2)) and Bacillus cereus phospholipase C (PLC) in Triton X-100 micelles. The activity of PLA(2) proved to be highly dependent on the length of the fatty acyl chain linker, the shorter compounds (3-10 atoms) being very poor substrates. In contrast, the PLC activity profile exhibited much less discrimination. In both cases, PCs with 16-21 atom chains at position sn-2 yielded optimal activity. We interpret these findings in terms of fatty acyl chain length-related steric hindrance caused by the terminal aromatic group, affecting the activity of PLA(2) and, to a smaller extent, that of PLC. This notion agrees with the more extended recognition of aliphatic chains inside the narrow channel leading to the catalytic site in the former case. Molecular models of these substrates bound to PLA(2) were built on the basis of the crystallographic structure of Naja naja atra PLA(2) complexed with a phospholipid analogue. Docking of these substrates necessarily requires the intrusion of the bulky 4-hydroxyphenyl group inside the binding pocket and also the failure of the amide group to form hydrogen bonds inside the hydrophobic substrate channel. PMID:10698694

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    2007-08-01

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

  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. 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.

  2. The electrostatic basis for the interfacial binding of secretory phospholipases A2.

    PubMed Central

    Scott, D L; Mandel, A M; Sigler, P B; Honig, B

    1994-01-01

    Biochemical and structural data suggest that electrostatic forces play a critical role in the binding of secretory phospholipases A2 to substrate aggregates (micelles, vesicles, monolayers, and membranes). This initial binding (adsorption) of the enzyme to the interface is kinetically distinct from the subsequent binding of substrate to the buried active site. Thus, in the absence of specific active-site interactions, electrostatic forces operating at the molecular surface may orient and hold the enzyme at the interface. We have calculated the electrostatic potentials for 10 species of secretory phospholipases A2 whose atomic coordinates have been determined by x-ray crystallography. Most of these enzymes show a marked electrostatic sidedness that is accentuated to a variable degree by the presence of the essential cofactor calcium ion. This asymmetry suggests a discrete interfacial binding region on the protein's surface, the location of which is in general agreement with proposals derived from the results of chemical modification, mutational, and crystallographic experiments. Images FIGURE 2 FIGURE 3 FIGURE 3 FIGURE 3 FIGURE 4 FIGURE 4 FIGURE 5 FIGURE 5 PMID:7948668

  3. Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria, and open their transition pores.

    PubMed

    Rigoni, Michela; Paoli, Marco; Milanesi, Eva; Caccin, Paola; Rasola, Andrea; Bernardi, Paolo; Montecucco, Cesare

    2008-12-05

    Snake presynaptic neurotoxins with phospholipase A(2) activity are potent inducers of paralysis through inhibition of the neuromuscular junction. These neurotoxins were recently shown to induce exocytosis of synaptic vesicles following the production of lysophospholipids and fatty acids and a sustained influx of Ca(2+) from the medium. Here, we show that these toxins are able to penetrate spinal cord motor neurons and cerebellar granule neurons and selectively bind to mitochondria. As a result of this interaction, mitochondria depolarize and undergo a profound shape change from elongated and spaghetti-like to round and swollen. We show that snake presynaptic phospholipase A(2) neurotoxins facilitate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. The relative potency of the snake neurotoxins was similar for the permeability transition pore opening and for the phospholipid hydrolysis activities, suggesting a causal relationship, which is also supported by the effect of phospholipid hydrolysis products, lysophospholipids and fatty acids, on mitochondrial pore opening. These findings contribute to define the cellular events that lead to intoxication of nerve terminals by these snake neurotoxins and suggest that mitochondrial impairment is an important determinant of their toxicity.

  4. Pivotal role of superoxides generated in the mitochondrial respiratory chain in peroxynitrite-dependent activation of phospholipase A2.

    PubMed Central

    Guidarelli, Andrea; Cantoni, Orazio

    2002-01-01

    Exposure of PC12 cells to reagent peroxynitrite promotes the release of arachidonic acid (AA) mediated by activation of phospholipase A(2) [Guidarelli, Palomba and Cantoni (2000) Br. J. Pharmacol. 129, 1539-1542]. We now present experimental evidence consistent with the notion that this response is not directly triggered by peroxynitrite but, rather, by reactive oxygen species generated at the level of complex III of the mitochondrial respiratory chain. In particular, superoxide (and not hydrogen peroxide) has a pivotal role in peroxynitrite-dependent activation of phospholipase A(2). This observation was confirmed by results showing that superoxide, or peroxynitrite, promotes release of AA in isolated mitochondria. Consistently, the release of AA elicited by either peroxynitrite or A23187 in intact cells was shown to be calcium-dependent and differentially affected by phospholipase A(2) inhibitors with different levels of specificity. In particular, the effects of peroxynitrite, unlike those of A23187, were both sensitive to low concentrations of two general phospholipase A(2) inhibitors and insensitive to arachidonyltrifluoromethyl ketone, which shows some selectivity towards cytosolic phospholipase A(2). In addition, peroxynitrite and A23187 synergistically enhanced the release of AA. Collectively, the above results demonstrate that peroxynitrite causes inhibition of complex III, followed by enforced formation of superoxides that stimulate the activity of a calcium-dependent PLA(2) isoform, probably localized in the mitochondria. PMID:11994047

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

    PubMed

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

    2014-01-01

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

  6. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-08-01

    Two myotoxic and noncatalytic Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A(2) (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56-2.05 Å and belonged to space groups P3(1)21 (braziliantoxin-II), P6(5)22 (braziliantoxin-III) and P2(1) (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A(2) braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A(2).

  9. Neuronal damage by secretory phospholipase A2: modulation by cytosolic phospholipase A2, platelet-activating factor, and cyclooxygenase-2 in neuronal cells in culture.

    PubMed

    Kolko, Miriam; Rodriguez de Turco, Elena B; Diemer, Nils H; Bazan, Nicolas G

    2003-02-27

    Activation of cytosolic phospholipase A(2) (cPLA(2)) is an early event in brain injury, which leads to the formation and accumulation of bioactive lipids: platelet-activating factor (PAF), free arachidonic acid, and eicosanoids. A cross-talk between secretory PLA(2) (sPLA(2)) and cPLA(2) in neural signal transduction has previously been suggested (J Biol Chem 271:32722; 1996). Here we show, using neuronal cell cultures, an up-regulation of cPLA(2) expression and an inhibition by the selective cPLA(2) inhibitor AACOCF3 after exposure to neurotoxic concentrations of sPLA(2)-OS2. Pretreatment of neuronal cultures with recombinant PAF acetylhydrolase (rPAF-AH) or the presynaptic PAF receptor antagonist, BN52021, partially blocked neuronal cell death induced by sPLA(2)-OS2. Furthermore, selective COX-2 inhibitors ameliorated sPLA(2)-OS2-induced neurotoxicity. We conclude that sPLA(2)-OS2 activates a neuronal signaling cascade that includes activation of cPLA(2), arachidonic acid release, PAF production, and induction of COX-2.

  10. Structure-activity relationship of 2-oxoamide inhibition of group IVA cytosolic phospholipase A2 and group V secreted phospholipase A2.

    PubMed

    Six, David A; Barbayianni, Efrosini; Loukas, Vassilios; Constantinou-Kokotou, Violetta; Hadjipavlou-Litina, Dimitra; Stephens, Daren; Wong, Alan C; Magrioti, Victoria; Moutevelis-Minakakis, Panagiota; Baker, Sharon F; Dennis, Edward A; Kokotos, George

    2007-08-23

    The Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is a key provider of substrates for the production of eicosanoids and platelet-activating factor. We explored the structure-activity relationship of 2-oxoamide-based compounds and GIVA cPLA2 inhibition. The most potent inhibitors are derived from delta- and gamma-amino acid-based 2-oxoamides. The optimal side-chain moiety is a short nonpolar aliphatic chain. All of the newly developed 2-oxoamides as well as those previously described have now been tested with the human Group V secreted PLA2 (GV sPLA2) and the human Group VIA calcium-independent PLA2 (GVIA iPLA2). Only one 2-oxoamide compound had appreciable inhibition of GV sPLA2, and none of the potent GIVA cPLA2 inhibitors inhibited either GV sPLA2 or GVIA iPLA2. Two of these specific GIVA cPLA2 inhibitors were also found to have potent therapeutic effects in animal models of pain and inflammation at dosages well below the control nonsteroidal anti-inflammatory drugs.

  11. OKT3-induced nephrotoxicity is associated with release of group II secretory phospholipase A2.

    PubMed

    Wever, P C; Roest, R W; Wolbink-Kamp, A M; Wolbink, G J; Weening, J J; Hack, C E; ten Berge, J M

    1996-10-01

    Administration of the murine IgG2a CD3 monoclonal antibody OKT3 exerts a transient nephrotoxic effect. Increased levels of group II secretory phospholipase A2 (sPLA2-II) might account for this nephrotoxicity as sPLA2-II induces the biosynthesis of prostaglandins, vasoactive lipid mediators that influence glomerular haemodynamics and renal function. Furthermore, extracellular phospholipases seem to be involved in proximal tubular cell injury. We studied plasma sPLA2-II levels in relation to circulating creatinine, tumour necrosis factor alpha, interleukin 6 and C-reactive protein levels in 15 renal allograft recipients receiving rejection treatment with OKT3. As a control group, we studied 15 renal allograft recipients receiving rejection treatment with methylprednisolone. A maximal fourfold increase in sPLA2-II levels was observed 48 h after the first OKT3 administration, preceded by increased tumour necrosis factor alpha and interleukin 6 levels and accompanied by increased C-reactive protein levels. Creatinine levels reached a maximal increase 72 h after initiation of treatment. During methylprednisolone treatment no increase in any of the studied parameters was observed. Thus, administration of OKT3 induces increased sPLA2-II levels, presumably via generation of cytokines. We hypothesize that sPLA2-II may contribute to the nephrotoxic effect of OKT3 by inducing vasoconstrictive prostaglandins and renal tubular cell injury.

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

    PubMed Central

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

    2004-01-01

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

  13. 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

  14. [The effect of the protease inhibitor FUT-175 on phospholipase A2, complement, prostaglandins and prekallikrein during endotoxin shock].

    PubMed

    Okuda, Y; Ogata, H

    1989-03-01

    This experiment was performed to investigate the effect of protease inhibitor FUT on the blood pressure, phospholipase A2, complement 3, CH50, thromboxane B2, 6-keto-PGF1 alpha and prekallikrein during endotoxin shock using 19 dogs. LPS was injected at a dose of 3mg.kg-1 in 11 dogs. Eight dogs were injected with FUT of 2mg.kg-1 before administration of LPS, and then infused continuously with 50 micrograms.kg-1.min-1 of FUT. FUT suppressed a decrease in the blood pressure, activations of phospholipase A2 and prekallikrein, but had no effect on serum TXB2, 6-keto-PGF1 alpha, C3 and CH50 compared with the LPS alone group. Suppression of the activation in phospholipase A2 and the falling of the blood pressure suggests that FUT has indirect effects such as inhibition of prekallikrein system.

  15. Racial variation in lipoprotein-associated phospholipase A2 in older adults

    PubMed Central

    2011-01-01

    Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a predictor of cardiovascular events that has been shown to vary with race. The objective of this study was to examine factors associated with this racial variation. Methods We measured Lp-PLA2 mass and activity in 714 healthy older adults with no clinical coronary heart disease and not taking dyslipidemia medication. We evaluated the association between race and Lp-PLA2 mass and activity levels after adjustment for various covariates using multivariable linear regression. These covariates included age, sex, diabetes, hypertension, body mass index, lipid measurements, C-reactive protein, smoking status, physical activity, diet, income, and education level. We further examined genetic covariates that included three single nucleotide polymorphisms shown to be associated with Lp-PLA2 activity levels. Results The mean age was 66 years. Whites had the highest Lp-PLA2 mass and activity levels, followed by Hispanics and Asians, and then African-Americans; in age and sex adjusted analyses, these differences were significant for each non-White race as compared to Whites (p < 0.0001). For example, African-Americans were predicted to have a 55.0 ng/ml lower Lp-PLA2 mass and 24.7 nmol/ml-min lower activity, compared with Whites, independent of age and sex (p < 0.0001). After adjustment for all covariates, race remained significantly correlated with Lp-PLA2 mass and activity levels (p < 0.001) with African-Americans having 44.8 ng/ml lower Lp-PLA2 mass and 17.3 nmol/ml-min lower activity compared with Whites (p < 0.0001). Conclusion Biological, lifestyle, demographic, and select genetic factors do not appear to explain variations in Lp-PLA2 mass and activity levels between Whites and non-Whites, suggesting that Lp-PLA2 mass and activity levels may need to be interpreted differently for various races. PMID:21714927

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

    PubMed Central

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

    2016-01-01

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

  17. Gabexate mesilate (FOY) inhibition of amylase and phospholipase A(2) activity in sow pancreatic juice.

    PubMed

    Caronna, Roberto; Loretta, Diana; Campedelli, Paolo; Catinelli, Stefania; Nofroni, Italo; Sibio, Simone; Sinibaldi, Giovanni; Chirletti, Piero

    2003-01-01

    We designed this study in sows to investigate the enzyme inhibitory action of gabexate mesylate (GM) directly in the pancreatic juice. We studied 16 sows, each weighing about 130 kg. The pancreatic duct was identified and cannulated to collect the pancreatic juice. Sows in the treated group received intravenous GM infusion at a dose of 1000 mg over 24 h. Control sows underwent the same sampling schedule while receiving physiological solution. GM inhibited the two pancreatic enzymes amylase and phospholipase A(2) (PA(2)) in pancreatic juice. Thus, the enzyme inhibition in the pancreatic gland itself and the central role of (PA(2)) inhibition in enzyme cascade responsible for activating other proteases confirm the therapeutic use of GM in acute pancreatitis.

  18. Cloning and functional expression of secreted phospholipases A(2) from Bothrops diporus (Yarará Chica).

    PubMed

    Yunes Quartino, Pablo Javier; Barra, José Luis; Fidelio, Gerardo Daniel

    2012-10-19

    Bothrops diporus is a very common viper in Argentina. At present, no complete sequence of secreted phospholipase A(2) (sPLA(2)) from this snake has been reported. We have cloned two sPLA(2) isoenzymes as well as a putative sPLA(2)-like myotoxin from venom gland. The two sPLA(2) were expressed as inclusion bodies in Escherichia coli with an N-terminal tag of ubiquitin. After in vitro renaturation and cleavage step, using an ubiquitin specific peptidase, the recombinants exhibited sPLA(2) activity when analyzed by means of Langmuir dilauroylphosphatidylcholine monolayers as substrate. Both enzymes have a similar surface pressure-activity profile when compared with non-recombinant purified isoforms. To our knowledge, this is the first time that analysis of optimal lateral pressure of substrate monolayers by using the surface barostat technique is performed on recombinant sPLA(2)s.

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

    PubMed Central

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

    2015-01-01

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

  20. 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

  1. New potent and selective polyfluoroalkyl ketone inhibitors of GVIA calcium-independent phospholipase A2.

    PubMed

    Magrioti, Victoria; Nikolaou, Aikaterini; Smyrniotou, Annetta; Shah, Ishita; Constantinou-Kokotou, Violetta; Dennis, Edward A; Kokotos, George

    2013-09-15

    Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA2 inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA2 inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA2 inhibitor ever reported with a XI(50) value of 0.0001, and with no significant inhibition against GIVA cPLA2 or GV sPLA2. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA2, GIVA cPLA2, and GV sPLA2. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. [Respiration of wheat roots during inhibition of phospholipase A2 by 4-bromphenacylbromide].

    PubMed

    Valitova, Iu N; Gordon, L Kh; Ogorodnikova, T I; Lygin, A V; Ruban, N F

    2001-01-01

    Dependence of oxygen consumption by wheat root cells on the activity of phospholipase A2 (PLA2) was studied. The treatment of excised roots with 4-bromophenacile bromide (BPB), a specific inhibitor of PLA2, caused a decrease in the content of free fatty acids (FFA) and in oxygen consumption of root cells. The latter was prevented by exogenous application of a mixture of FFA. A similar inhibitory effect was caused by BPB after the activation of root respiration by 2,4-dinitrophenol (DNP). These data suggest that FFA may be involved in the regulation of respiration through the formation of succinate. This is supported by the fact of reduction of DNP-induced stimulation of oxygen consumption by malonate, known to be an inhibitor of succinate dehydrogenase, and by stimulation of respiration by exogenous application of succinate.

  3. 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.

  4. Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A(2): an update.

    PubMed

    Šribar, Jernej; Oberčkal, Jernej; Križaj, Igor

    2014-10-01

    β-neurotoxins are enzymes, secreted phospholipases A2, that inhibit neurotransmission in neuromuscular synapses by poisoning the motoneuron. They were reviewed extensively several years ago (Pungerčar and Križaj, 2007). Here we present and critically discuss the most important experimental facts reported since then. Evidence has been presented for specific internalization of β-neurotoxins into the nerve endings of motoneurons, their in vivo binding to some cytosolic proteins, direct action on mitochondria, disruption of Ca(2+) homoeostasis and inhibition of amphiphysin function. New insights have led to a more confident interpretation of the action of these toxins at the molecular level. The most important questions that remain to be answered are listed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. 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.

  6. 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.

  7. The bacterium Xenorhabdus nematophila inhibits phospholipases A2 from insect, prokaryote, and vertebrate sources

    NASA Astrophysics Data System (ADS)

    Park, Youngjin; Kim, Yonggyun; Stanley, David

    The bacterium, Xenorhabdus nematophila, is a virulent insect pathogen. Part of its pathogenicity is due to impairing cellular immunity by blocking biosynthesis of eicosanoids, the major recognized signal transduction system in insect cellular immunity. X. nematophila inhibits the first step in eicosanoid biosynthesis, phospholipase A2 (PLA2). Here we report that the bacterium inhibits PLA2 from two insect immune tissues, hemocytes and fat body, as well as PLA2s selected to represent a wide range of organisms, including prokaryotes, insects, reptiles, and mammals. Our finding on a bacterial inhibitor of PLA2 activity contributes new insight into the chemical ecology of microbe-host interactions, which usually involve actions rather than inhibitors of PLA2s.

  8. Characterization of Serum Phospholipase A2 Activity in Three Diverse Species of West African Crocodiles

    PubMed Central

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

    2011-01-01

    Secretory phospholipase A2, an enzyme that exhibits substantial immunological activity, was measured in the serum of three species of diverse West African crocodiles. Incubation of different volumes of crocodile serum with bacteria labeled with a fluorescent fatty acid in the sn-2 position of membrane lipids resulted in a volume-dependent liberation of fluorescent probe. Serum from the Nile crocodile (Crocodylus niloticus) exhibited slightly higher activity than that of the slender-snouted crocodile (Mecistops cataphractus) and the African dwarf crocodile (Osteolaemus tetraspis). Product formation was inhibited by BPB, a specific PLA2 inhibitor, confirming that the activity was a direct result of the presence of serum PLA2. Kinetic analysis showed that C. niloticus serum produced product more rapidly than M. cataphractus or O. tetraspis. Serum from all three species exhibited temperature-dependent PLA2 activities but with slightly different thermal profiles. All three crocodilian species showed high levels of activity against eight different species of bacteria. PMID:22110960

  9. Hydrogen sulphide induces mouse paw oedema through activation of phospholipase A2

    PubMed Central

    di Villa Bianca, Roberta d'Emmanuele; Coletta, Ciro; Mitidieri, Emma; De Dominicis, Gianfranco; Rossi, Antonietta; Sautebin, Lidia; Cirino, Giuseppe; Bucci, Mariarosaria; Sorrentino, Raffaella

    2010-01-01

    BACKGROUND AND PURPOSE Hydrogen sulphide (H2S), considered as a novel gas transmitter, is produced endogenously in mammalian tissue from L-cysteine by two enzymes, cystathionine β-synthase and cystathionine γ-lyase. Recently, it has been reported that H2S contributes to the local and systemic inflammation in several experimental animal models. We conducted this study to investigate on the signalling involved in H2S-induced inflammation. EXPERIMENTAL APPROACH L-cysteine or sodium hydrogen sulphide (NaHS) was injected into the mouse hind paw and oedema formation was evaluated for 60 min. In order to investigate H2S-induced oedema formation, we used 5-HT and histamine receptor antagonists, and inhibitors of KATP channels or arachidonic acid cascade. Prostaglandin levels were determined in hind paw exudates by radioimmunoassay. Paws injected with L-cysteine or NaHS were examined by histological methods. KEY RESULTS Both NaHS and L-cysteine caused oedema characterized by a fast onset which peaked at 30 min. This oedematogenic action was not associated with histamine or 5-HT release or KATP channel activation. However, oedema formation was significantly inhibited by the inhibition of cyclooxygenases and selective inhibition of phospholipase A2. Prostaglandin levels were significantly increased in exudates of hind paw injected with NaHS or L-cysteine. The histological examination clearly showed an inflammatory state with a loss of tissue organization following NaHS or L-cysteine injection. CONCLUSIONS AND IMPLICATIONS Phospholipase A2 and prostaglandin production are involved in pro-inflammatory effects of H2S in mouse hind paws. The present study contributes to the understanding of the role of L-cysteine/H2S pathway in inflammatory disease. PMID:20825409

  10. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review.

    PubMed

    Garza, Carolina A; Montori, Victor M; McConnell, Joseph P; Somers, Virend K; Kullo, Iftikhar J; Lopez-Jimenez, Francisco

    2007-02-01

    To estimate the association between plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) levels and cardiovascular disease (CVD). We searched MEDLINE (January 1, 1985, through September 30, 2006), the Cochrane library (from inception through 2006), conference proceedings, and reference sections of obtained articles and contacted experts for unpublished studies. Eligible studies were cohorts with 1 year or more of follow-up or case-control designs that provided risk estimates for CVD according to blood levels of Lp-PLA2 that were unadjusted or adjusted for conventional CVD risk factors. We used random-effects meta-analysis to estimate the association between Lp-PLA2 and CVD risk and conducted preplanned subgroup analyses to identify risk-subgroup interactions that could explain between-study differences. We found 14 eligible studies (N = 20,549 patients) that reported either Lp-PLA2 plasma activity (n = 5) or an immunoassay that measured the plasma concentration (n = 9). The meta-analytic estimate from the unadjusted odds ratio for the association between elevated Lp-PLA2 levels and CVD risk was 1.51 (95% confidence interval, 1.30-1.75) and from the odds ratio adjusted for conventional CVD risk factors was 1.60 (95% confidence interval, 1.36-1.89). Differences in study methods explained differences in results across studies. Lipoprotein-associated phospholipase A2 is significantly associated with CVD. The risk estimate appears to be relatively unaffected by adjustment for conventional CVD risk factors. Measurement of Lp-PLA2 may be useful in CVD risk stratification. In addition, Lp-PLA2 may represent a potential therapeutic target for CVD risk reduction.

  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. Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

    PubMed Central

    Sato, Hiroyasu; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Yamamoto, Kei; Masuda, Seiko; Hosono, Tomohiko; Arata, Satoru; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Nakanishi, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Kudo, Ichiro; Murakami, Makoto

    2010-01-01

    Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction. PMID:20424323

  13. 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.

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

    PubMed Central

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

    2012-01-01

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

  15. Light controls phospholipase A2α and β gene expression in Citrus sinensis

    PubMed Central

    Liao, Hui-Ling; Burns, Jacqueline K.

    2010-01-01

    The low-molecular weight secretory phospholipase A2α (CssPLA2α) and β (CsPLA2β) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2α displayed distinct diurnal patterns in fruit tissues. CssPLA2α and CsPLA2β diurnal expression exhibited periods of approximately 24 h; CssPLA2α amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2β amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2α and CsPLA2β gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2α and CsPLA2β expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2α and CsPLA2β expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2α transcript expression in leaf blades of seedlings treated with low intensity blue light (24 μmol m−2 s−1). Compared with CssPLA2α basal expression, CsPLA2β expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action. PMID:20388744

  16. Light controls phospholipase A2alpha and beta gene expression in Citrus sinensis.

    PubMed

    Liao, Hui-Ling; Burns, Jacqueline K

    2010-05-01

    The low-molecular weight secretory phospholipase A2alpha (CssPLA2alpha) and beta (CsPLA2beta) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2alpha displayed distinct diurnal patterns in fruit tissues. CssPLA2alpha and CsPLA2beta diurnal expression exhibited periods of approximately 24 h; CssPLA2alpha amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2beta amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2alpha and CsPLA2beta gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2alpha and CsPLA2beta expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2alpha and CsPLA2beta expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2alpha transcript expression in leaf blades of seedlings treated with low intensity blue light (24 micromol m(-2) s(-1)). Compared with CssPLA2alpha basal expression, CsPLA2beta expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action.

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

    PubMed

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

    1991-07-25

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

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

    PubMed

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

    2001-08-31

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

  19. Activation of phospholipase A2 is associated with generation of placental lipid signals and fetal obesity.

    PubMed

    Varastehpour, Ali; Radaelli, Tatjana; Minium, Judi; Ortega, Henar; Herrera, Emilio; Catalano, Patrick; Hauguel-de Mouzon, Sylvie

    2006-01-01

    Obesity and diabetes during pregnancy are associated with increased insulin resistance and higher neonatal adiposity. In turn, insulin resistance triggers inflammatory pathways with accumulation of placental cytokines. To determine placental signals that translate into development of excess adipose tissue, we investigated the role of phospholipases A2 (PLA2) as targets of inflammatory mediators. The study was conducted at Case Western Reserve University, Department of Reproductive Biology. Volunteers gave informed written consent in accordance with the Institutional Review Board guidelines. Placenta and cord blood samples were obtained at the time of elective cesarean section in 15 term pregnancies. Neonatal anthropometric measurements were performed within 48 h of delivery. Placentas were grouped based on neonatal percentage body fat as obese (body fat > or = 16%) and lean control (body fat < or = 8%). The primary outcomes were placenta PLA2 expression and fatty acid concentration. Expression of PLA2G2A and PLA2G5, the main placenta phospholipases, was greater (P < 0.05) in placenta of obese compared with control neonates and was associated with increased 20:3 and 20:5 omega-3 polyunsaturated fatty acids. TNF-alpha and leptin content was increased 3-fold in placenta of obese neonates. TNF-alpha and leptin both induced a time-dependent activation of PLA2G2 and PLA2G5 in placental cells. Accumulation of omega-3 fatty acids through secretory PLA2 activation is associated with high neonatal adiposity. We propose that the generation of placental lipid mediators through TNF-alpha and leptin stimulation represents a key mechanism to favor excess fetal fat accretion.

  20. 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

  1. 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

  2. Mitochondrial dysfunction induced by pancreatic and crotalic (Crotalus durissus terrificus) phospholipases A2 on rabbit proximal tubules suspensions.

    PubMed

    Amora, Daniela N; Costa Martins, Alice M; Roeser, Nancy; Senter, Ruth; Ostrowsky, Tiffany; Weinberg, Joel M; Monteiro, Helena S A

    2008-12-15

    In the present study we show that phospholipases A2 isolated from porcine pancreas (PP-PLA2) and Crotalus durissus terrificus snake venom (SV-PLA2) induced dose-dependent increases of LDH release from rabbit proximal tubules in suspension. Both porcine and crotalic PLA(2)s induced increases in non-esterified fatty acid (NEFA) levels (microg of NEFA/mg of tubule protein). It was observed that the NEFA levels in the pellets were higher than in the supernatant for both PLA2, and were dose-dependent for the crotalic PLA2 group. Furthermore, snake venom PLA2 induced a decrease in mitochondrial membrane potential (DeltaPsi(m)) assessed by both JC-1 uptake and safranin O uptake. Porcine PLA2 produced no effects on JC-1 uptake with the highest concentrations and an unexpected increase in the group treated with the lowest concentration. In contrast, the safranin O method revealed decreases of energization with both phospholipases, so it had higher sensitivity to the presence of the increased NEFA levels. Addition of delipidated bovine serum albumin (dBSA) completely reversed the effects induced by phospholipases on DeltaPsi(m) measured with safranin O. Incubation with pancreatic and crotalic phospholipases A2 produced no changes on cell ATP levels. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic phospholipases disturbed membrane integrity as well as mitochondrial function. Specific early NEFA-mediated mitochondrial effects of the phospholipases used in the present study are indicated by the benefit provided by dBSA.

  3. Effects of lipoprotein-associated phospholipase A2 on arginase/nitric oxide pathway in hemodialysis patients.

    PubMed

    Tektaş, Ayşegül Korkmaz; Uslu, Sema; Yalçin, Ahmet Uğur; Sahin, Garip; Temiz, Gökhan; Kara, Mehmet; Temel, Halide Edip; Demirkan, Emine Sütken; Colak, Ertuğrul; Colak, Omer

    2012-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) and arginase are recently described inflammatory biomarkers associated with cardiovascular disease. In this study, we aimed to investigate the possible effects of serum Lp-PLA2 mass levels on arginase/nitric oxide (NO) pathway as a cardiovascular risk marker in hemodialysis (HD) patients. Forty-three HD patients and 15 healthy subjects were included in this study. Lipid profile, high sensitivity C-reactive protein (hs-CRP), albumin, creatinine, body mass index (BMI), Lp-PLA2 and total nitrite levels, and arginase activity were determined in serum samples from patients and control subjects. Lp-PLA2 levels were found to be positively correlated with arginase, triglycerides, total cholesterol, low-density lipoprotein-cholesterol, and age and negatively correlated with high-density lipoprotein-cholesterol and total nitrite levels, while there was no correlation with BMI and hs-CRP, albumin, and creatinine levels in HD patients. We conclude that elevated Lp-PLA2 mass levels may contribute to impaired arginase/NO pathway in HD patients and that increased the arginase activity and Lp-PLA2 mass levels with decreased total nitrite levels seem to be useful biochemical markers in terms of reflecting endothelial dysfunction and associated cardiovascular risks in HD patients.

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

    PubMed

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

    2015-04-02

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  8. Effect of Darapladib Treatment on Endarterectomy Carotid Plaque Lipoprotein-Associated Phospholipase A2 Activity: A Randomized, Controlled Trial

    PubMed Central

    Johnson, Joel L.; Shi, Yi; Snipes, Rose; Janmohamed, Salim; Rolfe, Timothy E.; Davis, Bill; Postle, Anthony; Macphee, Colin H.

    2014-01-01

    Background The aim of this study was to assess the effects of darapladib, a selective oral investigational lipoprotein-associated phospholipase A2 inhibitor, on both plasma and plaque lipoprotein-associated phospholipase A2 activity. Methods Patients undergoing elective carotid endarterectomy were randomized to darapladib 40 mg (n = 34), 80 mg (n = 34), or placebo (n = 34) for 14 days, followed by carotid endarterectomy 24 hours after the last dose of study medication. Results Darapladib 40 mg and 80 mg reduced plasma lipoprotein-associated phospholipase A2 activity by 52% and 81%, respectively, versus placebo (both P<0.001). Significant reductions in plaque lipoprotein-associated phospholipase A2 activity were also observed compared with placebo (P<0.0001), which equated to a 52% and 80% decrease compared with placebo. No significant differences were observed between groups in plaque lysophosphatidylcholine content or other biomarkers, although a dose-dependent decrease in plaque matrix metalloproteinase-9 mRNA expression was observed with darapladib 80 mg (P = 0.053 vs placebo). In a post-hoc analysis, plaque caspase-3 (P<0.001) and caspase-8 (P<0.05) activity were found to be significantly lower in the darapladib 80-mg group versus placebo. No major safety concerns were identified in the study. Conclusions Short-term treatment (14±4 days) with darapladib produced a robust, dose-dependent reduction in plasma lipoprotein-associated phospholipase A2 activity. More importantly, darapladib demonstrated placebo-corrected reductions in carotid plaque lipoprotein-associated phospholipase A2 activity of similar magnitude. Darapladib was generally well tolerated and no safety concerns were identified. Additional studies of longer duration are needed to explore whether these pharmacodynamic effects are associated with improved clinical outcomes, as might be hypothesized. Trial Registration Information Name of Registry 1: ClinicalTrials.gov Registry Number

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

    PubMed Central

    Hains, P G; Broady, K W

    2000-01-01

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

  10. Crystal forms of a lysine-49 phospholipase A 2 from the eastern cottonmouth snake

    NASA Astrophysics Data System (ADS)

    Clancy, Laura L.; Rydel, Timothy J.; Muchmore, Steven W.; Holland, Debra R.; Watenpaugh, Keith D.; Finzel, Barry C.; Einspahr, Howard M.

    1990-09-01

    As part of an effort to determine the structure of a lysine-49 variant phospholipase A 2 from the venom of a North American pit viper, the eastern cottonmouth ( Agkistrodon piscivorus piscivorus), we have produced five different crystal forms grown under a variety of crystallization conditions. They include an orthorthombic form (P2 12 12 1 or P2 12 12; a=87.8(3) Å, b=76.2(3) Å, c=57.4(4) Å), an hexagonal form (space group P6 122 or its enantiomer; a= b=62.22(3) Å, c=137.1(3) Å) and two tetragonal forms - a neutral pH form (space group P4 12 12 or its enantiomer; a = b = 81.99(3) Å) and high pH form (space group P4 12 12; a = b = 71.5(1) Å, c = 57.6(2) Å one molecule per asymmetric unit) — the latter of which was used for structure determination.

  11. 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

  12. Cloning, expression, and purification of lipoprotein-associated phospholipase A(2) in Pichia pastoris.

    PubMed

    Zhang, Fujun; Wang, Yiping

    2006-05-01

    Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) has been shown to play a crucial role in atherosclerosis, and has been proposed as a promising target for drug discovery. Here, we cloned the Lp-PLA(2) gene from differentiated THP-1 cells, and inserted a carboxy-terminal His(6)-tagged version of the gene into the pPIC9 Pichia expression vector. The Lp-PLA(2) fusion protein was successfully expressed in Pichia pastoris expression system and could be rapidly purified to apparent homogeneity using a single-step purification method. The activity of our recombinant Lp-PLA(2) was strong when [3H] PAF was used as a substrate, and the Lp-PLA(2) inhibitor SB435495 exhibited an inhibitory curve against the recombinant Lp-PLA2 (IC50 = 15.93 +/- 1 microM). This novel recombinant Lp-PLA(2) could prove useful as a screening model for Lp-PLA(2) inhibitors, and may facilitate further investigation of this protein in atherosclerosis.

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

    PubMed Central

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

    2015-01-01

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

  14. Amyloid-type fiber formation in control of enzyme action: interfacial activation of phospholipase A2.

    PubMed

    Code, Christian; Domanov, Yegor; Jutila, Arimatti; Kinnunen, Paavo K J

    2008-07-01

    The lag-burst behavior in the action of phospholipase A(2) (PLA(2)) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T(m) of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA(2), evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA(2), involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form "mature" fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.

  15. 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

  16. 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.

  17. Purification and characterization of a novel phospholipase A2 from king cobra (Ophiophagus hannah) venom.

    PubMed

    Chiou, J Y; Chang, L S; Chen, L N; Chang, C C

    1995-08-01

    A novel phospholipase A2, designated as Oh-DE-2, was isolated from the venom of Ophiophagus hannah (king cobra) by successive chromatography on SP-Sephadex C-25, DE-52, and Q-Sepharose columns. Oh-DE-2 with pI 5.1 showed an apparent molecular weight of 14 kD as revealed by SDS-PAGE and gel filtration. The amino acid sequence was homologous with those of PLA2S from Elapidae venoms. Oh-DE-2 was effectively inactivated by p-bromophenacyl bromide, indicating that the conserved His-48 is essential for its enzymatic activity. However, modification of the conserved Trp-19 did not cause a precipitous drop in the enzymatic activity of Oh-DE-2 as observed with PLA2S from Naja naja atra and Bungarus multicinctus venoms. A quenching study showed that the microenvironment of Trp in Oh-DE-2 was inaccessible to acrylamide, iodide, or cesium, a finding which was different from those observed with PLA2S from N. naja atra and B. multicinctus venoms. These results might suggest that, unlike other PLA2 enzymes, Trp-19 in Oh-DE-2 is not directly involved in its enzymatic mechanisms.

  18. 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

  19. Phospholipases A2 and neural membrane dynamics: Implications for Alzheimer's disease

    PubMed Central

    Lee, James C-M.; Simonyi, Agnes; Sun, Albert Y.; Sun, Grace Y.

    2010-01-01

    Phospholipases A2 (PLA2s) are essential enzymes in cells. They are not only responsible for maintaining the structural organization of cell membranes, but also play a pivotal role in the regulation of cell functions. Activation of PLA2s results in the release of fatty acids and lysophospholipids, products that are lipid mediators and compounds capable of altering membrane microdomains and physical properties. Although not fully understood, recent studies have linked aberrant PLA2 activity to oxidative signaling pathways involving NADPH oxidase that underlie the pathophysiology of a number of neurodegenerative diseases. In this paper, we review studies describing the involvement of cytosolic PLA2 (cPLA2) in oxidative signaling pathways leading to neuronal impairment and activation of glial cell inflammatory responses. In addition, this review also includes information on the role of cPLA2 and exogenous secretory PLA2 (sPLA2) on membrane physical properties, dynamics, and membrane proteins. Unraveling the mechanisms that regulate specific types of PLA2s and their effects on membrane dynamics are important prerequisites towards understanding their roles in the pathophysiology of AD, and in the development of novel therapeutics to retard progression of the disease. PMID:21214562

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

    SciTech Connect

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

    2016-07-01

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

  1. Secretory phospholipases A2 induce cytokine release from blood and synovial fluid monocytes.

    PubMed

    Triggiani, Massimo; Granata, Francescopaolo; Oriente, Alfonso; Gentile, Marco; Petraroli, Angelica; Balestrieri, Barbara; Marone, Gianni

    2002-01-01

    Secretory phospholipases A2 (sPLA2) are released in the blood of patients with various inflammatory diseases and exert proinflammatory activities by releasing arachidonic acid (AA), the precursor of eicosanoids. We examined the ability of four sPLA2 to activate blood and synovial fluid monocytes in vitro. Monocytes were purified from blood of healthy donors or from synovial fluid of patients with rheumatoid arthritis by negative immunoselection and by adherence to plastic dishes, respectively. The cells were incubated with group IA, IB, IIA and III sPLA2 and the release of TNF-alpha, IL-6 and IL-12 was determined by ELISA. Group IA, IB and IIA sPLA2 induced a concentration-dependent release of TNF-alpha and IL-6 from blood monocytes. These sPLA2 activated IL-12 production only in monocytes preincubated with IFN-gamma. Group IA and IIA sPLA2 also induced TNF-alpha and IL-6 release from synovial fluid monocytes. TNF-alpha and IL-6 release paralleled an increase in their mRNA expression and was independent from the capacity of sPLA2 to mobilize AA. These results indicate that sPLA2 stimulate cytokine release from blood and synovial fluid monocytes by a mechanism at least partially unrelated to their enzymatic activity. This effect may concur with the generation of AA in the proinflammatory activity of sPLA2 released during inflammatory diseases.

  2. Evaluation of Rhamnetin as an Inhibitor of the Pharmacological Effect of Secretory Phospholipase A2.

    PubMed

    Novo Belchor, Mariana; Hessel Gaeta, Henrique; Fabri Bittencourt Rodrigues, Caroline; Ramos da Cruz Costa, Caroline; de Oliveira Toyama, Daniela; Domingues Passero, Luiz Felipe; Dalastra Laurenti, Marcia; Hikari Toyama, Marcos

    2017-08-31

    Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammation; therefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.

  3. 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).

  4. Hair Follicular Expression and Function of Group X Secreted Phospholipase A2 in Mouse Skin*

    PubMed Central

    Yamamoto, Kei; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Sato, Hiroyasu; Masuda, Seiko; Nishito, Yasumasa; Morioka, Kiyokazu; Ishimoto, Yoshikazu; Suzuki, Noriko; Yokota, Yasunori; Hanasaki, Kohji; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Fukami, Kiyoko; Ikeda, Kazutaka; Nakanishi, Hiroki; Taguchi, Ryo; Murakami, Makoto

    2011-01-01

    Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A2 (PLA2) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA2 (sPLA2-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA2-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA2-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA2-X in hair follicles, the presence of skin-specific machinery leading to sPLA2-X activation, a functional link of sPLA2-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis. PMID:21266583

  5. Denervation Induces Cytosolic Phospholipase A2-mediated Fatty Acid Hydroperoxide Generation by Muscle Mitochondria*

    PubMed Central

    Bhattacharya, Arunabh; Muller, Florian L.; Liu, Yuhong; Sabia, Marian; Liang, Hanyu; Song, Wook; Jang, Youngmok C.; Ran, Qitao; Van Remmen, Holly

    2009-01-01

    Previously, we demonstrated that mitochondria from denervated muscle exhibited dramatically higher Amplex Red dependent fluorescence (thought to be highly specific for hydrogen peroxide) compared with control muscle mitochondria. We now demonstrate that catalase only partially inhibits the Amplex Red signal in mitochondria from denervated muscle. In contrast, ebselen (a glutathione peroxidase mimetic and inhibitor of fatty acid hydroperoxides) significantly inhibits the Amplex Red signal. This suggests that the majority of the Amplex Red signal in mitochondria from denervated muscle is not derived from hydrogen peroxide. Because Amplex Red cannot react with substrates in the lipid environment, we hypothesize that lipid hydroperoxides formed within the mitochondrial lipid bilayer are released as fatty acid hydroperoxides and react with the Amplex Red probe. We also suggest that the release of fatty acid hydroperoxides from denervated muscle mitochondria may be an important determinant of muscle atrophy. In support of this, muscle atrophy and the Amplex Red signal are inhibited in caloric restricted mice and in transgenic mice that overexpress the lipid hydroperoxide-detoxifying enzyme glutathione peroxidase 4. Finally, we propose that cytosolic phospholipase A2 may be a potential source of these hydroperoxides. PMID:19001413

  6. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid.

    PubMed Central

    Lin, L L; Lin, A Y; Knopf, J L

    1992-01-01

    Cytosolic phospholipase A2 (cPLA2) binds to natural membrane vesicles in a Ca(2+)-dependent fashion, resulting in the selective release of arachidonic acid, thus implicating cPLA2 in the hormonally regulated production of eicosanoids. Here we report that the treatment of Chinese hamster ovary (CHO) cells overexpressing cPLA2 with ATP or thrombin resulted in an increased release of arachidonic acid as compared with parental CHO cells, demonstrating the hormonal coupling of cPLA2. In contrast, CHO cells overexpressing a secreted form of mammalian PLA2 (sPLA2-II) failed to show any increased hormonal responsiveness. Interestingly, we have noted that the activation of cPLA2 with a wide variety of agents stimulates the phosphorylation of cPLA2 on serine residues. Pretreatment of cells with staurosporin blocked the ATP-mediated phosphorylation of cPLA2 and strongly inhibited the activation of the enzyme. Increased cPLA2 activity was also observed in lysates prepared from ATP-treated cells and was sensitive to phosphatase treatment. These results suggest that in addition to Ca2+, the phosphorylation of cPLA2 plays an important role in the agonist-induced activation of cPLA2. Images PMID:1631101

  7. Glutamine Prevents Late-Phase Anaphylaxis via MAPK Phosphatase 1-Dependent Cytosolic Phospholipase A2 Deactivation.

    PubMed

    Kim, Hae-Kyoung; Song, Chang-Ho; Bae, Yun-Soo; Im, Suhn-Young; Lee, Hern-Ku

    2016-01-01

    Cytosolic phospholipase A2 (cPLA2) plays a key role in the development of late-phase anaphylaxis. L-Glutamine (Gln), a nonessential amino acid, has anti-inflammatory activity via inhibiting cPLA2. We used a penicillin-induced murine model of anaphylaxis, and late-phase anaphylaxis was quantified by measuring the increase in the hematocrit (Ht) value. Various inhibitors, small interfering RNA, and knockout mice were used in inhibition experiments. Phosphorylation and protein expression of cPLA2, ERK, and MAPK phosphatase 1 (MKP-1) were detected by Western blotting. Leukotriene (LT) B4 was found to be another potent inducer of late-phase anaphylaxis besides the known mediator platelet-activating-factor (PAF). Gln efficiently prevented late-phase anaphylaxis when it was administered up to 3 h after challenge injection via inhibiting cPLA2. Inhibition studies indicated that p38 MAPK was the major upstream regulator of cPLA2. Gln dephosphorylated p38 and cPLA2 via up-regulating the negative regulator of p38 MAPK, i.e., MKP-1 protein. MKP-1 blockade abrogated all the effects of Gln. Of the cPLA2 metabolites, PAF and LTB4 play a key role in the development of late-phase anaphylaxis, and Gln prevents the reaction via MKP-1-dependent deactivation of cPLA2. © 2016 S. Karger AG, Basel.

  8. 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

  9. Secretory phospholipase A2 modified HDL rapidly and potently suppresses platelet activation.

    PubMed

    Curcic, Sanja; Holzer, Michael; Pasterk, Lisa; Knuplez, Eva; Eichmann, Thomas O; Frank, Saša; Zimmermann, Robert; Schicho, Rudolf; Heinemann, Akos; Marsche, Gunther

    2017-08-14

    Levels of secretory phospholipases A2 (sPLA2) highly increase under acute and chronic inflammatory conditions. sPLA2 is mainly associated with high-density lipoproteins (HDL) and generates bioactive lysophospholipids implicated in acute and chronic inflammatory processes. Unexpectedly, pharmacological inhibition of sPLA2 in patients with acute coronary syndrome was associated with an increased risk of myocardial infarction and stroke. Given that platelets are key players in thrombosis and inflammation, we hypothesized that sPLA2-induced hydrolysis of HDL-associated phospholipids (sPLA2-HDL) generates modified HDL particles that affect platelet function. We observed that sPLA2-HDL potently and rapidly inhibited platelet aggregation induced by several agonists, P-selectin expression, GPIIb/IIIa activation and superoxide production, whereas native HDL showed little effects. sPLA2-HDL suppressed the agonist-induced rise of intracellular Ca(2+) levels and phosphorylation of Akt and ERK1/2, which trigger key steps in promoting platelet activation. Importantly, sPLA2 in the absence of HDL showed no effects, whereas enrichment of HDL with lysophosphatidylcholines containing saturated fatty acids (the main sPLA2 products) mimicked sPLA2-HDL activities. Our findings suggest that sPLA2 generates lysophosphatidylcholine-enriched HDL particles that modulate platelet function under inflammatory conditions.

  10. 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

  11. 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

  12. Group IVA phospholipase A2 participates in the progression of hepatic fibrosis.

    PubMed

    Ishihara, Keiichi; Miyazaki, Akira; Nabe, Takeshi; Fushimi, Hideaki; Iriyama, Nao; Kanai, Shiho; Sato, Takashi; Uozumi, Naonori; Shimizu, Takao; Akiba, Satoshi

    2012-10-01

    Group IVA phospholipase A2 (IVA-PLA2) is an enzyme that intiates the arachidonic acid pathway and plays an important role in inflammation. We demonstrate that IVA-PLA2 deficiency suppresses lipid deposition in the liver, which was induced by administration of a high-fat and -cholesterol diet (HFCD) for 16 wk in mice. Herein, we performed 2-dimensional gel-based comparative proteomics to further define the suppressive effect of IVA-PLA2 deficiency on fatty liver formation. In comparisons among 4 groups, wild-type (WT)/normal diet (ND), IVA-PLA2-deficient knockout (KO)/ND, WT/HFCD, and KO/HFCD, 4 proteins, 3 of which are associated with hepatic fibrosis, were identified as molecules, of which altered expression by HFCD was suppressed in KO mice compared to WT mice. Therefore, we assessed the effect of IVA-PLA2 deficiency on hepatic fibrosis induced by HFCD or carbon tetrachloride (CCl4) in mouse models. Biochemical and histological analyses revealed that IVA-PLA2 deficiency markedly reduced overall collagen accumulation in the liver of HFCD- and CCl4-derived mouse models. We found that IVA-PLA2 deficiency prevented activation of hepatic stellate cells and infiltration of F4/80-positive macrophages without affecting other immunocytes such as CD8+ lymphocytes and natural killer cells. In summary, IVA-PLA2 deficiency attenuates not only lipid deposition in the liver but also hepatic fibrosis formation.

  13. Structure and function comparison of Micropechis ikaheka snake venom phospholipase A2 isoenzymes.

    PubMed

    Lok, Shee-Mei; Gao, Rong; Rouault, Morgane; Lambeau, Gerard; Gopalakrishnakone, Ponnampalam; Swaminathan, Kunchithapadam

    2005-03-01

    Comparison of the crystal structures of three Micropechis ikaheka phospholipase A2 isoenzymes (MiPLA2, MiPLA3 and MiPLA4, which exhibit different levels of pharmacological effects) shows that their C-terminus (residues 110-124) is the most variable. M-Type receptor binding affinity of the isoenzymes has also been investigated and MiPLA4 binds to the rabbit M-type receptor with high affinity. Examination of surface charges of the isoenzymes reveals a trend of increase in positive charges with potency. The isoenzymes are shown to oligomerize in a concentration-dependent manner in a semi-denaturing gel. The C-termini of the medium (MiPLA4) and highly potent (MiPLA2) isoenzyme molecules cluster together, forming a highly exposed area. A BLAST search using the sequence of the most potent MiPLA2 results in high similarity to Staphylococcus aureus clotting factor A and cadherin 11. This might explain the myotoxicity, anticoagulant and hemoglobinuria effects of MiPLA2s.

  14. 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.

  15. Involvement of Oxidative Pathways in Cytokine-induced Secretory Phospholipase A2-IIA in Astrocytes

    PubMed Central

    Jensen, Michael D.; Sheng, Wenwen; Simonyi, Agnes; Johnson, Gary S.; Sun, Albert Y.; Sun, Grace Y.

    2009-01-01

    Recent studies have suggested the involvement of secretory phospholipase A2-IIA (sPLA2-IIA) in neuroinflammatory diseases. Although sPLA2-IIA is transcriptionally induced through the NF-κB pathway by pro-inflammatory cytokines, whether this induction pathway is affected by other intracellular signaling pathways has not been investigated in detail. In this study, we demonstrated the induction of sPLA2-IIA mRNA and protein expression in astrocytes by cytokines and detected the protein in the culture medium after stimulation. We further investigated the effects of oxidative pathways and botanical antioxidants on the induction pathway and observed that IL-1β-induced sPLA2-IIA mRNA expression in astrocytes is dependent on ERK1/2 and PI-3 kinase, but not p38 MAPK. In addition to apocynin, a known NADPH oxidase inhibitor, botanical antioxidants, such as resveratrol and epigallocatechin gallate, also inhibited IL-1β-induced sPLA2-IIA mRNA expression. These compounds also suppressed IL-1β-induced ERK1/2 activation and translocation of the NADPH oxidase subunit p67 phox from cytosol to membrane fraction. Taken together, these results support the involvement of reactive oxygen species from NADPH oxidase in cytokine induction of sPLA2-IIA in astrocytes and promote the use of botanical antioxidants as protective agents for inhibition of inflammatory responses in these cells. PMID:19375465

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

    PubMed Central

    Soubeyrand, S; Lazure, C; Manjunath, P

    1998-01-01

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

  17. Effect of guggulsterone and cembranoids of Commiphora mukul on pancreatic phospholipase A(2): role in hypocholesterolemia.

    PubMed

    Yu, Bao-Zhu; Kaimal, Rajani; Bai, Shi; El Sayed, Khalid A; Tatulian, Suren A; Apitz, Rafael J; Jain, Mahendra K; Deng, Ruitang; Berg, Otto G

    2009-01-01

    Guggulsterone (7) and cembranoids (8-12) from Commiphora mukul stem bark resin guggul were shown to be specific modulators of two independent sites that are also modulated by bile salts (1-6) to control cholesterol absorption and catabolism. Guggulsterone (7) antagonized the chenodeoxycholic acid (3)-activated nuclear farnesoid X receptor (FXR), which regulates cholesterol metabolism in the liver. The cembranoids did not show a noticeable effect on FXR, but lowered the cholate (1)-activated rate of human pancreatic IB phospholipase A2 (hPLA2), which controls gastrointestinal absorption of fat and cholesterol. Analysis of the data using a kinetic model has suggested an allosteric mechanism for the rate increase of hPLA2 by cholate and also for the rate-lowering effect by certain bile salts or cembranoids on the cholate-activated hPLA2 hydrolysis of phosphatidylcholine vesicles. The allosteric inhibition of PLA2 by certain bile salts and cembranoids showed some structural specificity. Biophysical studies also showed specific interaction of the bile salts with the interface-bound cholate-activated PLA2. Since cholesterol homeostasis in mammals is regulated by FXR in the liver for metabolism and by PLA2 in the intestine for absorption, modulation of PLA2 and FXR by bile acids and selected guggul components suggests novel possibilities for hypolipidemic and hypocholesterolemic therapies.

  18. Phospholipase A2-modified low-density lipoprotein activates macrophage peroxisome proliferator-activated receptors.

    PubMed

    Namgaladze, Dmitry; Morbitzer, Daniel; von Knethen, Andreas; Brüne, Bernhard

    2010-02-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors modulating metabolic and inflammatory responses of phagocytes to stimuli such as fatty acids and their metabolites. We studied the role of PPARs in macrophages exposed to low-density lipoprotein (LDL) modified by secretory phospholipase A(2) (PLA). By analyzing PPAR ligand-binding domain luciferase reporter activation, we observed that PLA-LDL transactivates PPARalpha and PPARdelta, but not PPARgamma. We confirmed that PLA-LDL induced PPAR response element reporter activation by endogenous PPARalpha and PPARdelta in human THP-1 macrophages. By using THP-1 cells with a stable knockdown of PPARalpha and PPARdelta, we showed that PLA-LDL-activated PPARdelta altered macrophage gene expression related to lipid metabolism and lipid droplet formation. Although PPARalpha/delta silencing did not affect cholesterol and triglyceride accumulation in PLA-LDL-treated macrophages, PPARdelta activation by PLA-LDL attenuated macrophage inflammatory gene expression induced by interferon gamma and lipopolysaccharide. PPARdelta activation by PLA-LDL does not influence lipid accumulation in PLA-LDL-treated macrophages. However, it attenuates macrophage inflammatory responses, thus contributing to an anti-inflammatory cell phenotype.

  19. Phospholipase A2 Inhibitors Synthesized by Two Entomopathogenic Bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata

    PubMed Central

    Seo, Samyeol; Lee, Sunghong; Hong, Yongpyo

    2012-01-01

    The entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata suppress insect immune responses by inhibiting the catalytic activity of phospholipase A2 (PLA2), which results in preventing biosynthesis of immune-mediating eicosanoids. This study identified PLA2 inhibitors derived from culture broths of these two bacteria. Both X. nematophila and P. temperata subsp. temperata culture broths possessed significant PLA2-inhibitory activities. Fractionation of these bacterial metabolites in the culture broths using organic solvent and subsequent chromatography purified seven potent PLA2 inhibitors, three of which (benzylideneacetone [BZA], proline-tyrosine [PY], and acetylated phenylalanine-glycine-valine [FGV]) were reported in a previous study. Four other compounds (indole, oxindole, cis-cyclo-PY, and p-hydroxyphenyl propionic acid) were identified and shown to significantly inhibit PLA2. X. nematophila culture broth contained these seven compounds, while P. temperata subsp. temperata culture broth contained three compounds (BZA, acetylated FGV, and cis-cyclo-PY). BZA was detected in the largest amount among these PLA2 compounds in both bacterial culture broths. All seven bacterial metabolites also showed significant inhibitory activities against immune responses, such as phenoloxidase activity and hemocytic nodulation; BZA was the most potent. Finally, this study characterized these seven compounds for their insecticidal activities against the diamondback moth, Plutella xylostella. Even though these compounds showed relatively low toxicities to larvae, they significantly enhanced the pathogenicity of Bacillus thuringiensis. This study reports bacterial-origin PLA2 inhibitors, which would be applicable for developing novel insecticides. PMID:22447611

  20. Increase in phospholipase A2 activity towards lipopolymer-containing liposomes.

    PubMed

    Vermehren, C; Kiebler, T; Hylander, I; Callisen, T H; Jørgensen, K

    1998-08-14

    Phospholipase A2 (PLA2)-catalyzed hydrolysis of dipalmitoylphosphatidylcholine (DPPC) liposomes incorporated with submicellar concentrations of polyethyleneoxide covalently attached to dipalmitoylphosphatidylethanolamine (DPPE-PEG2000) has been studied in the gel-to-fluid transition region of the host DPPC lipid bilayer matrix. By means of fluorescence and light-scattering measurements, the characteristic PLA2 lag time has been determined as a function of lipopolymer concentration and temperature. The degree of lipid hydrolysis was followed using radioactive labeled lipids. Differential scanning calorimetry has been applied to characterize the thermodynamic phase behavior of the lipopolymer-containing liposomes. A remarkable lipopolymer concentration-dependent decrease in the lag time was observed over broad temperature ranges. The radioactive measurements demonstrate an increase in catalytic activity for increasing amounts of lipopolymers in the bilayer. Hence, the lipopolymers act as a promoter of PLA2 lipid hydrolysis resulting in a degradation of the bilayer structure and a concomitant destabilization of the liposomes. This behavior is in contrast to the generally observed protective and stabilization effect in biological fluids exerted by lipopolymers in polymer-grafted liposomes. It is proposed that the enhanced activity of the small water soluble and interfacially active enzyme may involve a non-uniform distribution of the lipopolymers in the lipid matrix due to a coupling between local lipid bilayer curvature and composition of the non-bilayer-preferring lipopolymers.

  1. 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.

  2. Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins.

    PubMed

    Shuaibu, M N; Kanbara, H; Yanagi, T; Ameh, D A; Bonire, J J; Nok, A J

    2001-11-01

    Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.

  3. Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex trunculus digestive cells.

    PubMed

    Zarai, Zied; Boulais, Nicholas; Karray, Aida; Misery, Laurent; Bezzine, Sofiane; Rebai, Tarek; Gargouri, Youssef; Mejdoub, Hafedh

    2011-06-01

    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. 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. 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.

  4. 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

  5. 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.

  6. Purification and characterization of a phospholipase A2-IIA from common stingray (Dasyatis pastinaca) intestine.

    PubMed

    Ben Bacha, Abir; Daihan, Sooad K; Moubayed, Nadine M S; Mejdoub, Hafedh

    2013-06-01

    A phospholipase A2 belonging to IIA group secretory PLA2 was isolated and purified to homogeneity from the intestine of common stingray (Dasyatis pastinaca) using acidic treatment (pH 1.5) and ammonium sulphate precipitation methods combined with single-column ion-exchange chromatography. The purified enzyme was found to be a glycosylated monomeric protein with a molecular mass of about 14 kDa. The stingray sPLA2-IIA had optimum activity at 45 degrees C, unlike known mammalian PLA2-IIAs, which show optimum activity at 37 degrees C. The purified enzyme exhibited a specific activity of 290 U/mg at optimal conditions (pH 9.5 and 45 degrees C) in the presence of 6 mM NaDC and 8 mM CaCl2 with egg yolk as substrate. The NH2-terminal sequence of the enzyme and some protein fragments obtained from its tryptic digestion were also determined. All sequences obtained were similar to those of sPLA2-IIA. The enzyme also showed good stability in the presence of organic solvents, acidic and alkaline pH media and high temperature conditions. Thus, the purified enzyme exhibited a number of unique and promising properties, making it a potential possible candidate for future applications in the treatment of phospholipid-rich industrial effluents and synthesis of useful preparations for the food production and processing industry.

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

    PubMed Central

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

    2013-01-01

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

  8. Enigma (partially) resolved: phospholipase A2 receptor is the cause of "idiopathic" membranous glomerulonephritis.

    PubMed

    Truong, Luan D; Seshan, Surya V

    2015-12-15

    Membranous glomerulonephritis (MGN) is a very significant kidney disease. It is one of the frequent causes of heavy protein excretion in urine. MGN is thought to be an immune-mediated disease caused by glomerular deposition of antigen-antibody complexes. The pathogenic antigen, however, has been an enigma until recently. It was discovered in 2009 that phospholipase A2 receptor (PLA2R), a normal transmembrane protein in podocyte plasma membrane, is the antigen causing MGN. Within 5 yr of its discovery, this seminal finding has leaded to novel insights into the treatment of this disease including diagnosis, therapy, and prediction of outcome. This finding also paves the way for fundamental studies on how and why autoimmunity against PLA2R develops. The discovery of PLA2A as the cause of "idiopathic" MGN after a half century of speculation, followed by further fundamental insights with such an expedient and successful application in patient care, embodies the elegance of science at its junction with society. This perspective traces the story of this remarkable discovery.

  9. Lipoprotein profile, lipoprotein-associated phospholipase A2 and cardiovascular risk in hemodialysis patients.

    PubMed

    Rolla, Roberta; De Mauri, Andreana; Valsesia, Ambra; Vidali, Matteo; Chiarinotti, Doriana; Bellomo, Giorgio

    2015-12-01

    Cardiovascular disease is the leading cause of morbidity and mortality in hemodialysis patients; the increased risk of cardiovascular disease is due to accelerated atherosclerosis, inflammation and impaired lipoprotein metabolism. We aimed to evaluate lipoprotein-associated phospholipase A2 (Lp-PLA2) and some pro-inflammatory aspects of the lipoprotein profile in dialyzed patients in order to evaluate the relationship with the accelerated atherosclerosis and vascular accidents. In 102 dialysis patients and 40 non-uremic controls, we investigated the lipoprotein plasma profile, high sensitivity C-reactive protein (CRP), ceruloplasmin and serum amyloid A protein (SAA), and followed patients for 1 year to analyze the risk of acute cardiovascular events. Total cholesterol, low-density lipoprotein and high-density lipoprotein plasma levels were significantly lower in uremic patients than controls, whereas CRP, SAA, ceruloplasmin, Lp-PLA2 and their ratio with apolipoprotein A1 were significantly higher. Patients with Lp-PLA2 levels >194 nmol/min/ml had more acute cardiovascular events than patients with lower values. Our results show that in dialysis subjects: (1) low-density lipoproteins show a more atherogenic phenotype than in the general population; (2) high-density lipoproteins are less anti-inflammatory; (3) Lp-PLA2 could potentially be used to evaluate cardiovascular risk.

  10. Aspirin induces its anti-inflammatory effects through its specific binding to phospholipase A2: crystal structure of the complex formed between phospholipase A2 and aspirin at 1.9 angstroms resolution.

    PubMed

    Singh, Rajendra Kumar; Ethayathulla, A S; Jabeen, Talat; Sharma, Sujata; Kaur, Punit; Singh, Tej P

    2005-02-01

    Phospholipase A2 is potentially an important target for structure-based rational drug design. In order to determine the involvement of phospholipase A2 in the action of non-steroidal anti-inflammatory drugs (NSAIDs), the crystal structure of the complex formed between phospholipase A2 and aspirin has been determined at 1.9 angstroms resolution. The structure contains 915 protein atoms, 1 calcium ion, 13 atoms of aspirin and 105 water molecules. The observed electron density of the aspirin molecule in the structure was of very high quality thus allowing the precise determination of its atomic coordinates leading to the clear description of its interactions with the enzyme. The structure of the complex clearly shows that aspirin is literally embedded in the hydrophobic environment of PLA2. It is so placed in the substrate binding channel that it forms several important attractive interactions with calcium ion, His 48 and Asp 49. Thus, the structure of the complex clearly shows that aspirin occupies a favourable place in the specific binding site of PLA2. The binding studies have shown that acetyl salicylate (aspirin) binds to PLA2 enzyme specifically with a dissociation constant of 6.4 x 10(-6) M. The structural details and binding data suggest that the inhibition of PLA2 by aspirin is of pharmacological

  11. Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

    PubMed Central

    Shmelzer, Zeev; Haddad, Nurit; Admon, Ester; Pessach, Itai; Leto, Thomas L.; Eitan-Hazan, Zahit; Hershfinkel, Michal; Levy, Rachel

    2003-01-01

    Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets. PMID:12913107

  12. Lipoprotein-associated phospholipase A2 (Lp-PLA(2)): a novel and promising biomarker for cardiovascular risks assessment.

    PubMed

    Cai, Anping; Zheng, Dongdan; Qiu, Ruofeng; Mai, Weiyi; Zhou, Yingling

    2013-01-01

    Atherosclerosis and its manifestations namely cardiovascular diseases (CVD) are still the leading cause of morbidity and mortality worldwide. Although intensified interventions have been applied, the residual cardiovascular (CV) risks are still very high. Lipoprotein-associated phospholipase A2 (Lp-PLA(2)) is a novel and unique biomarker highly specific for vascular inflammation and atherosclerosis. Both pro-atherogenic property of Lp-PLA(2) and positive correlation with CV events have already been demonstrated by a large number of scientific and clinical studies. Currently, in the Adult Treatment Panel III (ATP III) guideline, Lp-PLA(2) has been recommended as an adjunct to traditional risk factors in assessing future CV risks. Encouragingly, darapladib, an orally Lp-PLA(2) specific inhibitor, has been tested in basic research and preclinical trials and the outcomes are quite striking. Additionally, there are two phase III ongoing clinical trials in evaluating the efficacy and safety of darapladib on cardiovascular outcomes. With regard to the potential values of Lp-PLA(2) in risk stratification, therapeutic regimen establishment and prognosis evaluation in patients with moderate or high risk, our present review is going to summarize the relevant data about the bio-chemical characteristics of Lp-PLA(2), the actions of Lp-PLA(2) on atherosclerosis and the results of Lp-PLA(2) in scientific research and clinical studies.

  13. Anti-bactericidal properties of stingray Dasyatis pastinaca groups V, IIA, and IB phospholipases A2: a comparative study.

    PubMed

    Bacha, Abir Ben

    2014-10-01

    Group IIA secreted phospholipase A2 (group IIA sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of native stingray and dromedary groups V, IIA, and IB sPLA2s on several Gram-positive and Gram-negative strains. The rank order potency among both marine and mammal sPLA2s against Gram-positive bacteria is group IIA > V > IB, whereas Gram-negative bacteria exhibited a much higher resistance. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2.The bactericidal efficiency of groups V and IIA sPLA2s was shown to be dependent upon the presence of calcium ions and to a less extent Mg(2+) ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray and dromedary groups V, IIA, and IB sPLA2s present no cytotoxicity after their incubation with MDA-MB-231cells. stingray groups V and IIA sPLA2s, like mammal ones, may be considered as future therapeutic agents against bacterial infections.

  14. Competitive inhibition of cytosolic Ca2+-dependent phospholipase A2 by acteoside in RBL-2H3 cells.

    PubMed

    Song, Ho Sun; Choi, Mi Young; Ko, Myoung Soo; Jeong, Jae Min; Kim, Yong Ho; Jang, Beom Hyeon; Sung, Ji Hoon; Kim, Min Gyu; Whang, Wan Kyunn; Sim, Sang Soo

    2012-05-01

    The aim of this study was to investigate whether acteoside isolated from Clerodendron trichotomum Thunberg may act as a selective inhibitor of phospholipase A(2) in RBL-2H3 cells. Acteoside dose-dependently inhibited 0.5 μM melittin-induced release of [(3)H]arachidonic acid, which was due to the inhibition of cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)) rather than secretory PLA(2) (sPLA(2)). In Dixon plots, the apparent K ( i ) value of acteoside on cPLA(2) was 5.9 μM and the inhibitory pattern appeared to be a competitive inhibitor. The above data, suggests that acteoside acts as a competitive inhibitor of cPLA(2) in RBL-2H3 cells.

  15. Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae.

    PubMed

    Petrovic, Uros; Sribar, Jernej; Matis, Maja; Anderluh, Gregor; Peter-Katalinić, Jasna; Krizaj, Igor; Gubensek, Franc

    2005-10-15

    Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to g and e isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.

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

    PubMed Central

    Krug, H. F.

    1995-01-01

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

  17. Roles of various phospholipases A2 in providing lysophospholipid acceptors for fatty acid phospholipid incorporation and remodelling.

    PubMed Central

    Balsinde, Jesús

    2002-01-01

    In the present study the lysophospholipid sources for arachidonic (AA) and eicosapentaenoic acid (EPA) incorporation into and redistribution within the phospholipids of phorbol-ester-differentiated U937 cells was investigated. Initially, AA incorporated primarily into choline glycerophospholipids (PC), whereas EPA incorporated mainly into ethanolamine glycerophospholipids (PE). Bromoenol lactone (BEL), an inhibitor of the Group VI Ca2+-independent phospholipase A2 (iPLA2), diminished both lysophosphatidylcholine levels and the incorporation of AA into phospholipids. However BEL had little effect on EPA incorporation. In concanavalin A-activated cells, EPA, but not AA, incorporation was also affected by methyl arachidonyl fluorophosphonate (MAFP), suggesting an additional role for the group IV cytosolic phospholipase A2. In the activated cells AA and EPA did not compete with each other for incorporation, indicating that the pathways for AA and EPA incorporation are partially different. The AA and EPA initially incorporated into PC slowly moved to PE in a process that took several hours. The transfer of AA and EPA from PC to PE was not inhibited by BEL, MAFP or LY311727 [3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulphonic acid], raising the possibility that an as-yet-undetermined phospholipase A2 may be involved in fatty acid phospholipid remodelling. A strong candidate to be involved in these reactions is a novel Ca2+-independent phospholipase A2 that, unlike all known iPLA2s, is resistant to inhibition by BEL and also to MAFP and LY311727. The enzyme activity cleaves both PC and PE and is thus able to provide the lysoPC and lysoPE acceptors required for the fatty acid acylation reactions. PMID:12049633

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

    PubMed

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

    1998-04-01

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

  19. Prostaglandin synthesis is increased in selenium supplemented human mesangial cells despite suppression of phospholipase A2 - activity

    SciTech Connect

    Hampel, G.; Reinke, M.; Hren, J. )

    1991-01-01

    Antioxidants play an important role in the regulation of phospholipid hydrolysis and arachidonate metabolism. Supplementation of cultured human mesangial cells with selenium resulted in suppression of phospholipase A2 - activity and significantly increased production of three major prostaglandins. However, prostacyclin synthesis benefits most from selenium supplementation, suggesting that there is a specific action of selenium-dependent glutathione peroxidase on this pathway. Like in endothelial cells, production of platelet activating factor is significantly inhibited by selenium supplementation.

  20. Purification and characterization of a phospholipase A2 from the venom of the coral snake, Micrurus fulvius microgalbineus (Brown and Smith).

    PubMed Central

    Possani, L D; Alagòn, A C; Fletcher, P L; Varela, M J; Juliá, J Z

    1979-01-01

    A phospholipase A2 was purified from the Mexican coral snake Micrurus fulvius microgalbieus (Brown and Smith). Gel filtration of the soluble crude venom on Sephadex g-50 resolved five fractions, of which fraction II had 98% of the total phospholipase activity. This fraction was rechromatographed on a CM-cellulose column that resolved eight fractions, four of which had an important phospholipase activity. The first fraction (II-1) was homogeneous by polyacrylamide-gel electrophoresis and displayed a phospholipase specific activity of 920 units/mg of protein. The apparent molecular weight as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 14000. The amino acid analysis revealed the presence of 119 amino acid residues, with 12 half-cystines. the N-terminal sequence was shown to be Ser-Leu-Leu-Asx-Phe-Lys-Asx-Met-Ile-Glu-Ser-Thr..., which is homologous with that of phospholipases from other snake venoms. PMID:475771

  1. Association between lipoprotein associated phospholipase A2 mass and subclinical coronary and carotid atherosclerosis in Retired National Football League players.

    PubMed

    Pokharel, Yashashwi; Nambi, Vijay; Martin, Seth S; Hoogeveen, Ron C; Nasir, Khurram; Khera, Amit; Wong, Nathan D; Jones, Peter H; Boone, Jeffrey; Roberts, Arthur J; Ballantyne, Christie M; Virani, Salim S

    2014-10-01

    Retired National Football League (NFL) players were reported to have high prevalence of cardiovascular risk factors. Lipoprotein Associated Phospholipase A2 (LpPLA2) has shown to be associated with cardiovascular disease in the general population, but it is unknown whether such an association exists in retired NFL players. Our objective was to assess whether LpPLA2 mass was associated with coronary artery calcium (CAC) and carotid artery plaque (CAP) in retired NFL players. LpPLA2 mass was assessed using a dual monoclonal antibody immunoassay. CAC presence was defined as CAC score>0. CAP was defined as focal thickening ≥50% than that of the surrounding vessel wall with a minimal thickness of 1.2 mm on carotid ultrasound. In 832 NFL players, the median (IQR) age and LpPLA2 levels were 54 (45-63) years and 142 (109-181) ng/mL respectively. LpPLA2 mass was positively correlated with low-density lipoprotein (LDL) cholesterol and high-density lipoprotein cholesterol; negatively correlated with LDL particle concentration and body mass index; and not correlated with high-sensitivity C-reactive protein. CAC was present in 659 (79%) and CAP in 544 (65%) players. In a fully adjusted model, LpPLA2 was not associated with CAC (OR per 1-SD increase, 0.85; 95% CI 0.71-1.02) or CAP (0.90, 0.75-1.08). LpPLA2 was also not associated with CAC burden in those with CAC>0. Results were similar when highest and lowest LpPLA2 tertiles were compared, and also in various subgroups. LpPLA2 mass was not associated with coronary or carotid subclinical atherosclerosis in retired NFL players. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. Interaction of a trehalose lipid biosurfactant produced by Rhodococcus erythropolis 51T7 with a secretory phospholipase A2.

    PubMed

    Zaragoza, Ana; Teruel, José A; Aranda, Francisco J; Ortiz, Antonio

    2013-10-15

    Trehalose-containing glycolipid biosurfactants form an emerging group of interesting compounds, which alter the structure and properties of phospholipid membranes, and interact with enzymatic and non-enzymatic proteins. Phospholipases A2 constitute a class of enzymes that hydrolyze the sn-2 ester of glycerophospholipids, and are classified into secreted phospholipases A2 (sPLA2) and intracellular phospholipases A2. In this work, pancreatic sPLA2 was chosen as a model enzyme to study the effect of the trehalose lipid biosurfactant on enzymes acting on interfaces. By using this enzyme, it is possible to study the modulation of enzyme activity, either by direct interaction of the biosurfactant with the protein, or as a result of the incorporation of the glycolipid on the phospholipid target membrane. It is shown that the succinoyl trehalose lipid isolated from Rhodococcus erythropolis 51T7 interacts with porcine pancreatic sPLA2 and inhibits its catalytic activity. Two modes of inhibition are observed, which are clearly differentiated by its timescale. First, a slow inhibition of sPLA2 activity upon preincubation of the enzyme with trehalose lipid in the absence of substrate is described. Second, incorporation of trehalose lipid into the phospholipid target membrane gives rise to a fast enzyme inhibition. These results are discussed in the light of previous data on sPLA2 inhibitors and extend the list of interesting biological activities reported for this R. erythropolis trehalose lipid biosurfactant.

  3. Lipoprotein-associated phospholipase A2 as a predictive biomarker of sub-clinical inflammation in cardiovascular diseases.

    PubMed

    Cojocaru, Manole; Cojocaru, Inimioara Mihaela; Silosi, Isabela

    2010-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a predictor biomarker for incident atherosclerotic disease. Lp-PLA2 has been identified in atherosclerotic plaques, however, its role in atherosclerosis is still under investigation. Lp-PLA2 belongs to the superfamily of phospholipase A2 enzymes. It is produced by macrophages that appears to play a role in the atherosclerotic vessel wall. Emerging data seem to suggest that Lp-PLA2 may be proatherogenic, which is an effect thought to be mediated by lypophosphatidylcholine and oxidized nonesterified fatty acids, two mediators generated by Lp-PLA2. Phospholipase A2 plays an essential role in metabolism of membrane phospholipids, it is related to inflammatory reactions, secretion of amyloid precursor protein. Several studies have documented the strong association of Lp-PLA2 with coronary heart disease and stroke in the general population. Lp-PLA2 may be a stronger predictor of recurrent stroke risk. Inflammatory markers have been associated with ischemic stroke risk. Their relationship to prognosis after stroke is unsettled. The present review article focuses particularly on the characteristics of the Lp(a)-associated Lp-PLA2 and discusses the possible role of this enzyme in view of the new data.

  4. Lipoprotein-associated phospholipase A2 as a predictive biomarker of sub-clinical inflammation in cardiovascular diseases

    PubMed Central

    Cojocaru, Manole; Cojocaru, Inimioara Mihaela; Silosi, Isabela

    2010-01-01

    ABSTRACT Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a predictor biomarker for incident atherosclerotic disease. Lp-PLA2 has been identified in atherosclerotic plaques, however, its role in atherosclerosis is still under investigation. Lp-PLA2 belongs to the superfamily of phospholipase A2 enzymes. It is produced by macrophages that appears to play a role in the atherosclerotic vessel wall. Emerging data seem to suggest that Lp-PLA2 may be proatherogenic, which is an effect thought to be mediated by lypophosphatidylcholine and oxidized nonesterified fatty acids, two mediators generated by Lp-PLA2. Phospholipase A2 plays an essential role in metabolism of membrane phospholipids, it is related to inflammatory reactions, secretion of amyloid precursor protein. Several studies have documented the strong association of Lp-PLA2 with coronary heart disease and stroke in the general population. Lp-PLA2 may be a stronger predictor of recurrent stroke risk. Inflammatory markers have been associated with ischemic stroke risk. Their relationship to prognosis after stroke is unsettled. The present review article focuses particularly on the characteristics of the Lp(a)-associated Lp-PLA2 and discusses the possible role of this enzyme in view of the new data. PMID:21977119

  5. 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.

  6. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. In Vivo and In Vitro Studies of Cytosolic Phospholipase A2 Expression in Helicobacter pylori Infection

    PubMed Central

    Nardone, Gerardo; Holicky, Eileen L.; Uhl, James R.; Sabatino, Lina; Staibano, Stefania; Rocco, Alba; Colantuoni, Vittorio; Manzo, Barbara A.; Romano, Marco; Budillon, Gabriele; Cockerill, Franklin R.; Miller, Laurence J.

    2001-01-01

    Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H. pylori infection and no change in epithelial cell lines exposed to H. pylori. PMID:11500464

  8. Structural basis of the anionic interface preference and kcat* activation of pancreatic phospholipase A2.

    PubMed

    Yu, B Z; Poi, M J; Ramagopal, U A; Jain, R; Ramakumar, S; Berg, O G; Tsai, M D; Sekar, K; Jain, M K

    2000-10-10

    Pancreatic phospholipase A(2) (PLA2) shows a strong preference for the binding to the anionic interface and a consequent allosteric activation. In this paper, we show that virtually all the preference is mediated through 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancreatic PLA2. The lysine-to-methionine substitution enhances the binding of the enzyme to the zwitterionic interface, and for the K53,56,120M triple mutant at the zwitterionic interface is comparable to that for the wild type (WT) at the anionic interface. In the isomorphous crystal structure, the backbone folding of K53,56M K120,121A and WT are virtually identical, yet a significant change in the side chains of certain residues, away from the site of substitution, mostly at the putative contact site with the interface (i-face), is discernible. Such reciprocity, also supported by the spectroscopic results for the free and bound forms of the enzyme, is expected because a distal structural change that perturbs the interfacial binding could also affect the i-face. The results show that lysine-to-methionine substitution induces a structural change that promotes the binding of PLA2 to the interface as well as the substrate binding to the enzyme at the interface. The kinetic results are consistent with a model in which the interfacial Michaelis complex exists in two forms, and the complex that undergoes the chemical step is formed by the charge compensation of Lys-53 and -56. Analysis of the incremental changes in the kinetic parameters shows that the charge compensation of Lys-53 and -56 contributes to the activation and that of Lys-120 contributes only to the structural change that promotes the stability of the Michaelis complex at the interface. The charge compensation effects on these three residues also account for the differences in the anionic interface preference of the evolutionarily divergent secreted PLA2.

  9. Bile acids cause secretory phospholipase A2 activity enhancement, revertible by exogenous surfactant administration.

    PubMed

    De Luca, Daniele; Minucci, Angelo; Zecca, Enrico; Piastra, Marco; Pietrini, Domenico; Carnielli, Virgilio P; Zuppi, Cecilia; Tridente, Ascanio; Conti, Giorgio; Capoluongo, Ettore D

    2009-02-01

    Bile acids have been implicated in some forms of acute lung injury, including meconium aspiration and bile acid pneumonia in neonates, or aspiration related ARDS in adults. Secretory phospholipase A2 (sPLA2) is now known as a key enzyme in the lung injury pathways and is supposed to be responsible for surfactant dysfunction. Our aim was to investigate the interaction between bile acids and sPLA2 in an extracellular environment representing an in vitro model of aspiration. In vitro study using broncho-alveolar lavage (BAL) of 23 neonates/infants (<6 m) with healthy lungs. BAL supernatants were assayed for sPLA2 activity in basal condition and after addition of randomly assigned concentrations of bile acids (BA) or normal saline. Samples coming from neonates were then challenged with poractant-alfa up to a phospholipid concentration equal to that found in babies after the surfactant treatment for respiratory distress syndrome. sPLA2 activity was again measured, being corrected for serum/supernatant urea ratio and for confounding factors. High concentrations of BA (5 micromol/l) significantly increased (P = 0.012) sPLA2 activity, leading to increased surfactant catabolism. This finding was not observed with lower BA concentration and this is consistent with available literature data and may indicate an anionic activation of the enzyme by bile acids. Increased activity was significantly reverted by the addition of exogenous surfactant (P = 0.004) which was able to reduce sPLA2 activity almost to the baseline level. BA are likely to contribute to lung injury, causing surfactant inactivation through the increased sPLA2 activity. Other mechanisms cannot be excluded and require further studies to be clarified.

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

    PubMed

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

    2015-09-25

    Lipoprotein-associated phospholipase A 2 (Lp-PLA2) is associated with the risk of vascular disease. It circulates in human blood predominantly in association with low-density lipoprotein cholesterol (LDL-C) and hydrolyses oxidized phospholipids into pro-inflammatory products. However, in the mouse circulation, it predominantly binds to high-density lipoprotein cholesterol (HDL-C) and exhibits anti-inflammatory properties. To further investigate the effects of Lp-PLA2 in the circulation, we generated over-expressed Lp-PLA2 transgenic swine. The eukaryotic expression plasmid of porcine Lp-PLA2 which driven by EF1α promoter was constructed and generate transgenic swine via SCNT. The expression and activity of Lp-PLA2 in transgenic swine were evaluated, and the total cholesterol (TC), HDL-C, LDL-C and triglyceride (TG) levels in the fasting and fed states were also assessed. Compared with wild-type swine controls, the transgenic swine exhibited elevated Lp-PLA2 mRNA levels and activities, and the activity did not depend on the feeding state. The TC, HDL-C and LDL-C levels were not significantly increased. There was no change in the TG levels in the fasting state between transgenic and control pigs. However, in the fed state, the TG levels of transgenic swine were slightly increased compared with the control pigs and were significantly elevated compared with the fasting state. In addition, inflammatory gene (interleukin [IL]-6, monocyte chemotactic protein [MCP]-1 and tumor necrosis factor [TNF]-α) mRNA levels in peripheral blood mononuclear cells (PBMCs) were significantly increased. The results demonstrated that Lp-PLA2 is associated with triglycerides which may be helpful for understanding the relationship of this protein with cardiovascular disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Accelerated expansion of group IID-like phospholipase A2 genes in Bos taurus.

    PubMed

    Golik, Marina; Cohen-Zinder, Miri; Loor, Juan J; Drackley, James K; Band, Mark R; Lewin, Harris A; Weller, Joel I; Ron, Micha; Seroussi, Eyal

    2006-04-01

    Low-molecular-weight, calcium-dependent phospholipase A2 genes (PLA2s) that belong to the secreted type of PLA2s are clustered within a syntenic group on human 1p35-p36 and mouse 4qD3. We reassembled trace files available from the Whole Genome Sequencing (WGS) Project, obtaining an 86-kb contig with three tandem PLA2G2D duplications in the Hereford strain. We used mate-pair data to monitor the assembly and to exclude chimeric clones, demonstrating that the current WGS data may be assembled even in a highly repetitive region with a coverage exceeding fivefold. The genomic structure indicated that most of the PLA2G2D transcripts are formed by four exons. Two alternative first exons were present in all duplications. In two duplications insertions of satellite DNA in the third intron created a novel exon that gave rise to a two-exon product. Linkage and comparative mapping placed the bovine PLA2G2 locus on BTA2, indicating that it evolved from an ancestral PLA2G2D locus common to human, cattle, and rodents. Bovine PLA2G2D variants were capable of encoding 147-amino-acid polypeptides that consisted of putative signal peptide and metal-binding domains. Cysteine residues were conserved in positions analogous to those forming the seven disulfide bonds characteristic of PLA2G2 genes. Quantitative PCR analysis of bovine PLA2G2D transcripts indicated that their expression levels varied between the dry period and lactation in the mammary gland samples and that their expression was polymorphic in liver tissue. The recent burst of duplication and divergence of the bovine PLA2G2D genes and their polymorphic nature are typical of innate immune response genes.

  12. Evolution of a Rippled Membrane during Phospholipase A2 Hydrolysis Studied by Time-Resolved AFM

    PubMed Central

    Leidy, Chad; Mouritsen, Ole G.; Jørgensen, Kent; Peters, Günther H.

    2004-01-01

    The sensitivity of phospholipase A2 (PLA2) for lipid membrane curvature is explored by monitoring, through time-resolved atomic force microscopy, the hydrolysis of supported double bilayers in the ripple phase. The ripple phase presents a corrugated morphology. PLA2 is shown to have higher activity toward the ripple phase compared to the gel phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes, indicating its preference for this highly curved membrane morphology. Hydrolysis of the stable and metastable ripple structures is monitored for equimolar DMPC/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-supported double bilayers. As shown by high-performance liquid chromatography results, DSPC is resistant to hydrolysis at this temperature, resulting in a more gradual hydrolysis of the surface that leads to a change in membrane morphology without loss of membrane integrity. This is reflected in an increase in ripple spacing, followed by a sudden flattening of the lipid membrane during hydrolysis. Hydrolysis of the ripple phase results in anisotropic holes running parallel to the ripples, suggesting that the ripple phase has strip regions of higher sensitivity to enzymatic attack. Bulk high-performance liquid chromatography measurements indicate that PLA2 preferentially hydrolyzes DMPC in the DMPC/DSPC ripples. We suggest that this leads to the formation of a flat gel-phase lipid membrane due to enrichment in DSPC. The results point to the ability of PLA2 for inducing a compositional phase transition in multicomponent membranes through preferential hydrolysis while preserving membrane integrity. PMID:15240475

  13. Membrane Restructuring by Phospholipase A2 Is Regulated by the Presence of Lipid Domains

    PubMed Central

    Leidy, Chad; Ocampo, Jackson; Duelund, Lars; Mouritsen, Ole G.; Jørgensen, Kent; Peters, Günther H.

    2011-01-01

    Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (Lα/Lβ) lipid domains. In this work, we explore whether lipid domains can also direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA2. Specifically, we show that the presence of Lα/Lβ and Lα/Pβ′ phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) system results in the preferential hydrolysis of the shorter-chained lipid component in the mixture, leading to an enrichment in the longer-chained component. The restructuring process is monitored by atomic force microscopy on supported single and double bilayers formed by vesicle fusion. We observe that during preferential hydrolysis of the DMPC-rich Lα regions, the Lβ and Pβ′ regions grow and reseal, maintaining membrane integrity. This result indicates that a sharp reorganization of the membrane structure can occur during sPLA2 hydrolysis without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary. Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity in the hydrolytic activity of the enzyme, resulting in marked differences in the physical properties of the membrane end-product. PMID:21723818

  14. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Lymphoid tissue phospholipase A2 group IID resolves contact hypersensitivity by driving antiinflammatory lipid mediators

    PubMed Central

    Miki, Yoshimi; Yamamoto, Kei; Taketomi, Yoshitaka; Sato, Hiroyasu; Shimo, Kanako; Kobayashi, Tetsuyuki; Ishikawa, Yukio; Ishii, Toshiharu; Nakanishi, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Kabashima, Kenji; Arita, Makoto; Arai, Hiroyuki; Lambeau, Gérard; Bollinger, James M.; Hara, Shuntaro; Gelb, Michael H.

    2013-01-01

    Resolution of inflammation is an active process that is mediated in part by antiinflammatory lipid mediators. Although phospholipase A2 (PLA2) enzymes have been implicated in the promotion of inflammation through mobilizing lipid mediators, the molecular entity of PLA2 subtypes acting upstream of antiinflammatory lipid mediators remains unknown. Herein, we show that secreted PLA2 group IID (PLA2G2D) is preferentially expressed in CD11c+ dendritic cells (DCs) and macrophages and displays a pro-resolving function. In hapten-induced contact dermatitis, resolution, not propagation, of inflammation was compromised in skin and LNs of PLA2G2D-deficient mice (Pla2g2d−/−), in which the immune balance was shifted toward a proinflammatory state over an antiinflammatory state. Bone marrow-derived DCs from Pla2g2d−/− mice were hyperactivated and elicited skin inflammation after intravenous transfer into mice. Lipidomics analysis revealed that PLA2G2D in the LNs contributed to mobilization of a pool of polyunsaturated fatty acids that could serve as precursors for antiinflammatory/pro-resolving lipid mediators such as resolvin D1 and 15-deoxy-Δ12,14-prostaglandin J2, which reduced Th1 cytokine production and surface MHC class II expression in LN cells or DCs. Altogether, our results highlight PLA2G2D as a “resolving sPLA2” that ameliorates inflammation through mobilizing pro-resolving lipid mediators and points to a potential use of this enzyme for treatment of inflammatory disorders. PMID:23690440

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

    PubMed

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

    1997-02-01

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

  17. Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge.

    PubMed

    Shrestha, Sony; Park, Yoonseong; Stanley, David; Kim, Yonggyun

    2010-03-01

    We propose that expression of four genes encoding secretory phospholipases A(2) (sPLA(2)) 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 of which depends on PLA(2)-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA(2) activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA(2) and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA(2)s. The recombinant sPLA(2)s were inhibited by sPLA(2) inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA(2) genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA(2)s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA(2)s showed the presence of the sPLA(2) enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA(2) genes that mediate nodulation reactions strongly supports our hypothesis that sPLA(2)s are central enzymes in insect cellular immune reactions. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2009-01-01

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

  19. Phospholipases A2 and Inflammatory Responses in the Central Nervous System

    PubMed Central

    Sun, Grace Y.; Shelat, Phullara B.; Jensen, Michael B.; He, Yan; Sun, Albert Y.; Simonyi, Agnes

    2011-01-01

    Phospholipases A2 (PLA2s) belong to a superfamily of enzymes responsible for hydrolyzing the sn-2 fatty acids of membrane phospholipids. These enzymes are known to play multiple roles for maintenance of membrane phospholipid homeostasis and for production of a variety of lipid mediators. Over 20 different types of PLA2s are present in the mammalian cells, and in snake and bee venom. Despite their common function in hydrolyzing fatty acids of phospholipids, they are diversely encoded by a number of genes and express proteins that are regulated by different mechanisms. Recent studies have focused on the group IV calcium-dependent cytosolic cPLA2, the group VI calcium-independent iPLA2, and the group II small molecule secretory sPLA2. In the central nervous system (CNS), these PLA2s are distributed among neurons and glial cells. Although the physiological role of these PLA2s in regulating neural cell function has not yet been clearly elucidated, there is increasing evidence for their involvement in receptor signaling and transcriptional pathways that link oxidative events to inflammatory responses that underline many neurodegenerative diseases. Recent studies also reveal an important role of cPLA2 in modulating neuronal excitatory functions, sPLA2 in the inflammatory responses, and iPLA2 with childhood neurologic disorders associated with brain iron accumulation. The goal for this review is to better understand the structure and function of these PLA2s and to highlight specific types of PLA2s and their cross-talk mechanisms in these inflammatory responses under physiological and pathological conditions in the CNS. PMID:19855947

  20. No relationship between lipoprotein-associated phospholipase A2, proinflammatory cytokines, and neopterin in Alzheimer's disease.

    PubMed

    Savas, S; Kabaroglu, C; Alpman, A; Sarac, F; Yalcin, M A; Parıldar, Z; Ozkinay, F; Kumral, E; Akcicek, F

    2016-05-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a reported risk factor for dementia. However, the relationship between Alzheimer's disease (AD) and Lp-PLA2 is still debatable and, to the best of our knowledge, no study has evaluated the associations between levels of Lp-PLA2, proinflammatory cytokines, and neopterin in AD. In total, 59 patients with AD and 38 non-demented individuals were included in the case-control study. Fasting serum concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), neopterin, and Lp-PLA2 were determined using ELISA. The associations between AD and each of the variables were analyzed by logistic regression. The median Lp-PLA2 levels in AD and controls were similar (P=0.29, not significant). Median serum neopterin and IL-6 levels were significantly higher in patients with AD than in controls (P=0.0001 and P=0.03, respectively). In regression analyses, median neopterin levels, a lower level of education, and female gender were significantly associated with AD when compared with controls (OR, 31.44, 95% CI 3.59-275.28, P=0.002; OR, 4.35, 95% CI 1.13-16.61, P=0.032; OR, 7.25, 95% CI 1.88-28.00, P=0.004, respectively). In contrast to previous evidence suggesting its role in dementia and AD, Lp-PLA2 enzyme levels were higher in the controls, and no relationship between Lp-PLA2 and either proinflammatory cytokines or neopterin was identified in AD. Elevated neopterin levels may be considered inflammatory markers of AD. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Cytosolic phospholipase A(2) alpha mediates electrophysiologic responses of hippocampal pyramidal neurons to neurotoxic NMDA treatment.

    PubMed

    Shen, Ying; Kishimoto, Koji; Linden, David J; Sapirstein, Adam

    2007-04-03

    The arachidonic acid-generating enzyme cytosolic phospholipase A(2) alpha (cPLA(2)alpha) has been implicated in the progression of excitotoxic neuronal injury. However, the mechanisms of cPLA(2)alpha toxicity have yet to be determined. Here, we used a model system exposing mouse hippocampal slices to NMDA as an excitotoxic injury, in combination with simultaneous patch-clamp recording and confocal Ca(2+) imaging of CA1 pyramidal neurons. NMDA treatment caused significantly greater injury in wild-type (WT) than in cPLA(2)alpha null CA1 neurons. Bath application of NMDA evoked a slow inward current in voltage-clamped neurons (composed of both NMDA receptor-mediated and other conductances) that was smaller in cPLA(2)alpha null than in WT slices. This was not due to down-regulation of NMDA receptor function because NMDA receptor-mediated currents were equivalent in each genotype following brief photolysis of caged glutamate. Current-clamp recordings were made during and following NMDA exposure by eliciting a single action potential with a brief current injection. After NMDA exposure, WT CA1 neurons developed a spike-evoked plateau potential and an increased spike-evoked dendritic Ca(2+) transient. These effects were absent in CA1 neurons from cPLA(2)alpha null mice and WT neurons treated with a cPLA(2)alpha inhibitor. The Ca-sensitive K-channel toxins, apamin and paxilline, caused spike broadening and Ca(2+) enhancement in WT and cPLA(2)alpha null slices. NMDA application in WT and arachidonate applied to cPLA(2)alpha null cells occluded the effects of apamin/paxilline. These results indicate that cPLA(2)alpha activity is required for development of aberrant electrophysiologic events triggered by NMDA receptor activation, in part through attenuation of K-channel function.

  2. Role of Secretory Phospholipase A2 in CNS Inflammation: Implications in Traumatic Spinal Cord Injury

    PubMed Central

    Titsworth, W. Lee; Liu, Nai-Kui; Xu, Xiao-Ming

    2009-01-01

    Secretory phospholipases A2 (sPLA2s) are a subfamily of lipolytic enzymes which hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are precursors of bioactive eicosanoids and platelet-activating factor (PAF). The hydrolysis of membrane phospholipids by PLA2 is a rate-limiting step for generation of eicosanoids and PAF. To date, more than 10 isozymes of sPLA2 have been found in the mammalian central nervous system (CNS). Under physiological conditions, sPLA2s are involved in diverse cellular responses, including host defense, phospholipid digestion and metabolism. However, under pathological situations, increased sPLA2 activity and excessive production of free fatty acids and their metabolites may lead to inflammation, loss of membrane integrity, oxidative stress, and subsequent tissue injury. Emerging evidence suggests that sPLA2 plays a role in the secondary injury process after traumatic or ischemic injuries in the brain and spinal cord. Importantly, sPLA2 may act as a convergence molecule that mediates multiple key mechanisms involved in the secondary injury since it can be induced by multiple toxic factors such as inflammatory cytokines, free radicals, and excitatory amino acids, and its activation and metabolites can exacerbate the secondary injury. Blocking sPLA2 action may represent a novel and efficient strategy to block multiple injury pathways associated with the CNS secondary injury. This review outlines the current knowledge of sPLA2 in the CNS with emphasis placed on the possible roles of sPLA2 in mediating CNS injuries, particularly the traumatic and ischemic injuries in the brain and spinal cord. PMID:18673210

  3. Ca(2+)-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro.

    PubMed Central

    Nagao, T; Kubo, T; Fujimoto, R; Nishio, H; Takeuchi, T; Hata, F

    1995-01-01

    The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused with the granules within 30 s, and induced amylase release by reacting with the membranes of granules, whereas without this pretreatment they had no significant effect. The fusion process accompanied by amylase release was induced in the presence of 10 mM EGTA, and therefore was apparently Ca(2+)-independent. On the other hand, the presence of EGTA or 100 microM quinacrine, an inhibitor of PLA2, during treatment of plasma membranes with PLA2 inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA2 treatment. The presence of albumin, an adsorbent of fatty acids, during the treatment also inhibited the activity. Pretreatment of the membranes with arachidonic acid or linoleic acid did not have any effect, but the presence of exogenously added arachidonic acid during PLA2 treatment enhanced the membrane-fusion-inducing effect of PLA2. Pretreatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA2 and the presence of arachidonic acid or linoleic acid produced by PLA2 are important in the process of fusion of secretory granules with the plasma membranes of rat parotid acinar cells and that the fusion process itself is independent of Ca2+. PMID:7537492

  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. 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.

  6. 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.

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

    PubMed

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

    2014-03-01

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

  8. Study on the activation of phospholipases A2 by purinergic agonists in rat submandibular ductal cells.

    PubMed

    Kabré, E; Chaïb, N; Boussard, P; Mérino, G; Devleeschouwer, M; Dehaye, J P

    1999-01-04

    Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of [3H]arachidonic acid ([3H]AA) from prelabeled rat submandibular gland (RSMG) ductal cells respectively two- and threefold. Both agonists also increased the release of [3H]AA from acini but at a lower level (+50% and +100% respectively). Carbachol had no significant effect on either cellular population. In ductal cells phorbol myristate acetate, an activator of protein kinase C, slightly increased the basal release of [3H]AA but did not affect the release of [3H]AA in response to ATP. Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines. The removal of calcium from the extracellular medium decreased the response to ATP and Bz-ATP. Only barium could partly substitute for calcium to restore the purinergic response. Zinc inhibited the release of [3H]AA. Permeabilization of the cells with streptolysin O (SLO) activated the calcium-independent phospholipase A2 activity (iPLA2). The iPLA2, not the calcium-dependent PLA2 (cPLA2), released [3H]oleic acid ([3H]OA) from RSMG ductal cells. It is concluded that RSMG ducts have a higher PLA2 activity when compared to acini. This activity is accounted for by iPLA2 and cPLA2. Both enzymes are activated by P2X agonists by a staurosporine-sensitive mechanism. Cells permeabilized with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes. In intact RSMG ductal cells the two activities can be distinguished by rather specific inhibitors, by different ionic conditions and also by the fatty acid used to label the cells.

  9. Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

    PubMed Central

    Manevich, Yefim; Shuvaeva, Tea; Dodia, Chandra; Kazi, Altaf; Feinstein, Sheldon I.; Fisher, Aron B.

    2010-01-01

    Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A2 (PLA2) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA2 activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO2(3) antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes. PMID:19236840

  10. New quinoxalinone inhibitors targeting secreted phospholipase A2 and α-glucosidase.

    PubMed

    Alasmary, Fatmah A S; Alnahdi, Fatima S; Ben Bacha, Abir; El-Araby, Amr M; Moubayed, Nadine; Alafeefy, Ahmed M; El-Araby, Moustafa E

    2017-12-01

    Elevated blood glucose and increased activities of secreted phospholipase A2 (sPLA2) are strongly linked to coronary heart disease. In this report, our goal was to develop small heterocyclic compound that inhibit sPLA2. The title compounds were also tested against α-glucosidase and α-amylase. This array of enzymes was selected due to their implication in blood glucose regulation and diabetic cardiovascular complications. Therefore, two distinct series of quinoxalinone derivatives were synthesised; 3-[N'-(substituted-benzylidene)-hydrazino]-1H-quinoxalin-2-ones 3a-f and 1-(substituted-phenyl)-5H-[1,2,4]triazolo[4,3-a]quinoxalin-4-ones 4a-f. Four compounds showed promising enzyme inhibitory effect, compounds 3f and 4b-d potently inhibited the catalytic activities of all of the studied proinflammatory sPLA2. Compound 3e inhibited α-glucosidase (IC50 = 9.99 ± 0.18 µM); which is comparable to quercetin (IC50 = 9.93 ± 0.66 µM), a known inhibitor of this enzyme. Unfortunately, all compounds showed weak activity against α-amylase (IC50 > 200 µM). Structure-based molecular modelling tools were utilised to rationalise the SAR compared to co-crystal structures with sPLA2-GX as well as α-glucosidase. This report introduces novel compounds with dual activities on biochemically unrelated enzymes mutually involved in diabetes and its complications.

  11. Carvacrol attenuates serum levels of total protein, phospholipase A2 and histamine in asthmatic guinea pig

    PubMed Central

    Boskabady, Mohammad Hossein; Jalali, Sedigheh; Yahyazadeh, Negin; Boskabady, Mostafa

    2016-01-01

    Objective: Pharmacological effects of carvacrol such as its anti-inflammatory activities have been shows. In this study the effects of carvacrol on serum levels of total protein (TP), phospholipase A2 (PLA2) and histamine in sensitized guinea pigs was evaluated. Materials and Methods: Sensitized guinea pigs were given drinking water alone (group S), drinking water containing three concentrations of carvacrol (40, 80 and 160 µg/ml) or dexamethasone. Serum levels of TP, PLA2 and histamine were examined I all sensitized groups as well as a non-sensitized control group (n=6 for each group). Results: In sensitized animals, serum levels of TP, PLA2 and histamine were significantly increased compared to control animals (p<0.05 to p<0.001). Significant reduction in TP, PLA2 and histamine levels were observed in treated groups with the two higher concentrations of carvacrol but dexamethasone treatment only decreased serum level of PLA2 (p<0.05 to p<0.001). Although the effect of the lowest concentration of the extract was less than that of dexamethasone (p<0.05 for TP and p<0.001 for PLA2), the effects of the two higher concentrations on PLA2 were similar to dexamethasone and on TP (p<0.01) and histamine (p<0.001) were higher than those of dexamethasone. Conclusion: These results showed that carvacrol reduced serum levels of TP, PLA2 and histamine in sensitized guinea pigs which may indicate an anti-inflammatory effect of this agent in inflammatory disorders such as asthma. PMID:28078244

  12. Differential roles of phospholipases A2 in neuronal death and neurogenesis: implications for Alzheimer disease.

    PubMed

    Schaeffer, Evelin L; da Silva, Emanuelle R; Novaes, Barbara de A; Skaf, Heni D; Gattaz, Wagner F

    2010-12-01

    The involvement of phospholipase A(2) (PLA(2)) in Alzheimer disease (AD) was first investigated nearly 15 years ago. Over the years, several PLA(2) isoforms have been detected in brain tissue: calcium-dependent secreted PLA(2) or sPLA(2) (IIA, IIC, IIE, V, X, and XII), calcium-dependent cytosolic PLA(2) or cPLA(2) (IVA, IVB, and IVC), and calcium-independent PLA(2) or iPLA(2) (VIA and VIB). Additionally, numerous in vivo and in vitro studies have suggested the role of different brain PLA(2) in both physiological and pathological events. This review aimed to summarize the findings in the literature relating the different brain PLA(2) isoforms with alterations found in AD, such as neuronal cell death and impaired neurogenesis process. The review showed that sPLA(2)-IIA, sPLA(2)-V and cPLA(2)-IVA are involved in neuronal death, whereas sPLA(2)-III and sPLA(2)-X are related to the process of neurogenesis, and that the cPLA(2) and iPLA(2) groups can be involved in both neuronal death and neurogenesis. In AD, there are reports of reduced activity of the cPLA(2) and iPLA(2) groups and increased expression of sPLA(2)-IIA and cPLA(2)-IVA. The findings suggest that the inhibition of cPLA(2) and iPLA(2) isoforms (yet to be determined) might contribute to impaired neurogenesis, whereas stimulation of sPLA(2)-IIA and cPLA(2)-IVA might contribute to neurodegeneration in AD.

  13. 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.

  14. Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells.

    PubMed Central

    Rossini, G P; Fayard, J M; Tessier, C; Laugier, C

    1996-01-01

    The cellular uptake of extracellular type-I phospholipase A2 (PLA2) was investigated in rat uterine stromal cells (UIII) in culture, which were found to express the high-affinity binding site for mammalian type-I PLA2, with a measured KD of 6.4 nM, a Bmax of 0.1-1 pmol/mg of DNA at 4 degrees C, and a molecular mass of about 200 kDa. When UIII cells were treated with type-I PLA2 at 37 degrees C, the ligand specifically associated with the cells increased, reaching a plateau after 90 min of incubation, whose level was about 5-fold higher than that measured if cells were maintained at 4 degrees C. We could determine that the PLA2 was bound to plasma membrane receptors which were responsible for internalization of the ligand, and that the binding sites were still suitable for binding at the level of plasma membrane during UIII cell incubation at 37 degrees C. Proteolysis of internalized PLA2 could be clearly detected only after 90 min of UIII cell incubation with the ligand at 37 degrees C, and most of the intracellular PLA2 consisted of the apparently intact 14 kDa enzyme. By cross-linking studies, we found that most of the internalized PLA2 was not associated with the receptor, supporting the conclusion that in our experimental system a single pool of membrane receptors for mammalian type-I PLA2 undergoes cycles of ligand binding, intracellular transfer and release of PLA2, followed by restoration of binding sites on the plasma membrane. We calculated that the rate of internalization of the ligand by one receptor molecule in UIII cells at 37 degrees C is about three molecules of type-I PLA2 per h. PMID:8645137

  15. 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

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

    PubMed

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

    2002-01-01

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

  17. Hypotensive and vascular relaxant effects of phospholipase A2 toxins from Papuan taipan (Oxyuranus scutellatus) venom.

    PubMed

    Chaisakul, Janeyuth; Isbister, Geoffrey K; Tare, Marianne; Parkington, Helena C; Hodgson, Wayne C

    2014-01-15

    Phospholipase A2 (PLA2) toxins are common and abundant components of Australasian elapid venoms. These toxins are associated with a range of activities including neurotoxicity, myotoxicity and coagulation disturbances. We have recently reported that sudden cardiovascular collapse induced by Papuan taipan (Oxyuranus scutellatus) venom involves a combination of the release of dilator autacoids and a direct effect on the smooth muscle. In this study, we aimed to isolate PLA2 components from Papuan taipan venom and investigate their contribution to the hypotensive action of this venom. O. scutellatus venom was fractionated using size-exclusion high performance liquid chromatography (HPLC), and fractions screened for activity in anaesthetized rats. Fraction three from O. scutellatus venom (i.e. OSC3, 14.2±1.0% of whole venom) produced a 64% decrease in mean arterial pressure. Reverse-phase HPLC indicated that OSC3 consisted of two major components (i.e. OSC3a and OSC3b). OSC3a and OSC3b produced a significant hypotensive response in anaesthetized rats which were attenuated by prior administration of indomethacin or the combination of mepyramine and heparin. N-terminal analysis indicated that OSC3a and b displayed sequence homology to PLA2 toxins isolated from coastal taipan (O. scutellatus scutellatus) venom. These findings indicate that PLA2 components may play an important role in the development of hypotension and vascular relaxation which may contribute to the effects observed after envenoming by these Australasian elapids. © 2013 Published by Elsevier B.V.

  18. Creating air-stable supported lipid bilayers by physical confinement induced by phospholipase A2.

    PubMed

    Han, Chung-Ta; Chao, Ling

    2014-05-14

    Supported lipid bilayer platforms have been used for various biological applications. However, the lipid bilayers easily delaminate and lose their natural structure after being exposed to an air-water interface. In this study, for the first time, we demonstrated that physical confinement can be used instead of chemical modifications to create air-stable membranes. Physical confinement was generated by the obstacle network induced by a peripheral enzyme, phospholipase A2. The enzyme and reacted lipids could be washed away from the obstacle network, which was detergent-resistant and strongly bonded to the solid support. On the basis of these properties, the obstacle framework on the solid support was reusable and lipid bilayers with the desired composition could be refilled and formed in the region confined by the obstacle framework. The results of fluorescence recovery after photobleaching (FRAP) indicate that the diffusivities of the lipid bilayers before drying and after rehydration were comparable, indicating the air stability of the physically confined membrane. In addition, we observed that the obstacles could trap a thin layer of water after the air-water interface passed through the lipid bilayer. Because the obstacles were demonstrated to be several times higher than a typical lipid membrane on a support, the obstacles may act as container walls, which can trap water above the lipid membrane. The water layer may have prevented the air-water interface from directly contacting the lipid membrane and, therefore, buffered the interfacial force, which could cause membrane delamination. The results suggest the possibility of using physical confinement to create air-stable membranes without changing local membrane rigidity or covering the membrane with protecting molecules.

  19. Molecular determinants of bacterial sensitivity and resistance to mammalian Group IIA phospholipase A2.

    PubMed

    Weiss, Jerrold P

    2015-11-01

    Group IIA secretory phospholipase A2 (sPLA(2)-IIA) of mammalian species is unique among the many structurally and functionally related mammalian sPLA(2) in their high net positive charge and potent (nM) antibacterial activity. Toward the Gram-positive bacteria tested thus far, the global cationic properties of sPLA(2)-IIA are necessary for optimal binding to intact bacteria and penetration of the multi-layered thick cell wall, but not for the degradation of membrane phospholipids that is essential for bacterial killing. Various Gram-positive bacterial species can differ as much as 1000-fold in sPLA(2)-IIA sensitivity despite similar intrinsic enzymatic activity of sPLA(2)-IIA toward the membrane phospholipids of various bacteria. d-alanylation of wall- and lipo-teichoic acids in Staphylococcus aureus and sortase function in Streptococcus pyogenes increase bacterial resistance to sPLA(2)-IIA by up to 100-fold apparently by affecting translocation of bound sPLA(2)-IIA to the cell membrane. Action of the sPLA(2)-IIA and other related sPLA(2) against Gram-negative bacteria is more dependent on cationic properties of the enzyme near the amino-terminus of the protein and collaboration with other host defense proteins that produce alterations of the unique Gram-negative bacterial outer membrane that normally represents a barrier to sPLA(2)-IIA action. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

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

    PubMed Central

    Cavigiolio, Giorgio; Jayaraman, Shobini

    2014-01-01

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

  1. Rickettsial phospholipase A2 as a pathogenic mechanism in a model of cell injury by typhus and spotted fever group rickettsiae.

    PubMed

    Walker, D H; Feng, H M; Popov, V L

    2001-12-01

    Phospholipase A2 activity by typhus group rickettsiae causes hemolysis in vitro. Rickettsial phospholipase A2 has been proposed to mediate entry into the host cell, escape from the phagosome, and cause injury to host cells by both typhus and spotted fever group rickettsiae. In a rickettsial contact-associated cytotoxicity model, the interaction of Rickettsia prowazekii or R. conorii with Vero cells caused temperature-dependent release of 51Cr from the cells. Treatment of rickettsiae, but not the cells, with a phospholipase A2 inhibitor (bromophenacyl bromide) or with antibody to king cobra venom inhibited cell injury. Rickettsial treatment with bromophenacyl bromide inhibited the release of free fatty acids from the host cell. Neither the inhibitor nor antivenom impaired rickettsial active transport of L-lysine. Thus, host cell injury was mediated by a rickettsial phospholipase A2-dependent mechanism.

  2. Elevated Type II Secretory Phospholipase A2 Increases the Risk of Early Atherosclerosis in Patients with Newly Diagnosed Metabolic Syndrome

    PubMed Central

    Sun, Chang-Qing; Zhong, Chun-Yan; Sun, Wei-Wei; Xiao, Hua; Zhu, Ping; Lin, Yi-Zhang; Zhang, Chen-Liang; Gao, Hao; Song, Zhi-Yuan

    2016-01-01

    A critical association between type II secretory phospholipase A2 (sPLA2-IIa) and established atherosclerotic cardiovascular disease has been demonstrated. However, the contribution of sPLA2-IIa to early atherosclerosis remains unknown. This study investigated the association between early-stage atherosclerosis and sPLA2-IIa in metabolic syndrome (MetS) patients. One hundred and thirty-six MetS patients and 120 age- and gender-matched subjects without MetS were included. Serum sPLA2-IIa protein levels and activity were measured using commercial kits. Circulating endothelial activation molecules (vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin), and carotid intima-media thickness (cIMT), were measured as parameters of vascular endothelial dysfunction and early atherosclerosis. MetS patients exhibited significantly higher sPLA2-IIa protein and activity levels than the controls. Both correlated positively with fasting blood glucose and waist circumference in MetS patients. Additionally, MetS patients exhibited strikingly higher levels of endothelial activation molecules and increased cIMT than controls. These levels correlated positively with serum sPLA2-IIa protein levels and activity. Moreover, multivariate analysis showed that high sPLA2-IIa protein and activity levels were independent risk factors of early atherosclerosis in MetS patients. This study demonstrates an independent association between early-stage atherosclerosis and increased levels of sPLA2-IIa, implying that increased sPLA2-IIa may predict early-stage atherosclerosis in MetS patients. PMID:27941821

  3. Characterisation and expression of secretory phospholipase A2 group IB during ontogeny of Atlantic cod ( Gadus morhua).

    PubMed

    Sæle, Øystein; Nordgreen, Andreas; Olsvik, Pål A; Hamre, Kristin

    2011-01-01

    The pancreatic enzyme secretory phospholipase A2 group IB (sPLA2 IB) hydrolyses phospholipids at the sn-2 position, resulting in a NEFA and a lyso-phospholipid, which are then absorbed by the enterocytes. The sPLA2 IB is a member of a family of nineteen enzymes sharing the same catalytic ability, of which nine are cytosolic and ten are secretory. Presently, there are no pharmacological tools to separate between the different secretory enzymes when measuring the enzymatic activity. Thus, it is important to support activity data with more precise techniques when isolation of intestinal content is not possible for analysis, as in the case of small teleost larvae, where the whole animal is sometimes analysed. In the present study, we characterise the sPLA2 IB gene in Atlantic cod (Gadus morhua) and describe its ontogeny at the genetic and protein level and compare this to the total sPLA2 activity level. A positive correlation was found between the expression of sPLA2 IB mRNA and protein. Both remained stable and low during the larval stage followed by an increase from day 62 posthatch, coinciding with the development of the pyloric ceaca. Meanwhile, total sPLA2 enzyme activity in cod was stable and relatively high during the early stages when larvae were fed live prey, followed by a decrease in activity when the fish were weaned to a formulated diet. Thus, the expression of sPLA2 IB mRNA and protein did not correlate with total sPLA2 activity.

  4. Plasma levels of lipoprotein-associated phospholipase A2 are increased in patients with β-thalassemia

    PubMed Central

    Tselepis, Alexandros D.; Hahalis, George; Tellis, Constantinos C.; Papavasiliou, Eleni C.; Mylona, Panagiota T.; Kourakli, Alexandra; Alexopoulos, Dimitrios C.

    2010-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an independent cardiovascular risk factor. We investigated the plasma levels of Lp-PLA2 activity and mass as a function of plasma lipid levels, LDL subclass profile, and oxidative stress in patients with β-thalassemia. Thirty-five patients with β-thalassemia major (β-TM) and 25 patients with β-thalassemia intermedia (β-TI) participated in the study. Lp-PLA2 activity and mass were measured in total plasma, in apolipoprotein (apo)B-depleted plasma (HDL-Lp-PLA2), and in LDL subclasses. Lp-PLA2 activity produced and secreted from peripheral blood monocytes in culture was also determined. Patients with β-thalassemia are characterized by a predominance of small-dense LDL particles, increased oxidative stress, and very high plasma levels of Lp-PLA2 mass and activity, despite low LDL-cholesterol levels. A significant positive correlation between plasma Lp-PLA2 activity or mass and 8-isoprostane (8-epiPGF2a) and ferritin levels as well as intima-media thickness (IMT) values was observed. An increase in secreted and cell-associated Lp-PLA2 activity from monocytes in culture was observed in both patient groups. The HDL-Lp-PLA2 activity and mass as well as the ratio of HDL-Lp-PLA2/plasma Lp-PLA2 were significantly higher in both patient groups compared with the control group. In conclusion, patients with β-thalassemia exhibit high plasma Lp-PLA2 levels, attributed to increased enzyme secretion from monocytes/macrophages and to the predominance of sdLDL particles in plasma. Plasma Lp-PLA2 is correlated with carotid IMT, suggesting that this enzyme may be implicated in premature carotid atherosclerosis observed in β-thalassemia. PMID:20625038

  5. [Meta-analysis for the relationship between lipoprotein-associated phospholipase A2 and ischemic stroke].

    PubMed

    Xia, Wencui; Hu, Zhongyang; Song, Zhi

    2017-02-28

    目的:用Meta分析的方法探讨脂蛋白相关磷脂酶A2(lipoprotein-associated phospholipase A2,Lp-PLA2)与缺血性脑卒中发生的相关性。方法:在线检索PubMed,Web of Science,Embase和Cochrane Library以及中国生物医学文献数据库、中国期刊全文数据库、重庆维普和万方数字化期刊全文数据库中关于Lp-PLA2与缺血性脑卒中相关性的病例对照研究或队列研究,采用Stata 12.0软件对入选研究进行Meta分析。结果:共有8篇文献46 034例符合纳入标准。Meta分析结果显示:Lp-PLA2水平与缺血性脑卒中发生的合并效应量的危险比(95%可信区间)[RR(95% CI)]为1.04(0.98~1.11),提示Lp-PLA2水平与缺血性卒中发生无相关性。Lp-PLA2 活性与缺血性脑卒中发生的相关性合并效应量RR(95% CI)为1.03(0.96~1.10),提示Lp-PLA2活性与缺血性卒中发生无相关性。结论:目前不能认为Lp-PLA2水平及Lp-PLA2活性可以预测缺血性脑卒中的发生,高Lp-PLA2水平及高Lp-PLA2活性是缺血性脑卒中发病的危险因素的结论有待进一步论证。.

  6. Lipoprotein-associated phospholipase A2 is associated with atherosclerotic stroke risk: the Northern Manhattan Study.

    PubMed

    Katan, Mira; Moon, Yeseon P; Paik, Myunghee C; Wolfert, Robert L; Sacco, Ralph L; Elkind, Mitchell S V

    2014-01-01

    Lipoprotein-associated phospholipase A2 (LpPLA2) levels are associated with stroke, though whether this extends to all populations and stroke subtypes is unknown. Serum samples from stroke-free community participants in the Northern Manhattan Study were assayed for LpPLA2 mass and activity. Participants were followed annually for stroke. Cox-proportional-hazard models were fitted to estimate hazard-ratios and 95% confidence intervals (HR, 95% CI) for the association of LpPLA2 levels with ischemic stroke (IS), after adjusting for demographic and medical risk factors. Serum samples were available in 1946 participants, of whom 151 (7.8%) experienced a first IS during median follow-up 11 years. Mean age was 69 (SD 10), 35.6% were men, 20% non-Hispanic Whites, 22% non-Hispanic Blacks, and 55% Hispanics. LpPLA2 mass and activity levels were not associated with overall IS risk. LpPLA2 mass but not activity levels were associated with strokes due to large artery atherosclerosis (LAA; adjusted HR per SD 1.55, 95% CI 1.17-2.04). There was a dose-response relationship with LAA (compared to first quartile, 2nd quartile HR = 1.43, 95% CI 0.23-8.64; 3rd quartile HR = 4.47, 95% CI 0.93-21.54; 4th quartile HR = 5.07, 95% CI 1.07-24.06). The associations between LpPLA2-mass and LAA-stroke risk differed by race-ethnicity (p = 0.01); LpPLA2-mass was associated with increased risk of LAA among non-Hispanic Whites (adjusted HR per SD 1.44, 95% CI 0.98-2.11), but not other race-ethnic groups. LpPLA2-mass levels were associated with risk of atherosclerotic stroke among non-Hispanic White participants, but not in other race-ethnic groups in the cohort. Further study is needed to confirm these race-ethnic differences and the reasons for them.

  7. 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

  8. Taiwanese female vegetarians have lower lipoprotein-associated phospholipase A2 compared with omnivores.

    PubMed

    Chen, Chih-Wei; Lin, Chih-Ta; Lin, Ying-Lung; Lin, Tin-Kwang; Lin, Chin-Lon

    2011-01-01

    Many studies supported that vegetarians have a lower risk of cardiac diseases and mortality, partly due to better blood pressure and serum cholesterol profiles. However, the inflammatory markers, especially lipoprotein-associated phospholipase A2 (Lp-PLA2), have not been well-studied. This study aimed to compare inflammatory markers and conventional risk factors between vegetarians and omnivores. One hundred and seventy-three vegetarians and 190 omnivores were studied. Fasting blood samples were obtained to compare levels of glucose, total cholesterol, triacylglycerol, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, homocysteine, Lp-PLA2 activity, and high-sensitivity C-reactive protein (hs-CRP). Vegetarians had higher serum levels of the following markers: hs-CRP (1.8 ± 3.4 vs. 1.2 1.8 mg/L, respectively; p = 0.05), homocysteine (9.39 ± 3.22 vs. 7.62 ± 2.41 μmol/L, respectively; p < 0.01), and triacylglycerol (96.91 ± 59.56 vs. 84.66 ± 43.24 mg/dL, respectively; p < 0.05). Vegetarians also had lower levels of Lp-PLA2 (18.32 ± 7.19 10-3 μmol/min/mL vs. 20.22 8.13 10-3 μmol/min/mL; p < 0.05), total cholesterol (180.62 ± 36.55 mg/dL vs. 192.73 ± 36.57 mg/dL; p < 0.01), LDL cholesterol (118.15 ± 32.8 vs. 126.41 ± 34.28 mg/dL; p < 0.05), and HDL cholesterol (55.59 ± 13.30 vs. 62.09 ± 14.52 mg/dL, p < 0.01). Multivariate analyses demonstrated that a vegetarian diet increases the chances for high serum hs-CRP and low Lp-PLA2 activity. In addition to lower total cholesterol, LDL-cholesterol, and HDL-cholesterol, Taiwanese female vegetarians have lower serum Lp-PLA2 activity but higher levels of hs-CRP, homocysteine, and triacylglyerol. It might be due to geographic differences of vegetarian diets, and further studies are needed.

  9. 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

  10. Taiwanese Female Vegetarians Have Lower Lipoprotein-Associated Phospholipase A2 Compared with Omnivores

    PubMed Central

    Chen, Chih-Wei; Lin, Chih-Ta; Lin, Ying-Lung; Lin, Tin-Kwang

    2011-01-01

    Purpose Many studies supported that vegetarians have a lower risk of cardiac diseases and mortality, partly due to better blood pressure and serum cholesterol profiles. However, the inflammatory markers, especially lipoprotein-associated phospholipase A2 (Lp-PLA2), have not been well-studied. This study aimed to compare inflammatory markers and conventional risk factors between vegetarians and omnivores. Materials and Methods One hundred and seventy-three vegetarians and 190 omnivores were studied. Fasting blood samples were obtained to compare levels of glucose, total cholesterol, triacylglycerol, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, homocysteine, Lp-PLA2 activity, and high-sensitivity C-reactive protein (hs-CRP). Results Vegetarians had higher serum levels of the following markers: hs-CRP (1.8 ± 3.4 vs. 1.2 1.8 mg/L, respectively; p = 0.05), homocysteine (9.39 ± 3.22 vs. 7.62 ± 2.41 µmol/L, respectively; p < 0.01), and triacylglycerol (96.91 ± 59.56 vs. 84.66 ± 43.24 mg/dL, respectively; p < 0.05). Vegetarians also had lower levels of Lp-PLA2 (18.32 ± 7.19 10-3 µmol/min/mL vs. 20.22 8.13 10-3 µmol/min/mL; p < 0.05), total cholesterol (180.62 ± 36.55 mg/dL vs. 192.73 ± 36.57 mg/dL; p < 0.01), LDL cholesterol (118.15 ± 32.8 vs. 126.41 ± 34.28 mg/dL; p < 0.05), and HDL cholesterol (55.59 ± 13.30 vs. 62.09 ± 14.52 mg/dL, p < 0.01). Multivariate analyses demonstrated that a vegetarian diet increases the chances for high serum hs-CRP and low Lp-PLA2 activity. Conclusion In addition to lower total cholesterol, LDL-cholesterol, and HDL-cholesterol, Taiwanese female vegetarians have lower serum Lp-PLA2 activity but higher levels of hs-CRP, homocysteine, and triacylglyerol. It might be due to geographic differences of vegetarian diets, and further studies are needed. PMID:21155029

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

    PubMed

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

    2010-02-01

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

  12. 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

  13. Cloning and recombinant expression of a novel mouse-secreted phospholipase A2.

    PubMed

    Valentin, E; Koduri, R S; Scimeca, J C; Carle, G; Gelb, M H; Lazdunski, M; Lambeau, G

    1999-07-02

    Secreted phospholipases A2 (sPLA2s) form a class of structurally related enzymes that are involved in a variety of physiological and pathological effects including inflammation and associated diseases, cell proliferation, cell adhesion, and cancer, and are now known to bind to specific membrane receptors. Here, we report the cloning and expression of a novel sPLA2 isolated from mouse thymus. Based on its structural features, this sPLA2 is most similar to the previously cloned mouse group IIA sPLA2 (mGIIA sPLA2). As for mGIIA sPLA2, the novel sPLA2 is made up of 125 amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been mapped to mouse chromosome 4. However, the novel sPLA2 has only 48% identity with mGIIA and displays similar levels of identity with the other mouse group IIC and V sPLA2s, indicating that the novel sPLA2 is not an isoform of mGIIA sPLA2. This novel sPLA2 has thus been called mouse group IID (mGIID) sPLA2. In further contrast with mGIIA, which is found mainly in intestine, transcripts coding for mGIID sPLA2 are found in several tissues including pancreas, spleen, thymus, skin, lung, and ovary, suggesting distinct functions for the two enzymes. Recombinant expression of mGIID sPLA2 in Escherichia coli indicates that the cloned sPLA2 is an active enzyme that has much lower specific activity than mGIIA and displays a distinct specificity for binding to various phospholipid vesicles. Finally, recombinant mGIID sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA was previously found to bind to this receptor with high affinity.

  14. Surfactant protein B inhibits secretory phospholipase A2 hydrolysis of surfactant phospholipids

    PubMed Central

    Grier, Bonnie L.; Waite, B. Moseley; Veldhuizen, Ruud A.; Possmayer, Fred; Yao, Li-Juan; Seeds, Michael C.

    2012-01-01

    Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A2 (sPLA2) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA2 exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA2-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA2-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1–5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (DPPC:POPC:POPG, 40:40:20) prepared as vesicles and monomolecular films in the presence or absence of SP-B. Hydrolysis of PG and PC by Group IB sPLA2 (PLA2G1A) was significantly lower in the extracted OS, which contains SP-B, compared with Survanta (P = 0.005), which is SP-B poor. Hydrolysis of PG and PC in nonextracted OS, which contains all SPs, was lower than both Survanta and extracted OS. When Survanta was supplemented with 1% SP-B, PG and PC hydrolysis by PLA2G1B was significantly lower (P < 0.001) than in Survanta alone. When supplemented into pure lipid vesicles and monomolecular films composed of PG and PC mixtures, SP-B also inhibited hydrolysis by both PLA2G1B and Group IIA sPLA2 (PLA2G2A). In films, PLA2G1B hydrolyzed surfactant PL monolayers at surface pressures ≤30 mN/m (P < 0.01), and SP-B lowered the surface pressure range at which hydrolysis can occur. These results suggest the hydrophobic SP, SP-B, protects alveolar surfactant PL from

  15. Eosinophil Cysteinyl Leukotriene Synthesis Mediated by Exogenous Secreted Phospholipase A2 Group X*

    PubMed Central

    Lai, Ying; Oslund, Rob C.; Bollinger, James G.; Henderson, William R.; Santana, Luis F.; Altemeier, William A.; Gelb, Michael H.; Hallstrand, Teal S.

    2010-01-01

    Secreted phospholipase A2 group X (sPLA2-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C4, D4, and E4) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA2-X. We found that recombinant sPLA2-X caused marked AA release and a rapid onset of CysLT synthesis in human eosinophils that was blocked by a selective sPLA2-X inhibitor. Exogenous sPLA2-X released lysophospholipid species that arise from phospholipids enriched in AA in eosinophils, including phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine as well as plasmenyl phosphatidylcholine and phosphatidylethanolamine. CysLT synthesis mediated by sPLA2-X but not AA release could be suppressed by inhibition of cPLA2α. Exogenous sPLA2-X initiated Ser505 phosphorylation of cPLA2α, an intracellular Ca2+ flux, and translocation of cPLA2α and 5-lipoxygenase in eosinophils. Synthesis of CysLTs in response to sPLA2-X or lysophosphatidylcholine was inhibited by p38 or JNK inhibitors but not by a MEK 1/2 inhibitor. A further increase in CysLT synthesis was induced by the addition of sPLA2-X to eosinophils under conditions of N-formyl-methionyl-leucyl-phenylalanine-mediated cPLA2α activation. These results indicate that sPLA2-X participates in AA and lysophospholipid release, resulting in CysLT synthesis in eosinophils through a mechanism involving p38 and JNK MAPK, cPLA2α, and 5-lipoxygenase activation and resulting in the amplification of CysLT synthesis during cPLA2α activation. Transactivation of eosinophils by sPLA2-X may be an important mechanism leading to CysLT formation in the airways of patients with asthma. PMID:20974857

  16. “Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints

    PubMed Central

    Heinze, Michael; Brandt, Wolfgang; Marillonnet, Sylvestre; Roos, Werner

    2015-01-01

    The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins. PMID:25670767

  17. Design of specific peptide inhibitors of phospholipase A2: structure of a complex formed between Russell's viper phospholipase A2 and a designed peptide Leu-Ala-Ile-Tyr-Ser (LAIYS).

    PubMed

    Chandra, Vikas; Jasti, Jayasankar; Kaur, Punit; Dey, Sharmistha; Srinivasan, A; Betzel, Ch; Singh, T P

    2002-10-01

    Phospholipase A(2) (EC 3.1.1.4) is a key enzyme of the cascade mechanism involved in the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to membrane surfaces and the hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. In order to regulate the production of proinflammatory compounds, a specific peptide inhibitor of PLA(2), Leu-Ala-Ile-Tyr-Ser, has been designed. Phospholipase A(2) from Daboia russelli pulchella (DPLA(2)) and peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) have been co-crystallized. The structure of the complex has been determined and refined to 2.0 A resolution. The structure contains two crystallographically independent molecules of DPLA(2), with one molecule of peptide specifically bound to one of them. The overall conformations of the two molecules are essentially similar except in three regions; namely, the calcium-binding loop including Trp31 (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Of these, the most striking difference pertains to the orientation of Trp31 in the two molecules. The conformation of Trp31 in molecule A was suitable to allow the binding of peptide LAIYS, while that in molecule B prevented the entry of the ligand into the hydrophobic channel. The structure of the complex clearly showed that the OH group of Tyr of the inhibitor formed hydrogen bonds with both His48 N(delta1) and Asp49 O(delta1), while O(gamma)H of Ser was involved in a hydrogen bond with Trp31. Other peptide backbone atoms interact with protein through water molecules, while Leu, Ala and Ile form strong hydrophobic interactions with the residues of the hydrophobic channel.

  18. Group V Secretory Phospholipase A2 Is Involved in Tubular Integrity and Sodium Handling in the Kidney

    PubMed Central

    Moraes-Santos, Felipe; Landgraf, Sharon Schilling; Silva, Leandro Souza; Sirtoli, Gabriela Modenesi; Zamith-Miranda, Daniel; Takiya, Christina Maeda; Pinheiro, Ana Acacia Sá; Diaz, Bruno Lourenço; Caruso-Neves, Celso

    2016-01-01

    Group V (GV) phospholipase A2 (PLA2) is a member of the family of secreted PLA2 (sPLA2) enzymes. This enzyme has been identified in several organs, including the kidney. However, the physiologic role of GV sPLA2 in the maintenance of renal function remains unclear. We used mice lacking the gene encoding GV sPLA2 (Pla2g5−/−) and wild-type breeding pairs in the experiments. Mice were individually housed in metabolic cages and 48-h urine was collected for biochemical assays. Kidney samples were evaluated for glomerular morphology, renal fibrosis, and expression/activity of the (Na+ + K+)-ATPase α1 subunit. We observed that plasma creatinine levels were increased in Pla2g5−/− mice following by a decrease in creatinine clearance. The levels of urinary protein were higher in Pla2g5−/− mice than in the control group. Markers of tubular integrity and function such as γ-glutamyl transpeptidase, lactate dehydrogenase, and sodium excretion fraction (FENa+) were also increased in Pla2g5−/− mice. The increased FENa+ observed in Pla2g5−/− mice was correlated to alterations in cortical (Na+ + K+) ATPase activity/ expression. In addition, the kidney from Pla2g5−/− mice showed accumulation of matrix in corticomedullary glomeruli and tubulointerstitial fibrosis. These data suggest GV sPLA2 is involved in the maintenance of tubular cell function and integrity, promoting sodium retention through increased cortical (Na+ + K+)-ATPase expression and activity. PMID:26820468

  19. Effects of Biological Oxidants on the Catalytic Activity and Structure of Group VIA Phospholipase A2

    PubMed Central

    Song, Haowei; Bao, Shunzhong; Ramanadham, Sasanka; Turk, John

    2007-01-01

    Group VIA phospholipase A2 (iPLA2β) is expressed in phagocytes, vascular cells, pancreatic islet β-cells, neurons, and other cells and plays roles in transcriptional regulation, cell proliferation, apoptosis, secretion, and other events. A bromoenol lactone (BEL) suicide substrate used to study iPLA2β functions inactivates iPLA2β by alkylating Cys thiols. Because thiol redox reactions are important in signaling and some cells that express iPLA2β produce biological oxidants, iPLA2β might be subject to redox regulation. We report that biological concentrations of H2O2, NO, and HOCl inactivate iPLA2β, and this can be partially reversed by dithiothreitol (DTT). Oxidant-treated iPLA2β modifications were studied by LC–MS/MS analyses of tryptic digests and included DTT-reversible events, e.g., formation of disulfide bonds and sulfenic acids, and others not so reversed, e.g., formation of sulfonic acids, Trp oxides, and Met sulfoxides. W460 oxidation could cause irreversible inactivation because it is near the lipase consensus sequence (463GTSTG467), and site-directed mutagenesis of W460 yields active mutant enzymes that exhibit no DTT-irreversible oxidative inactivation. Cys651-sulfenic acid formation could be one DTT-reversible inactivation event because Cys651 modification correlates closely with activity loss and its mutagenesis reduces sensitivity to inhibition. Intermolecular disulfide bond formation might also cause reversible inactivation because oxidant-treated iPLA2β contains DTT-reducible oligomers, and oligomerization occurs with time- and temperature-dependent iPLA2β inactivation that is attenuated by DTT or ATP. Subjecting insulinoma cells to oxidative stress induces iPLA2β oligomerization, loss of activity, and subcellular redistribution and reduces the rate of release of arachidonate from phospholipids. These findings raise the possibility that redox reactions affect iPLA2β functions. PMID:16700550

  20. Anti-phospholipase A2 receptor antibody levels at diagnosis predicts spontaneous remission of idiopathic membranous nephropathy

    PubMed Central

    Jullien, Perrine; Seitz Polski, Barbara; Maillard, Nicolas; Thibaudin, Damien; Laurent, Blandine; Ollier, Edouard; Alamartine, Eric; Lambeau, Gérard

    2017-01-01

    Abstract Background: The diagnostic role of circulating anti-phospholipase A2 receptor antibodies (anti-PLA2R Abs) is now well recognized in idiopathic membranous nephropathy (iMN). These Abs could also be interesting as predictors of clinical outcome. In this study, we explored the prognostic value of anti-PLA2R Abs measured in a cohort of iMN patients, with a special focus on their ability to detect patients achieving spontaneous remission. Methods: All adult patients with biopsy-proven iMN diagnosed between 1978 and 2007 were retrospectively screened in our centre. Using a validated enzyme-linked immunosorbent assay, levels of anti-PLA2R Abs were measured from serum samples obtained at the time of renal biopsy and stored at −80°C until processing. Clinical data on disease activity, treatments and outcomes were collected by reviewing patients’ medical records. The association between anti-PLA2R Ab titres and clinical activity/outcome was assessed by Cox proportional hazard and Kaplan–Meier methods. Results: In this retrospective study, 68 patients were included in the final analysis (median follow-up of 81 months). No significant association was found between anti-PLA2R Ab titres at diagnosis with baseline proteinuria, baseline estimated glomerular filtration rate or chronic kidney disease progression. Spontaneous remission was observed in 22% of patients. Ab titres were significantly and gradually correlated in a dose–response manner with the likelihood of spontaneous remission. Conclusions: While Ab titres measured at diagnosis were not found to predict the activity of iMN, evaluation of anti-PLA2R Ab titres might prove useful in the early identification of patients likely to achieve spontaneous remission.

  1. High plasma phospholipase A2 activity, inflammation markers, and LDL alterations in obesity with or without type 2 diabetes.

    PubMed

    Garces, Fatima; López, Flor; Niño, Cladimar; Fernandez, Anazita; Chacin, Luis; Hurt-Camejo, Eva; Camejo, Germán; Apitz-Castro, Rafael

    2010-10-01

    Plasma phospholipases A(2) (PLA(2)) hydrolyze phospholipids of circulating lipoproteins or deposited in arteries producing bioactive lipids believed to contribute to the atherosclerotic inflammatory response. PLA(2)(s) are elevated in obesity and type 2 diabetes (T2D) but it is not clear which of these conditions is the cause since they frequently coexist. This study attempts to evaluate if high plasma PLA(2)(s) activities and markers of their effects in lipoproteins are associated with obesity or T2D diabetes, or with both. Total PLA(2) and Ca(2+)-dependent and -independent activities, lipids, lipoproteins, apoAI, and apoB apolipoproteins and affinity of apoB-lipoproteins for arterial proteoglycans were measured, as well as Inflammation markers. These parameters were evaluated in plasma samples of four groups: (i) apparently healthy controls with normal BMI (nBMI), (ii) obese subjects with no T2D, (iii) patients with T2D but with nBMI, and (iv) obese patients with T2D. PLA(2) activities were measured in the presence and absence of Ca(2+) and in the presence of specific inhibitors. Obese subjects, with or without T2D, had high activities of total PLA(2) and of Ca(2+)-dependent and Ca(2+)-independent enzymes. The activities were correlated with inflammation markers in obese subjects with and without diabetes and with alterations of low-density lipoproteins (LDLs) that increased their affinity for arterial proteoglycans. Ca(2+)-dependent secretory (sPLA(2)) enzymes were the main responsible of the obesity-associated high activity. We speculate that augmented PLA(2)(s) activity that increases affinity of circulating LDL for arterial intima proteoglycans could be another atherogenic component of obesity.

  2. Lipoprotein-associated phospholipase A2 levels are associated with erectile dysfunction in patients without known coronary artery disease.

    PubMed

    Otunctemur, A; Sahin, S; Ozbek, E; Cekmen, M; İnal, A; Tulubas, F; Dursun, M; Besiroglu, H; Koklu, I

    2015-08-01

    Endothelial dysfunction and microvascular damage play a crucial role in the pathogenesis of erectile dysfunction (ED). Lp-PLA2 is a calcium-independent member of the phospholipase A2 family and hydrolyses oxidised phospholipids on low-density lipoprotein (LDL) particles that plays a pivotal role in ox-LDL-induced endothelial dysfunction. The purpose of the current study was to determine the association between Lp-PLA2 levels and ED in patients without known coronary artery disease (CAD). All patients were evaluated for ED and divided into two groups: 88 patients suffering from ED for >1 year were enrolled as an experimental group and 88 patients without ED were enrolled as a control group in this study. Diagnosis of ED was based on the International Index of Erectile Function Score-5. Levels of Lp-PLA2 were measured in serum by colorimetric assay. The relationship between Lp-PLA2 levels and ED in patients was evaluated statistically. The mean age of patients with ED group was 59.4 ± 11.32 and 55.8 ± 9.67 in the control group. Plasma Lp-PLA2 levels were significantly higher in ED than in the control group (220.3 ± 66.90 and 174.8 ± 58.83 pg ml(-1) , respectively, P < 0.001). The Lp-PLA2 levels were negatively correlated with score of ED (r = -0.482, P < 0.05). In logistic regression analysis, enhanced plasma Lp-PLA2 levels result in approximately 1.2-fold increase in ED [1.22 (1.25-2.76)]. In this study, serum Lp-PLA2 levels were found to be associated with endothelial dysfunction predictive of ED. Serum Lp-PLA2 level appears to be a specific predictor of ED, and it may be used in early prediction of ED in the male population.

  3. SKELETAL MUSCLE GROUP VIA PHOSPHOLIPASE A2 (iPLA2β): EXPRESSION AND ROLE IN FATTY ACID OXIDATION†

    PubMed Central

    Carper, Michael J.; Zhang, Sheng; Turk, John; Ramanadham, Sasanka

    2009-01-01

    Among the phospholipases A2 (PLA2s) are the Group VI Ca2+-independent PLA2s (iPLA2s) and expression of multiple transcripts of iPLA2 in skeletal muscle has been reported. In the present study, phospholipase activity and sequential ATP and calmodulin affinity column chromatography analyses reveal that skeletal muscle iPLA2 exhibits properties characteristic of the iPLA2β isoform. The phospholipase activity of iPLA2β has been demonstrated to participate in signal transduction, cell proliferation, and apoptosis. We also report here that skeletal muscle from iPLA2β-null mice, relative to wild type muscle, exhibits a reduced capacity to oxidize palmitate but not palmitoyl-CoA or acetyl-CoA in the absence of changes in fatty acid transporters CD36 and CPT1 or β-hydroxyacyl-CoA dehydrogenase activity. Recently, purified iPLA2β was demonstrated to manifest a thioesterase activity which catalyzes hydrolysis of fatty acyl-CoAs. The liberated CoA-SH facilitates fatty acid transport into the mitochondria. In this regard, we find that fractions eluted from the ATP column and containing iPLA2β phospholipase activity also contained acyl-CoA thioesterase activity that was inhibited by the bromoenol lactone (BEL) suicide inhibitor of iPLA2β. We further find that acyl-CoA thioesterase activity in skeletal muscle preparations from iPLA2β-null mice is significantly reduced, relative to WT activity. These findings suggest that the absence of acyl-CoA thioesterase activity of iPLA2β can lead to reduced fatty acyl-CoA generation and impair fatty acid oxidation in iPLA2β-null mice. Our findings therefore reveal a novel function of iPLA2β, related not to its phospholipase activity but to its thioesterase activity, which contributes to optimal fatty acid oxidation in skeletal muscle. PMID:18937505

  4. Pulmonary toxicity of trichloroethylene: induction of changes in surfactant phospholipids and phospholipase A2 activity in the mouse lung.

    PubMed

    Scott, J E; Forkert, P G; Oulton, M; Rasmusson, M G; Temple, S; Fraser, M O; Whitefield, S

    1988-08-01

    Trichloroethylene (TCE) is a common organic solvent in use as a dry cleaning agent as well as an inhalant anesthetic. Nevertheless the effects of this material on the pulmonary surfactant which prevents alveolar collapse at maximal expiration is not known. Therefore, we have examined the effect of TCE on the intra- and extracellular surfactant pools and the activity of phospholipase A2, an enzyme which controls the remodeling of phosphatidylcholine to dipalmitoylphosphatidylcholine, the primary constituent of the pulmonary surfactant. Male CD-1 mice were treated ip with 2500 or 3000 mg/kg TCE. Twenty-four hours later mice were anesthetized and the lungs lavaged. Mice were then killed, the lungs perfused and excised, and subcellular fractions including lamellar bodies prepared. Some lungs were prepared for ultrastructural examination. Phospholipase A2 was assayed in all subcellular fractions. Phospholipid was assayed in the lavage (extracellular surfactant) and the lamellar bodies (intracellular surfactant). TCE (2500 mg/kg) caused selective exfoliation of Clara cells. However, only the dose of 3000 mg/kg TCE produced a significant decrease in the intracellular surfactant phospholipid. Minimal changes occurred in the phospholipid profiles. Phospholipase A2 specific activity was significantly decreased at both dosages within the lung microsomal fraction. In addition after treatment with 3000 mg/kg TCE the enzyme activity in the lamellar body fraction was significantly increased. These data suggest that inhalation of TCE may damage the enzymes which are responsible for synthesizing the pulmonary surfactant resulting in lower amounts of surfactant being stored and available for secretion into the alveolus.

  5. Ginger phenylpropanoids inhibit IL-1beta and prostanoid secretion and disrupt arachidonate-phospholipid remodeling by targeting phospholipases A2.

    PubMed

    Nievergelt, Andreas; Marazzi, Janine; Schoop, Roland; Altmann, Karl-Heinz; Gertsch, Jürg

    2011-10-15

    The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A(2) (iPLA(2))-triggered maturation and the cytosolic phospholipase A(2) (cPLA(2))-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA(2) assay, most major ginger phenylpropanoids directly inhibited i/cPLA(2) from U937 macrophages, but not hog pancreas secretory phospholipase A(2). The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA(2) and methyl arachidonyl fluorophosphonate for cPLA(2), with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE(2) and thromboxane B2 (>50%) and partially also leukotriene B(4) in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA(2) and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA(2), the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.

  6. The First Potent Inhibitor of Mammalian Group X Secreted Phospholipase A2: Elucidation of Sites for Enhanced Binding

    PubMed Central

    Smart, Brian P.; Oslund, Rob C.; Walsh, Laura A.; Gelb, Michael H.

    2010-01-01

    Using the X-ray structure of human group X secreted phospholipase A2 (hGX), we carried out structure-based design of indole-based inhibitors and prepared the compounds using a new synthetic route. The most potent compound inhibited hGX and the mouse orthologue with an IC50 of 75 nM. This compound is the most potent hGX inhibitor reported to date and was also found to inhibit a subset of the other mouse and human sPLA2s. PMID:16686528

  7. Crystallization and preliminary X-ray analysis of cardiotoxic phospholipase A2 from Ophiophagus hannah (king cobra).

    PubMed

    Wang, Z; Zhuang, M; Shu, Y; Zhang, H; Song, S; Lin, Z

    2001-05-01

    An acidic phospholipase A2 exhibiting cardiotoxicity, myotoxicity and anti-platelet activity was isolated from Ophiophagus hannah (king cobra) from Guangxi, China. It contains an unusual 'pancreatic loop'. The enzyme was purified to homogeneity and crystallized using polyethylene glycol and ethylene glycol as precipitants. The crystal belongs to space group C2, with unit-cell parameters a = 117.92, b = 62.94, c = 57.16 A, beta = 100.93 degrees. Diffraction data were collected to 2.6 A.

  8. Structural and Thermodynamic Characterization of Protein-Ligand Interactions Formed between Lipoprotein-Associated Phospholipase A2 and Inhibitors.

    PubMed

    Liu, Qiufeng; Chen, Xinde; Chen, Wuyan; Yuan, Xiaojing; Su, Haixia; Shen, Jianhua; Xu, Yechun

    2016-05-26

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) represents a promising therapeutic target for atherosclerosis and Alzheimer's disease. Here we reported the first crystal structures of Lp-PLA2 bound with reversible inhibitors and the thermodynamic characterization of complexes. High rigidity of Lp-PLA2 structure and similar binding modes of inhibitors with completely different scaffolds are revealed. It not only provides the molecular basis for inhibitory activity but also sheds light on the essential features of Lp-PLA2 recognition with reversible inhibitors.

  9. Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship.

    PubMed

    Elsbach, P; Weiss, J; Franson, R C; Beckerdite-Quagliata, S; Schneider, A; Harris, L

    1979-11-10

    Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible gram-negative bacteria and a phospholipase A2, have been purified to near homogeneity by ion exchange, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bactericidal/permeability-increasing protein has an approximate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bacterial species tested are insensitive to high concentrations of either rabbit or human protein. The phospholipase A2, purified by hydrophobic interaction chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity comparable to that of purified phospholipases A2 from other sources. Separation of the phospholipase A2 from the bactericidal/permeability-increasing protein has no noticeable effect on the bactericidal and permeability-increasing activities of the purified bactericidal protein, but removes the ability of the phospholipase A2 to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein, the phospholipase A2 regains its activity toward the phospholipids of intact E. coli suggesting that these two antibacterial leukocyte proteins act in concert.

  10. Lipoprotein-associated phospholipase A2 is associated with postpartum hypertension in women with history of preeclampsia.

    PubMed

    Zhou, Yuheng; Niu, Jianmin; Duan, Dongmei; Lei, Qiong; Wen, Jiying; Lin, Xiaohong; Lv, Lijuan; Chen, Longding

    2015-07-01

    Both hypertension and preeclampsia (PE) are considered as inflammatory diseases. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an inflammatory marker associated with lipid metabolism. We aimed to study the correlation and predictive value of Lp-PLA2 in postpartum hypertension after PE. A group of 160 PE patients (PE group) and a separate group of 160 normal pregnant women (control group) were recruited from January 2010 to October 2011. The average age in the PE group was 28.4 ± 4.5 years and the average gestational age was 34.7 ± 1.1 weeks. The average age in the control group was 27.8 ± 4.5 years and the average gestational age was 35.5 ± 1.2 weeks. General information (including age, gestational age, parity, history of metabolic disease, family history of high blood pressure, height, body weight before childbirth, and blood pressure) and blood samples were collected for measuring Lp-PLA2 and lipid parameters. From February to April in 2013, 153 cases in the PE group and 132 in the control group were re-called. We assessed their postpartum health, pregnancy, height, weight, and blood pressure. Serum mass of Lp-PLA2 in the PE group (210.67 ± 17.98 ng/mL) was significantly higher compared with that in the control group (174.72 ± 30.26 ng/mL) (P < 0.01). The pro-gestation BMI, systolic blood pressure (SBP), diastolic blood pressure, total cholesterol, triglyceride, and low-density lipoprotein-cholesterol (LDL-C) were also significantly higher. Correlation analysis showed that the level of Lp-PLA2 and SBP (r = 0.31), LDL-C (r = 0.37) were positively correlated. The incidence of postpartum hypertension in the PE group was higher than that in the normal control group. Logistic regression analysis showed that prenatal Lp-PLA2 mass was an independent risk factor for PE postpartum hypertension (OR 1.134,95 % CI 1.086-1.185). ROC curve analysis showed that the sensitivity of predicting postpartum hypertension was 73.2% and the specific degree was 86.6%, with

  11. Bilobalide, a constituent of Ginkgo biloba, inhibits NMDA-induced phospholipase A2 activation and phospholipid breakdown in rat hippocampus.

    PubMed

    Weichel, O; Hilgert, M; Chatterjee, S S; Lehr, M; Klein, J

    1999-12-01

    In rat hippocampal slices superfused with magnesium-free buffer, glutamate (1 mM) caused the release of large amounts of choline due to phospholipid breakdown. This phenomenon was mimicked by N-methyl-D-aspartate (NMDA) in a calcium-sensitive manner and was blocked by NMDA receptor antagonists such as MK-801 and 7-chlorokynurenate. The NMDA-induced release of choline was not caused by activation of phospholipase D but was mediated by phospholipase A2 (PLA2) activation as the release of choline was accompanied by the formation of lyso-phosphatidylcholine (lyso-PC) and glycerophospho-choline (GPCh) and was blocked by 5-[2-(2-carboxyethyl)-4-dodecanoyl-3,5-dimethylpyrrol-1-yl]pentano ic acid, a PLA2 inhibitor. Bilobalide, a constituent of Ginkgo biloba, inhibited the NMDA-induced efflux of choline with an IC50 value of 2.3 microM and also prevented the formation of lyso-PC and GPCh. NMDA also caused a release of choline in vivo when infused into the hippocampus of freely moving rats by retrograde dialysis. Again, the effect was completely inhibited by bilobalide which was administered systemically (20 mg/kg i.p.). Interestingly, convulsions which were observed in the NMDA-treated rats were almost totally suppressed by bilobalide. We conclude that release of choline is a sensitive marker for NMDA-induced phospholipase A2 activation and phospholipid breakdown. Bilobalide inhibited the glutamatergic excitotoxic membrane breakdown both in vitro and in vivo, an effect which may be beneficial in the treatment of brain hypoxia and/or neuronal hyperactivity.

  12. Increased phospholipase A2 and lyso-phosphatidylcholine levels are associated with surfactant dysfunction in lung contusion injury in mice.

    PubMed

    Machado-Aranda, David; Wang, Zhengdong; Yu, Bi; Suresh, M V; Notter, Robert H; Raghavendran, Krishnan

    2013-01-01

    Surfactant dysfunction is an important pathologic disturbance in various forms of acute inflammatory lung injury. Previously we reported the presence of marked alterations in the composition and activity of pulmonary surfactant in bilateral lung contusions (LC) injury induced by blunt trauma in rats. This is extended here to a mouse model of unilateral LC with a focus on compositional and functional changes in surfactant associated with permeability injury and increases in activity of secretory phospholipase A2. Surfactant-associated gene expression was not altered in mice with unilateral LC injury on the basis of Affymetrix analysis. LC mice had significant permeability injury with increased albumin and total protein in bronchoalveolar lavage at 5, 24, 48, and 72 hours after insult compared with uninjured controls. The percent content of large surfactant aggregates was depleted at all postinjury times, and pulmonary pressure-volume (P-V) mechanics and compliance were abnormal during this period. Surfactant dysfunction was evaluated in 24 hours, when permeability injury and P-V changes were most prominent. At this time, activity levels of secretory phospholipase A2 were increased in bronchoalveolar lavage, and chromatographic analysis showed that large surfactant aggregates had decreased levels of phosphatidylcholine and increased levels of lyso-phosphatidylcholine. These changes were accompanied by severe detriments in large aggregate surface activity by pulsating bubble surfactometry. Large aggregates from LC mice at 24 hours had minimum surface tensions of only 12.6 ± 1.1 mN/m after prolonged bubble pulsation (20 min) compared with 0.7 ± 0.03 mN/m for uninjured controls. These results document important detriments in the composition and activity of pulmonary surfactant in LC injury in mice and suggest that active synthetic phospholipase-resistant exogenous surfactants may have utility in treating surfactant dysfunction in this clinically important condition

  13. Increased phospholipase A2 and lyso-phosphatidylcholine levels are associated with surfactant dysfunction in lung contusion injury in mice

    PubMed Central

    Machado-Aranda, David; Wang, Zhengdong; Yu, Bi; Suresh, M V; Notter, Robert H.; Raghavendran, Krishnan

    2012-01-01

    Objective Surfactant dysfunction is an important pathological disturbance in various forms of acute inflammatory lung injury. Previously we reported the presence of significant alterations in the composition and activity of pulmonary surfactant in blunt trauma-induced bilateral lung contusion (LC) injury in rats. This is extended here to a mouse model of unilateral LC, with a focus on compositional and functional surfactant changes associated with permeability injury and increases in activity of secretory phospholipase A2. Results Surfactant-associated gene expression was not significantly altered in mice with unilateral LC injury based on Affymetrix analysis. LC mice had significant permeability injury with increased albumin and total protein in bronchoalveolar lavage (BAL) at 5, 24, 48 and 72 h post-insult compared to uninjured controls. The percent content of large surfactant aggregates was significantly depleted at all post-injury times, and pulmonary pressure-volume (P-V) mechanics and compliance were abnormal over this period. Surfactant dysfunction was evaluated in mechanistic detail at 24 h, when permeability injury and P-V changes were most prominent. At this time, activity levels of secretory phospholipase A2 (PLA2) were increased in BAL, and chromatographic analysis showed that large surfactant aggregates had decreased levels of phosphatidylcholine (PC) and increased levels of lyso-PC. These changes were accompanied by severe detriments in large aggregate surface activity by pulsating bubble surfactometry. Large aggregates from LC mice at 24 h had minimum surface tensions of only 12.6±1.1 mN/m after prolonged bubble pulsation (20 min) compared to 0.7±0.03 mN/m for uninjured controls. Conclusion These results document significant detriments in the composition and activity of pulmonary surfactant in LC injury in mice, and suggest that active synthetic phospholipase-resistant exogenous surfactants may have future utility in treating surfactant dysfunction

  14. Synthesis of mixed-chain phosphatidylcholines including coumarin fluorophores for FRET-based kinetic studies of phospholipase A(2) enzymes.

    PubMed

    Wang, Manlin; Pinnamaraju, Susmitha; Ranganathan, Radha; Hajdu, Joseph

    2013-01-01

    Phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the sn-2 ester linkage of glycerophospholipids to produce fatty acids and lysophospholipids. A significant number of mammalian phospholipases comprise a family of secreted PLA2 enzymes, found in specific tissues and cellular locations, exhibiting unique enzymatic properties and distinct biological functions. Development of new real-time spectrofluorimetric PLA2 assays should facilitate the kinetic characterization and mechanistic elucidation of the isozymes in vitro, with the potential applicability to detect and measure catalytic PLA2 activity in tissues and cellular locations. Here we report a new synthesis of double-labeled phosphatidylcholine analogs with chain-terminal reporter groups including coumarin fluorophores for fluorescence resonance energy transfer (FRET)-based kinetic studies of PLA2 enzymes. The use of coumarin derivatives as fluorescent labels provides reporter groups with substantially decreased size compared to the first generation of donor-acceptor pairs of fluorescent phospholipids. The key advantage of the design is to interfere less with the physicochemical properties of the acyl chains, thereby improving the substrate quality of the synthetic probes. In order to assess the impact of the fluorophore substituents on the catalytic hydrolysis and on the phospholipid packing in the lipid-water interface of the assay, we used the experimentally determined specific activity of bee-venom phospholipase A2 as a model for the secretory PLA2 enzymes. Specifically, the rate of PLA2 hydrolysis of the coumarin labeled phosphatidylcholine analogs was less than three times slower than the natural substrate dipalmitoyl phosphatidylcholine (DPPC) under the same experimental conditions. Furthermore, variation of the mole fraction of the synthetic phosphatidylcholine vs. that of the natural DPPC substrate showed nearly ideal mixing behavior in the phospholipid-surfactant aggregates of the assay. The

  15. 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

  16. 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.

  17. Auxins action on Glycine max secretory phospholipase A2 is mediated by the interfacial properties imposed by the phytohormones.

    PubMed

    Mariani, María Elisa; Madoery, Ricardo Román; Fidelio, Gerardo Daniel

    2015-07-01

    Secretory phospholipase A2 (sPLA2) are soluble enzymes that catalyze the conversion of phospholipids to lysophospholipids and free fatty acids at membrane interfaces. The effect of IAA and IPA auxins over the activity of recombinant sPLA2 isoforms from Glycine max was studied using membrane model systems including mixed micelles and Langmuir lipid monolayers. Both phytohormones stimulate the activity of both plant sPLA2 using DLPC/Triton mixed micelles as substrate. To elucidate the mechanism of action of the phytohormones, we showed that both auxins are able to self-penetrate lipid monolayers and cause an increment in surface pressure and an expansion of lipid/phytohormone mixed interfaces. The stimulating effect of auxins over phospholipase A2 activity was still present when using Langmuir mixed monolayers as organized substrate regardless of sPLA2 source (plant or animal). All the data suggest that the stimulating effect of auxins over sPLA2 is due to a more favorable interfacial environment rather to a direct effect over the enzyme. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2012-03-01

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

  19. Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes.

    PubMed

    Picelli, Carina G; Borges, Rafael J; Fernandes, Carlos A H; Matioli, Fabio M; Fernandes, Carla F C; Sobrinho, Juliana C; Holanda, Rudson J; Ozaki, Luiz S; Kayano, Anderson M; Calderon, Leonardo A; Fontes, Marcos R M; Stábeli, Rodrigo G; Soares, Andreimar M

    2017-10-01

    Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIβ and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107-116) may be responsible for PLA2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Activity of mammalian secreted phospholipase A(2) from inflammatory peritoneal fluid towards PEG-liposomes. Early indications.

    PubMed

    Vermehren, C; Jørgensen, K; Schiffelers, R; Frokjaer, S

    2001-02-19

    Due to an increase in the activity of phospholipase A(2) (PLA(2)) in various inflammatory diseases, this enzyme may play a key role in the degradation of liposomes and the subsequent release of drug when PEG-liposomes passively target inflammatory tissue. The activity of mammalian secreted phospholipase A(2) (sPLA(2)) in casein stimulated peritoneal fluid was tested toward liposomes of different compositions. Early results indicate only a slight degradation of conventional dipalmitoylphosphatidylcholine (DPPC) liposomes as well as DPPC liposomes incorporated with different concentrations of PEG(2000). However, the DPPC degradation increased to 7% when inclusion of 30 mol% phosphatidylethanolamine (PE) in the lipid bilayer. The increase in degradation may be due to an improvement of the substrate - as it is well known, that PE is a better substrate for the mammalian sPLA(2) than PC. Incorporation of PE into the bilayer may increase the binding properties of the bilayer resulting in improved conditions for the enzymatic attack by sPLA(2). In addition, inhibitory zones of Staphylococcus aureus in an agar diffusion test showed that PLA(2) from Crotalus atrox venom was able to catalyze the release of gentamicin from PEG-liposomes. In conclusion, this study suggest that degradation of the lipid bilayer of PEG-liposomes by PLA(2) result in release of incapsulated drug, e.g. gentamicin and inclusion of PE in the liposomal bilayer, may enhance the activity of the mammalian sPLA(2) toward liposomes composed of DPPC.

  1. Intracellular- and extracellular-derived Ca2+ influence phospholipase A2-mediated fatty acid release from brain phospholipids

    PubMed Central

    Rosa, Angelo O.; Rapoport, Stanley I.

    2009-01-01

    Summary Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca2+-dependent cytosolic phospholipase A2 (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca2+, even though iPLA2 has been thought to be Ca2+-independent. The source of Ca2+ for activation of cPLA2 is largely extracellular, whereas Ca2+ released from the endoplasmic reticulum can activate iPLA2 by a number of mechanisms. This review focuses on the role of Ca2+ in modulating cPLA2 and iPLA2 activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in brain, depending on the primary source of the Ca2+ signal. PMID:19327408

  2. Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa.

    PubMed

    Lessig, Jacqueline; Reibetanz, Uta; Arnhold, Jurgen; Glander, Hans-Jurgen

    2008-11-01

    To determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. Although sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality. The ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality. (c) 2008, Asian Journal of Andrology, SIMM and SJTU. All rights reserved.

  3. Lipoprotein-Associated Phospholipase A2 Is an Independent Predictor of Incident Coronary Heart Disease in an Apparently Healthy Older Population

    PubMed Central

    Daniels, Lori B.; Laughlin, Gail A.; Sarno, Mark J.; Bettencourt, Ricki; Wolfert, Robert L.; Barrett-Connor, Elizabeth

    2011-01-01

    Objectives Lipoprotein-associated phospholipase A2 (Lp-PLA2) levels predict incident coronary heart disease (CHD) in adults without known CHD, independent of heart disease risk factors. We examined whether the independent association was apparent in older adults. Background Serum levels of Lp-PLA2, an enzyme that hydrolyzes oxidized phospholipids to yield potentially proatherogenic particles, have been associated with CHD and may help predict cardiovascular risk. Methods Participants were 1,077 community-dwelling men and women, median age 72 years, who had no known CHD at baseline (1984 to 1987) when blood samples and risk factor data were collected. Participants were followed for CHD events for a mean of 16 years, through 2002. Cox proportional hazards regression models were used to examine the association of serum Lp-PLA2 with incident CHD (myocardial infarction, angina, or coronary revascularization). Results The Lp-PLA2 levels positively correlated with age (r = 0.09), body mass index (r = 0.11), low-density lipoprotein (r = 0.37), triglycerides (r = 0.25), and C-reactive protein (r = 0.10), and negatively correlated with high-density lipoprotein (r = −0.27) (all p < 0.05). During follow-up, 228 participants had incident CHD events. Lipoprotein-associated phospholipase A2 levels in the second, third, and fourth quartiles predicted an increased risk of CHD compared with the lowest quartile (hazard ratios 1.66, 1.80, and 1.89, respectively; p < 0.05 for each). This association persisted after adjusting for C-reactive protein and other CHD risk factors. Conclusions Elevated Lp-PLA2 levels predict CHD events in apparently healthy older adults, independent of CHD risk factors. PMID:18308160

  4. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. 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

  6. Surface Dilution Kinetics Using Substrate Analog-Enantiomers as Diluents: Enzymatic Lipolysis by Bee-Venom Phospholipase A2

    PubMed Central

    Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

    2010-01-01

    A novel assay employing D-enantiomers of phospholipids as diluents for characterizing surface kinetics of lipid hydrolysis by phospholipases is introduced. The rationale of the method are: (i) D-enantiomers resist hydrolysis because of the stereoselectivity of the enzymes toward L-enantiomers and (ii) mixtures of L+D-lipids at various L:D ratios but constant L+D-lipid concentrations yield a surface dilution series of variable L-lipid concentration with constant medium properties. Kinetic characterization of bee-venom phospholipase A2 activity at bile salt + phospholipid aggregate-water interfaces was performed using the mixed L+D-lipid surface dilution assay and interface kinetic parameters were obtained. The assay applies to bio-membrane models as well. Activity was measured by pH-Stat methods. Aggregation numbers and interface hydration/microviscosity measured by time resolved fluorescence quenching and electron spin resonance respectively confirmed that interface properties were indeed invariant in a surface dilution series, supporting rationale (ii) and were used to calculate substrate concentrations. Activity data show excellent agreement with a kinetic model derived with D-enantiomers as diluents and also that D-phospholipids bind to the enzyme but resist hydrolysis; underscoring rationale (i). The assay is significant to enabling determination of interface specific kinetic parameters for the first time and thereby characterization of interface specificity of lipolytic enzymes. PMID:20727845

  7. Identification of the Major Ubiquitin-binding Domain of the Pseudomonas aeruginosa ExoU A2 Phospholipase*

    PubMed Central

    Anderson, David M.; Feix, Jimmy B.; Monroe, Andrew L.; Peterson, Francis C.; Volkman, Brian F.; Haas, Arthur L.; Frank, Dara W.

    2013-01-01

    Numerous Gram-negative bacterial pathogens use type III secretion systems to deliver effector molecules into the cytoplasm of a host cell. Many of these effectors have evolved to manipulate the host ubiquitin system to alter host cell physiology or the location, stability, or function of the effector itself. ExoU is a potent A2 phospholipase used by Pseudomonas aeruginosa to destroy membranes of infected cells. The enzyme is held in an inactive state inside of the bacterium due to the absence of a required eukaryotic activator, which was recently identified as ubiquitin. This study sought to identify the region of ExoU required to mediate this interaction and determine the properties of ubiquitin important for binding, ExoU activation, or both. Biochemical and biophysical approaches were used to map the ubiquitin-binding domain to a C-terminal four-helix bundle of ExoU. The hydrophobic patch of ubiquitin is required for full binding affinity and activation. Binding and activation were uncoupled by introducing an L8R substitution in ubiquitin. Purified L8R demonstrated a parental binding phenotype to ExoU but did not activate the phospholipase in vitro. Utilizing these new biochemical data and intermolecular distance measurements by double electron-electron resonance, we propose a model for an ExoU-monoubiquitin complex. PMID:23908356

  8. 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

  9. 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

  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

    2014-10-02

    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.

  11. Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

    PubMed

    Furtado, Juliana L; Oliveira, George A; Pontes, Adriana S; Setúbal, Sulamita da S; Xavier, Caroline V; Lacouth-Silva, Fabianne; Lima, Beatriz F; Zaqueo, Kayena D; Kayano, Anderson M; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Zuliani, Juliana P

    2014-01-01

    In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

  12. Polyunsaturated omega-3 fatty acids reduce lipoprotein-associated phospholipase A(2) in patients with stable angina.

    PubMed

    Gajos, Grzegorz; Zalewski, Jaroslaw; Mostowik, Magdalena; Konduracka, Ewa; Nessler, Jadwiga; Undas, Anetta

    2014-04-01

    Increased consumption of omega-3 polyunsaturated fatty acids (PUFA) together with lifestyle measures and medications is recommended for the prevention of cardiovascular diseases. However, the exact mechanisms underlying observed benefits are not well defined. To this aim, we evaluated the effects of omega-3 PUFA in stable coronary artery disease (CAD) patients undergoing percutaneous coronary intervention (PCI) on lipoprotein associated phospholipase A2 (Lp-PLA2) mass and activity and their relation to oxidized low-density lipoproteins (oxy-LDL). In a prospective, double-blind, placebo-controlled, randomized study Lp-PLA2, oxy-LDL, myeloperoxidase and interleukin-6 were determined at baseline, 3-5 days and 30 days during administration of omega-3 PUFA 1 g/day (n = 30) or placebo (n = 24). Treatment with omega-3 PUFA resulted in reduction of Lp-PLA2 mass by 10.7%, activity by 9.3 (p = 0.026 for both) and oxy-LDL by 10.9% (p = 0.014) at 30 days, with no change in myeloperoxidase and interleukin-6. Compared with placebo, patients receiving omega-3 PUFA had lower Lp-PLA2 mass by 9.42%, activity by 9.2 (p = 0.041 for both) and oxy-LDL by 12.3% (p = 0.10) after one month, but not at 3-5 days. There were no correlations between Lp-PLA2 and both myeloperoxidase and oxy-LDL throughout the study. The multivariate model showed that only treatment with omega-3 PUFA and baseline myeloperoxidase levels were independent predictors of Lp-PLA2 mass changes at one month (R(2) = 0.37, P = 0.005). Administration of omega-3 PUFA can decrease Lp-PLA2 in patients with stable angina undergoing PCI. This novel effect may contribute to the benefits derived from omega-3 PUF. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Kinetics of C-reactive protein, interleukin-6 and -10, and phospholipase A2-II in severely traumatized septic patients.

    PubMed

    Lausević, Zeljko; Vuković, Goran; Stojimirović, Biljana; Trbojević-Stanković, Jasna; Resanović, Vladimir; Lausevic, Mirjana

    2010-11-01

    Injury-induced anergy is one of the key factors contributing to trauma victims' high susceptibility to sepsis. This group of patients is mostly of young age and it is therefore essential to be able to predict as accurately as possible the development of septic complications, so appropriate treatment could be provided. The aim of this study was to assess kinetics of interleukin (IL)-6 and -10, phospholipase A2-II and C-reactive protein (CRP) in severely traumatized patients and explore the possibilities for early detection of potentially septic patients. This prospective study included 65 traumatized patients with injury severity score (ISS) > 18, requiring treatment at surgical intensive care units, divided into two groups: 24 patients without sepsis and 41 patients with sepsis. C-reactive protein, IL-6 and -10 and phospholipase A2 group II, were determined within the first 24 hours, and on the second, third and seventh day of hospitalization. Mean values of IL-6 and phospholipase A2-II in the patients with and without sepsis did not show a statistically significant difference on any assessed time points. In the septic patients with ISS 29-35 and > 35 on the days two and seven a statistically significantly lower level of IL-10 was found, compared with those without sepsis and with the same ISS. C-reactive protein levels were significantly higher in septic patients with ISS 18-28 on the first day. On the second, third and seventh day CRP levels were significantly lower in the groups of septic patients with ISS 29-35 and > 35, than in those with the same ISS but without sepsis. Mean levels of CRP on the first day after the injury may be useful predictor of sepsis development in traumatized patients with ISS score 18-28. Mean levels of CRP on the days two, three and seven after the injury may be a useful predictor of sepsis development in traumatized patients with ISS score more than 28. Mean levels of IL-10 on the second and seventh day after the injury may be a

  14. 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.

  15. Functional and Structural Characterization of a Thermostable Phospholipase A2 from a Sparidae Fish (Diplodus annularis).

    PubMed

    Smichi, Nabil; Othman, Houcemeddine; Achouri, Neila; Noiriel, Alexandre; Arondel, Vincent; Srairi-Abid, Najet; Abousalham, Abdelkarim; Gargouri, Youssef; Miled, Nabil; Fendri, Ahmed

    2017-03-22

    Novel phospholipase (PLA2) genes from the Sparidae family were cloned. The sequenced PLA2 revealed an identity with pancreatic PLA2 group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA2 (DaPLA2) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA2 was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA2 was constructed to gain insights into the functional properties of sparidae PLA2. Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA2. Using the monolayer technique, the purified DaPLA2 was found to be active on various phospholipids ranging from 10 to 20 mN·m(-1), which explained the absence of the hemolytic activity for DaPLA2.

  16. Group IVA phospholipase A(2) deficiency prevents CCl4-induced hepatic cell death through the enhancement of autophagy.

    PubMed

    Ishihara, Keiichi; Kanai, Shiho; Tanaka, Kikuko; Kawashita, Eri; Akiba, Satoshi

    2016-02-26

    Group IVA phospholipase A2 (IVA-PLA2), which generates arachidonate, plays a role in inflammation. IVA-PLA2-deficiency reduced hepatotoxicity and hepatocyte cell death in mice that received a single dose of carbon tetrachloride (CCl4) without any inhibitory effects on CCl4-induced lipid peroxidation. An immunoblot analysis of extracts from wild-type mouse- and IVA-PLA2 KO mouse-derived primary hepatocytes that transiently expressed microtubule-associated protein 1 light chain 3B (LC3) revealed a higher amount of LC3-II, a typical index of autophagosome formation, in IVA-PLA2-deficient cells, suggesting the enhancement of constitutive autophagy. IVA-PLA2 may promote CCl4-induced cell death through the suppression of constitutive autophagy in hepatocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Amino acid sequence of a neurotoxic phospholipase A2 enzyme from common death adder (Acanthophis antracticus) venom.

    PubMed

    van der Weyden, L; Hains, P; Broady, K; Shaw, D; Milburn, P

    2001-02-01

    The amino acid sequence of the first neurotoxic phospholipase A2, acanthoxin A1, purified from the venom of the Common death adder (Acanthophis antarcticus) was determined. Acanthoxin A1 shows high homology with other Australian elapid PLA2 neurotoxins, in particular Acanthin-I and -II, also from Death adder, Pseudexin A from the Red-bellied black snake (Pseudechis porphyriacus), and Pa-12a and Pa-9c from the King brown snake (Pseudechis australis). Acanthoxin A1 is a single-chain 118 amino acid residue PLA2, including 14 half cystine residues and the essential residues forming the ubiquitous calcium binding pocket and catalytic site. Critical analysis of the residues hypothesized to be important for neurotoxicity is presented.

  18. 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.

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

    PubMed

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

    2014-10-22

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

  20. Inhibition of vacuolar adenosine triphosphatase antagonizes the effects of clostridial neurotoxins but not phospholipase A2 neurotoxins.

    PubMed

    Simpson, L L; Coffield, J A; Bakry, N

    1994-04-01

    Bafilomycin A1, an inhibitor of vacuolar adenosine triphosphatase, was tested for its ability to antagonize botulinum neurotoxins (serotypes A-G), tetanus toxin and phospholipase A2 neurotoxins (notexin, beta-bungarotoxin, taipoxin and textilotoxin) on the mouse phrenic nerve-hemidiaphragm preparation. Bafilomycin itself produced concentration-dependent blockade of neuromuscular transmission without blocking nerve action potentials or muscle action potentials. This effect may have been due to inhibition of the proton pump that regulates acetylcholine transport into vesicles. At submaximal concentrations, bafilomycin was very effective in delaying the onset of paralysis due to all clostridial neurotoxins, but it had no protective effect against phospholipase A2 neurotoxins. Experiments were done to determine which of the three steps in clostridial neurotoxin action was antagonized by bafilomycin (e.g., binding, internalization and intracellular poisoning). Both pharmacological experiments and ligand-binding experiments showed that the drug did not block toxin binding to the plasma membrane. Similarly, pharmacological experiments on the time-dependent effects of bafilomycin showed that the drug did not antagonize the intracellular actions of toxins. The data indicated that bafilomycin acted at the intermediate step of internalization. This is in keeping with the facts that: 1) bafilomycin inhibits vacuolar adenosine triphosphatase, which in turn leads to inhibition of acidification in endosomes and 2) clostridial neurotoxins depend upon acidification of endosomes for translocation to the cytosol. The finding that bafilomycin antagonizes tetanus toxin may provide important clues for understanding how this toxin can act locally to produce flaccid paralysis. The finding that bafilomycin is a universal antagonist that protects against all clostridial neurotoxins may have important implications for developing therapeutic drugs.

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

    PubMed

    Franson, R C; Rosenthal, M D

    1997-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2.

    PubMed

    Ji, Jung Eun; Kim, Seok Kyun; Ahn, Kyong Hoon; Choi, Jong Min; Jung, Sung Yun; Jung, Kwang Mook; Jeon, Hyung Jun; Kim, Dae Kyong

    2011-04-01

    Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2). Copyright © 2011. Published by Elsevier Inc.

  5. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. 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.

  7. Polycyclic aromatic hydrocarbons present in cigarette smoke cause endothelial cell apoptosis by a phospholipase A2-dependent mechanism.

    PubMed

    Tithof, Patricia K; Elgayyar, Mona; Cho, Yeesook; Guan, Wei; Fisher, Aron B; Peters-Golden, Marc

    2002-09-01

    Smoking is a major risk factor for endothelial cell injury and subsequent coronary artery disease. Epidemiological studies implicate the phospholipase A2/arachidonic acid cascade in the mechanism by which smoking causes heart disease. However, specific components of cigarette smoke that activate this pathway have not been identified. The purpose of this study was to investigate the effects of polycyclic aromatic hydrocarbons contained in cigarette smoke on phospholipase A2 (PLA2) activity and apoptosis of human coronary artery endothelial cells. 1-methylanthracene (1-MA), phenanthrene (PA), and benzo(a)pyrene (B(a)P) caused significant release of 3H-arachidonate from endothelial cells. 1-MA and PA, but not B(a)P, also caused significant release of 3H-linoleic acid. Release of fatty acids from membrane phospholipids preceded the onset of apoptosis. 3H-arachidonate release and apoptosis induced by 1-MA, B(a)P, and PA were inhibited by methylarachidonoyl-fluorophosphonate, an inhibitor of Groups IV and VI PLA2s. Bromoenol lactone, an inhibitor of Group VI enzymes, inhibited both 3H-arachidonate release and apoptosis induced by 1-MA and PA, but not B(a)P. MJ33, an inhibitor of the acidic calcium-independent PLA2, attenuated 3H-arachidonate release and apoptosis by PA, but not 1-MA or B(a)P. The presence of Groups IV and VI and the acidic iPLA2 in endothelial cells was demonstrated by reverse transcriptase-polymerase chain reaction and Western analysis. These data suggest that 1-MA, B(a)P and PA induce apoptosis of endothelial cells by a mechanism that involves activation of these three distinct isoforms of PLA2.

  8. Increased activity of group II phospholipase A2 in plasma in rat sodium deoxycholate induced acute pancreatitis

    PubMed Central

    Furue, S; Hori, Y; Kuwabara, K; Ikeuchi, J; Onoyama, H; Yamamoto, M; Tanaka, K

    1997-01-01

    Background—Two different types of secretory phospholipase A2 (PLA2), pancreatic group I (PLA2-I) and non-pancreatic group II (PLA2-II), have been identified and postulated to be associated with the pathogenesis of various diseases, such as acute pancreatitis, septic shock, and multiple organ failure. 
Aims—To investigate the type of secretory PLA2 responsible for its catalytic activity found in plasma and ascites of experimental acute pancreatitis. 
Methods—Acute pancreatitis of differing severity was induced by the injection of different concentrations (1% or 10%) of sodium deoxycholate (DCA) into the common biliopancreatic duct in rats, and catalytic PLA2 activity in plasma and ascites were differentiated by anti-PLA2-I antibody and specific inhibitor of PLA2-II. Survival rate and plasma amylase, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were also measured.
Results—In 1% and 10% DCA induced acute pancreatitis, plasma amylase values as well as PLA2 activity in ascites were greatly increased. PLA2 activity in plasma was also notably increased in 10% DCA induced acute pancreatitis, but not in 1% DCA induced acute pancreatitis. PLA2-I specific polyclonal antibody significantly inhibited PLA2 activity in ascites but not that in plasma. In contrast, plasma PLA2 activity was completely suppressed by PLA2-II specific inhibitor. In addition, a high mortality (93% at five hours) and a significant increase in plasma AST and ALT were noted in 10% DCA induced pancreatitis. 
Conclusion—Ascites PLA2 activity is mainly derived from PLA2-I, whereas plasma PLA2 activity is mostly derived from PLA2-II in severe acute pancreatitis, suggesting that increased plasma PLA2-II activity might be implicated in hepatic failure arising after severe acute pancreatitis. 

 Keywords: acute pancreatitis; phospholipase A2; sodium deoxycholate pancreatitis; hepatic failure PMID:9462218

  9. Cholinergic and glutamatergic alterations beginning at the early stages of Alzheimer disease: participation of the phospholipase A2 enzyme.

    PubMed

    Schaeffer, Evelin L; Gattaz, Wagner F

    2008-05-01

    Alzheimer disease (AD), a progressive neurodegenerative disorder, is the leading cause of dementia in the elderly. A combination of cholinergic and glutamatergic dysfunction appears to underlie the symptomatology of AD, and thus, treatment strategies should address impairments in both systems. Evidence suggests the involvement of phospholipase A(2) (PLA(2)) enzyme in memory impairment and neurodegeneration in AD via actions on both cholinergic and glutamatergic systems. To review cholinergic and glutamatergic alterations underlying cognitive impairment and neuropathology in AD and attempt to link PLA(2) with such alterations. Medline databases were searched (no date restrictions) for published articles with links among the terms Alzheimer disease (mild, moderate, severe), mild cognitive impairment, choline acetyltransferase, acetylcholinesterase, NGF, NGF receptor, muscarinic receptor, nicotinic receptor, NMDA, AMPA, metabotropic glutamate receptor, atrophy, glucose metabolism, phospholipid metabolism, sphingolipid, membrane fluidity, phospholipase A(2), arachidonic acid, attention, memory, long-term potentiation, beta-amyloid, tau, inflammation, and reactive species. Reference lists of the identified articles were checked to identify additional studies of interest. Overall, results suggest the hypothesis that persistent inhibition of cPLA(2) and iPLA(2) isoforms at early stages of AD may play a central role in memory deficits and beta-amyloid production through down-regulation of cholinergic and glutamate receptors. As the disease progresses, beta-amyloid induced up-regulation of cPLA(2) and sPLA(2) isoforms may play critical roles in inflammation and oxidative stress, thus participating in the neurodegenerative process. Activation and inhibition of specific PLA(2) isoforms at different stages of AD could be of therapeutic importance and delay cognitive dysfunction and neurodegeneration.

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

    PubMed

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

    1997-07-25

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

  11. 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.

  12. Activation of J77A.1 Macrophages by Three Phospholipases A2 Isolated from Bothrops atrox Snake Venom

    PubMed Central

    Furtado, Juliana L.; Oliveira, George A.; Pontes, Adriana S.; Setúbal, Sulamita da S.; Xavier, Caroline V.; Lacouth-Silva, Fabianne; Lima, Beatriz F.; Zaqueo, Kayena D.; Kayano, Anderson M.; Calderon, Leonardo A.; Stábeli, Rodrigo G.; Soares, Andreimar M.; Zuliani, Juliana P.

    2014-01-01

    In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects. PMID:24592395

  13. Effect of phospholipid hydrolysis by phospholipase A2 on the kinetics of antagonist binding to cardiac muscarinic receptors.

    PubMed

    Rauch, B; Niroomand, F; Messineo, F C; Weis, A; Kübler, W; Hasselbach, W

    1994-09-15

    Activation of phospholipases during prolonged myocardial ischemia could contribute to the functional derangement of myocardial cells by altering the phospholipid environment of a number of membrane bound proteins including receptors. The present study examined the kinetics of muscarinic receptor antagonist [3H]quinuclidinyl benzilate binding ([3H]QNB) to muscarinic receptors of highly purified sarcolemmal membranes under control conditions and after treatment with phospholipase A2 (PLA2; EC 3.1.1.4). Initial binding rates of QNB exhibited saturation kinetics, when plotted against the ligand concentration in control and PLA2 treated sarcolemmal membranes. This kinetic behaviour of QNB-binding is consistent with at least a two step binding mechanism. According to this two step binding hypothesis an unstable intermediate receptor-QNB complex (R*QNB) forms rapidly, and this form undergoes a slow conversion to the high affinity ligand-receptor complex R-QNB. The Michaelis constant Km of R-QNB formation was 1.8 nM, whereas the dissociation constant Kd obtained from equilibrium measurements was 0.062 nM. After 5 min exposure of sarcolemmal membranes to PLA2QNB binding capacity (Bmax) was reduced by 62%, and the affinity of the remaining receptor sites was decreased by 47% (Kd = 0.116 nM). This PLA2-induced increase of Kd was accompanied by a corresponding increase of Km, whereas the rate constants k2 and k-2 of the hypothetical slow conversion step (second reaction step) remained unchanged. These results suggest that binding of QNB to cardiac muscarinic receptors induces a transition in the receptor-ligand configuration, which is necessary for the formation of the final high affinity R-QNB complex. PLA2-induced changes of the lipid environment result in the inability of a part of the receptor population to undergo this transition, thereby inhibiting high affinity QNB-binding.

  14. 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

  15. Effects of phospholipase A2 inhibitors on coupling of alpha 2-adrenoceptors to inwardly rectifying potassium currents in guinea-pig submucosal neurones.

    PubMed Central

    Evans, R. J.; Surprenant, A.

    1993-01-01

    1. Noradrenaline hyperpolarizes guinea-pig submucosal neurones by opening inwardly rectifying potassium channels. Intracellular recordings were made from submucosal neurones and the possible involvement of the phospholipase A2 pathway in this response was examined. 2. The non-specific phospholipase A2 inhibitors, quinacrine (10 microM) and 4-bromophenacyl bromide (4-BPB, 10 microM) inhibited nerve-evoked inhibitory synaptic potentials (i.p.s.ps) and hyperpolarizations to somatostatin and UK 14304. Quinacrine and 4-BPB also blocked the inward rectification present in current-voltage curves in the absence of somatostatin or UK 14304. 3. The more selective phospholipase A2 inhibitor, cyclosporin A (10 microM) and the lipoxygenase and cyclo-oxygenase inhibitor, eicosatetraynoic acid (ETYA, 20 microM) and nordihydroguairetic acid (NDGA, 20 microM) did not alter i.p.s.ps or hyperpolarizations to UK 14304. 4. Exogenously applied arachidonic acid (1-300 microM) did not mimic the i.p.s.p. or the hyperpolarization to UK 14304. 5. We conclude that arachidonic acid or its eicosanoid metabolites produced by phospholipase A2 stimulation are unlikely to be involved in the receptor G-protein coupled activation of potassium currents in submucosal neurones. The inhibition of the noradrenaline-induced hyperpolarization by quinacrine and 4-BPB is most likely due primarily to blockade of the basal inwardly rectifying potassium conductance present in these neurones. PMID:7902174

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2012-05-01

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

  18. Inhibition of Cytosolic Phospholipase A2α (cPLA2α) by Medicinal Plants in Relation to Their Phenolic Content.

    PubMed

    Arnold, Eva; Benz, Thorsten; Zapp, Cornelia; Wink, Michael

    2015-08-17

    The cytosolic phospholipase A2α(cPLA2α) is one of the potential targets for anti-inflammatory drugs, since this enzyme plays a key role in the inflammation processes seen in health disorders, like asthma, allergic reactions, arthritis and neuronal diseases. In this study, cPLA2α inhibition by 43 methanol extracts from medicinal plants rich in polyphenols was determined. The eight most active extracts were derived from Ribes nigrum (IC50 of 27.7 μg/mL), Ononis spinosa (IC50 of 39.4 μg/mL), Urtica dioica (IC50 of 44.32 μg/mL), Betula sp. (IC50 of 58.02 μg/mL), Sanguisorba officinalis (IC50 of 76.25 μg/mL), Orthosiphon stamineus (IC50 of 78.83 μg/mL), Petasites hybridus (IC50 of 81.02 μg/mL) and Tussilago farfara (IC50 of 123.28 μg/mL). Additionally, the antioxidant activities of these extracts were determined with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and their phenolic content with the Folin-Ciocalteu reagent. Antioxidant activity showed a non-linear, positive correlation to the phenolic content, but no correlation of PLA2 inhibition with phenolic content could be established. This study provides evidence that cPLA2α may be a relevant target for anti-inflammatory agents.

  19. 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

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

    PubMed Central

    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

    2016-01-01

    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

  1. The significance of the altered expression of lysophosphatidic acid receptors, autotaxin and phospholipase A2 as the potential biomarkers in type 1 endometrial cancer biology.

    PubMed

    Wasniewski, Tomasz; Woclawek-Potocka, Izabela; Boruszewska, Dorota; Kowalczyk-Zieba, Ilona; Sinderewicz, Emilia; Grycmacher, Katarzyna

    2015-11-01

    In order to study lysophosphatidic acid (LPA) signaling associated with type 1 endometrial carcinoma (EC), we evaluated the LPA receptors (LPARs), autotaxin (ATX) and phospholipase A2 (PLA2) expression in EC and normal endometrium with correlation to clinicopathological features. We investigated LPAR1, LPAR2, LPAR3, LPAR4, ATX and PLA2 expression at mRNA and protein levels using quantitative real-time PCR and western blot analyses in 37 ECs and 10 normal endometria. All the examined LPARs (except for LPAR3 protein), ATX and PLA2 were overexpressed in cancerous compared to healthy endometrium. The studied ECs showed the highest LPAR2 and LPAR1 expression. Statistically positive correlations were found between depth of myoinvasion and levels of LPAR1, LPAR2 and PLA2 transcripts and proteins. We also found positive correlations between LPAR1, LPAR2, LPAR4 and PLA2 expression with the International Federation of Gynecology and Obstetrics (FIGO) stage. The expression of LPAR1, LPAR2 and PLA2 was positively associated with the age of patients. Positive correlations were found between the expression of LPAR1 mRNA, LPAR2 mRNA and protein and LPAR3 mRNA and body mass index (BMI) of the examined patients. We found no association between the expression levels of the studied factors and diabetes or hypertension among the examined patients. Owing to the highest LPAR2 and LPAR1 expression in EC and positive correlations of these two receptors with the depth of myoinvasion and the FIGO stage, we believe that LPAR2 and LPAR1 show promise as predictors of the EC progression as well as the main receptors responsible for LPA action in the EC tissue.

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

    PubMed

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

    1999-03-01

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

  3. 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

  4. Divergent functional profiles of acidic and basic phospholipases A2 in the venom of the snake Porthidium lansbergii lansbergii.

    PubMed

    Jiménez-Charris, Eliécer; Montealegre-Sánchez, Leonel; Solano-Redondo, Luis; Castro-Herrera, Fernando; Fierro-Pérez, Leonardo; Lomonte, Bruno

    2016-09-01

    The Lansberg's hognose pitviper, Porthidium lansbergii lansbergii, inhabits northern Colombia. A recent proteomic characterization of its venom (J. Proteomics [2015] 114, 287-299) revealed the presence of phospholipases A2 (PLA2) accounting for 16.2% of its proteins. The two most abundant PLA2s were biochemically and functionally characterized. Pllans-I is a basic, dimeric enzyme with a monomer mass of 14,136 Da, while Pllans-II is an acidic, monomeric enzyme of 13,901 Da. Both have Asp49 in their partial amino acid sequences and, accordingly, are catalytically active upon natural or synthetic substrates. Nevertheless, these two enzymes differ markedly in their bioactivities. Pllans-I induces myonecrosis, edema, and is lethal by intracerebro-ventricular injection in mice, as well as cytolytic and anticoagulant in vitro. In contrast, Pllans-II is devoid of these effects, except for the induction of a moderate edema. In spite of lacking myotoxicity, Pllans-II enhances the muscle damaging action of Pllans-I in vivo. Altogether, results further illustrate the divergent functional profiles of basic and acidic PLA2s in viperid venoms, and suggest that Pllans-I plays a myotoxic role in envenomings by P. l. lansbergii, whereas Pllans-II, apparently devoid of toxicity, enhances muscle damage caused by Pllans-I. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Lipid metabolites of the phospholipase A2 pathway and inflammatory cytokines are associated with brain volume in paediatric cerebral malaria.

    PubMed

    Pappa, Vasiliki; Seydel, Karl; Gupta, Sanchit; Feintuch, Catherine M; Potchen, Michael J; Kampondeni, Samuel; Goldman-Yassen, Adam; Veenstra, Mike; Lopez, Lillie; Kim, Ryung S; Berman, Joan W; Taylor, Terrie; Daily, Johanna P

    2015-12-21

    Cerebral malaria (CM) remains a significant cause of morbidity and mortality in children in sub-Saharan Africa. CM mortality has been associated with increased brain volume, seen on neuroimaging studies. To examine the potential role of blood metabolites and inflammatory mediators in increased brain volume in Malawian children with CM, an association study was performed between plasma metabolites, cytokine levels and phospholipase A2 (PLA2) activity with brain volume. The metabolomics analysis demonstrated arachidonic acid and other lysophospholipids to be positively associated with brain swelling. These lipids are products of the PLA2 enzyme and an association of plasma PLA2 enzymatic activity with brain swelling was confirmed. TNFα, which can upregulate PLA2 activity, was associated with brain volume. In addition, CCL2 and IL-8 were also associated with brain volume. Some of these cytokines can alter endothelial cell tight junction proteins and increase blood brain barrier permeability. Taken together, paediatric CM brain volume was associated with products of the PLA2 pathway and inflammatory cytokines. Their role in causality is unknown. These molecules will need to undergo testing in vitro and in animal models to understand their role in processes of increased brain volume. These observations provide novel data on host physiology associated with paediatric CM brain swelling, and may both inform pathogenesis models and suggest adjunct therapies that could improve the morbidity and mortality associated with paediatric CM.

  6. Activation of phospholipase A2 by temporin B: formation of antimicrobial peptide-enzyme amyloid-type cofibrils.

    PubMed

    Code, Christian; Domanov, Yegor A; Killian, J Antoinette; Kinnunen, Paavo K J

    2009-05-01

    Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at >8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.

  7. Cytosolic phospholipase A2 α has a crucial role in the pathogenesis of DSS-induced colitis in mice.

    PubMed

    Rosengarten, Marina; Hadad, Nurit; Solomonov, Yulia; Lamprecht, Sergio; Levy, Rachel

    2016-02-01

    Colitis, an inflammation of the colon, is a well-characterized massive tissue injury. Cytosolic phospholipase A2 α (cPLA2 α) upregulation plays an important role in the development of several inflammatory diseases. The aim of the present study was to define the role of cPLA2 α upregulation in the development of colitis. We used a mouse model of dextran sulfate sodium induced colitis. Immunoblotting analysis showed that cPLA2 α and NF-κB were upregulated and activated in the colon from day 2 of colitis induction. This molecular event preceded the development of the disease, as determined by Disease Activity Index score, body weight, colon length, and the expression of colonic inflammatory markers, including neutrophil infiltration detected by myeloperoxidase and by NIMP-R14, ICAM-1, COX-2, iNOS upregulation and LTB4 and TNF-α secretion. Prevention of cPLA2 α upregulation and activity in the colon by i.v. administration of specific antisense oligonucleotides against cPLA2 α 1 day prior and every day of exposure to dextran sulfate sodium significantly impeded the development of the disease and prevented NF-κB activation, neutrophils infiltration into the colonic mucosa, and expression of proinflammatory proteins in the colon. Our results demonstrate a critical role of cPLA2 α upregulation in inflammation and development of murine colitis.

  8. Activation of phospholipase A2 by 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine in vitro.

    PubMed

    Code, Christian; Mahalka, Ajay K; Bry, Kristian; Kinnunen, Paavo K J

    2010-08-01

    Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90 degrees light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22 microM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy.

  9. Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons.

    PubMed

    Wei, S; Ong, W Y; Thwin, M M; Fong, C W; Farooqui, A A; Gopalakrishnakone, P; Hong, W

    2003-01-01

    Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.

  10. cAMP-Inhibits Cytoplasmic Phospholipase A2 and Protects Neurons against Amyloid-β-Induced Synapse Damage

    PubMed Central

    Bate, Clive; Williams, Alun

    2015-01-01

    A key event in Alzheimer’s disease (AD) is the production of amyloid-β (Aβ) peptides and the loss of synapses. In cultured neurons Aβ triggered synapse damage as measured by the loss of synaptic proteins. α-synuclein (αSN), aggregates of which accumulate in Parkinson’s disease, also caused synapse damage. Synapse damage was associated with activation of cytoplasmic phospholipase A2 (cPLA2), an enzyme that regulates synapse function and structure, and the production of prostaglandin (PG) E2. In synaptosomes PGE2 increased concentrations of cyclic adenosine monophosphate (cAMP) which suppressed the activation of cPLA2 demonstrating an inhibitory feedback system. Thus, Aβ/αSN-induced activated cPLA2 produces PGE2 which increases cAMP which in turn suppresses cPLA2 and, hence, its own production. Neurons pre-treated with pentoxifylline and caffeine (broad spectrum phosphodiesterase (PDE) inhibitors) or the PDE4 specific inhibitor rolipram significantly increased the Aβ/αSN-induced increase in cAMP and consequently protected neurons against synapse damage. The addition of cAMP analogues also inhibited cPLA2 and protected neurons against synapse damage. These results suggest that drugs that inhibit Aβ-induced activation of cPLA2 and cross the blood–brain barrier may reduce synapse damage in AD. PMID:26389963

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

    PubMed Central

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

    2010-01-01

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

  12. Transgenic mosquitoes expressing a phospholipase A(2) gene have a fitness advantage when fed Plasmodium falciparum-infected blood.

    PubMed

    Smith, Ryan C; Kizito, Christopher; Rasgon, Jason L; Jacobs-Lorena, Marcelo

    2013-01-01

    Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development. We have created two transgenic lines of Anophelesstephensi, a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A2 (mPLA2) into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA2 significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium-infected blood. Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control.

  13. 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.

  14. A Novel Phospholipase A2 (D49) from the Venom of the Crotalus oreganus abyssus (North American Grand Canyon Rattlesnake)

    PubMed Central

    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

  15. An ultraviolet photoacoustic spectroscopy study of the interaction between Lys49-phospholipase A2 and amphiphilic molecules.

    PubMed

    Bugs, Milton Roque; Bortoleto-Bugs, Raquel Kely; Cornélio, Marinônio Lopes; Ward, Richard John

    2007-02-23

    We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A2 from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios>10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios<8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios>10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant.

  16. Expression and regulation of phospholipase A2 in venom gland of the chinese honeybee, Apis cerana cerana.

    PubMed

    Li, Jiang-Hong; Zhang, Chuan-Xi; Shen, Li-rong; Tang, Zhen-Hua; Cheng, Jia-An

    2005-09-01

    Phospholipase A(2) (PLA(2)) is one of the components of bee venom with a wide range of pharmacological functions. It operates as a major allergen working with other venom components to defend the colony from intruder. In the present study, the cDNA sequence of the Ac-pla(2) gene from cDNA library of the venom gland of Apis cerana was compared with the amplified corresponding region of genomic DNA. The result showed that the Ac-pla(2) gene consisted of four exons and three introns. Southern blot showed that the Ac-pla(2) gene was a single copy per haploid genome. The most active transcription period was during the first 8 days of adults, which correspondingly was the period of sharp increase of PLA(2) protein. ELISA analysis revealed that the PLA(2) was undetectable in pupal stage and the newly eclosed adult, but increased sharply to a maximum of 10-12 mug per honeybee by 8-10 days of adult life, followed by a gradual decrease to 8 mug for the rest of adult life. Transcriptional or post transcriptional regulation is the key step for Ac-pla(2) expression. The early secreted Ac-PLA(2) showed a low degree of post-translational modification; with increasing age, glycosylation was detected by Western blot and glycoprotein staining analysis. Different post-translational modifications were found among different individuals in A. cerana when compared to A. mellifera.

  17. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-04-03

    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. 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. 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). 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.

  20. Transgenic Mosquitoes Expressing a Phospholipase A2 Gene Have a Fitness Advantage When Fed Plasmodium falciparum-Infected Blood

    PubMed Central

    Smith, Ryan C.; Kizito, Christopher; Rasgon, Jason L.; Jacobs-Lorena, Marcelo

    2013-01-01

    Background Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development. Results We have created two transgenic lines of Anophelesstephensi, a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A2 (mPLA2) into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA2 significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium-infected blood. Conclusions Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control. PMID:24098427

  1. 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

  2. Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates.

    PubMed

    Mouchlis, Varnavas D; Bucher, Denis; McCammon, J Andrew; Dennis, Edward A

    2015-02-10

    Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein-lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein-lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme-substrate-membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme's interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Cytoplasmic Phospholipase A2 Modulation of Adolescent Rat Ethanol-Induced Protein Kinase C Translocation and Behavior

    PubMed Central

    Santerre, J. L.; Kolitz, E. B.; Pal, R.; Rogow, J. A.; Werner, D. F.

    2015-01-01

    Ethanol consumption typically begins during adolescence, a developmental period which exhibits many age-dependent differences in ethanol behavioral sensitivity. Protein kinase C (PKC) activity is largely implicated in ethanol-behaviors, and our previous work indicates that regulation of novel PKC isoforms likely contributes to decreased high-dose ethanol sensitivity during adolescence. The cytoplasmic Phospholipase A2 (cPLA2) signaling cascade selectivity modulates novel and atypical PKC isoform activity, as well as adolescent ethanol hypnotic sensitivity. Therefore, the current study was designed to ascertain adolescent cPLA2 activity both basally and in response to ethanol, as well as it's involvement in ethanol-induced PKC isoform translocation patterns. cPLA2 expression was elevated during adolescence, and activity was increased only in adolescents following high-dose ethanol administration. Novel, but not atypical PKC isoforms translocate to cytosolic regions following high-dose ethanol administration. Inhibiting cPLA2 with AACOCF3 blocked ethanol-induced PKC cytosolic translocation. Finally, inhibition of novel, but not atypical, PKC isoforms when cPLA2 activity was elevated, modulated adolescent high-dose ethanol-sensitivity. These data suggest that the cPLA2/PKC pathway contributes to the acute behavioral effects of ethanol during adolescence. PMID:25791059

  6. C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes[S

    PubMed Central

    Ward, Katherine E.; Ropa, James P.; Adu-Gyamfi, Emmanuel; Stahelin, Robert V.

    2012-01-01

    Group IVA cytosolic phospholipase A2 (cPLA2α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA2α binds zwitterionic lipids such as phosphatidylcholine in a Ca2+-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA2α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA2α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA2α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA2α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA2α. PMID:22991194

  7. Snake Venom Cytotoxins, Phospholipase A2s, and Zn2+-dependent Metalloproteinases: Mechanisms of Action and Pharmacological Relevance

    PubMed Central

    Gasanov, Sardar E; Dagda, Ruben K; Rael, Eppie D

    2014-01-01

    Snake venom toxins are responsible for causing severe pathology and toxicity following envenomation including necrosis, apoptosis, neurotoxicity, myotoxicity, cardiotoxicity, profuse hemorrhage, and disruption of blood homeostasis. Clinically, snake venom toxins therefore represent a significant hazard to snakebite victims which underscores the need to produce more efficient anti-venom. Some snake venom toxins, however, have great potential as drugs for treating human diseases. In this review, we discuss the biochemistry, structure/function, and pathology induced by snake venom toxins on human tissue. We provide a broad overview of cobra venom cytotoxins, catalytically active and inactive phospholipase A2s (PLA2s), and Zn2+-dependent metalloproteinases. We also propose biomedical applications whereby snake venom toxins can be employed for treating human diseases. Cobra venom cytotoxins, for example, may be utilized as anti-cancer agents since they are efficient at destroying certain types of cancer cells including leukemia. Additionally, increasing our understanding of the molecular mechanism(s) by which snake venom PLA2s promote hydrolysis of cell membrane phospholipids can give insight into the underlying biomedical implications for treating autoimmune disorders that are caused by dysregulated endogenous PLA2 activity. Lastly, we provide an exhaustive overview of snake venom Zn2+-dependent metalloproteinases and suggest ways by which these enzymes can be engineered for treating deep vein thrombosis and neurodegenerative disorders. PMID:24949227

  8. Effects of an acidic phospholipase A2 purified from Ophiophagus hannah (king cobra) venom on rat heart.

    PubMed

    Huang, M Z; Wang, Q C; Liu, G F

    1993-05-01

    An acidic phospholipase A2 (OHV A-PLA2) purified from Ophiophagus hannah venom had a cardiotoxic action on rat heart. In rats OHV A-PLA2 (2-4 mg/kg) caused ECG abnormalities including decreased heart rate, prolonged P-R interval, widened QRS complex and complete A-V block. When tested on isolated rat right atria, OHV A-PLA2 (10-20 micrograms/ml) produced a positive chronotropic effect. When tested on isolated rat left atria or papillary muscle preparations, OHV A-PLA2 (2.5-20 micrograms/ml) caused positive inotropic effect, followed by contracture. The positive inotropic effects could be abolished by high Ca2+ and enhanced by low Ca2+; both treatments accelerated contracture. The contracture could be inhibited in Mn2+ (5 mM)-containing medium and abolished by Ca(2+)-free bath solution containing 1 mM EDTA. The cardiotoxic action of OHV A-PLA2 was not influenced by verapamil, tetrodotoxin, propranolol, phentolamine, atropine or indomethacin. It is suggested that the cardiotoxic effects of OHV A-PLA2 may result from increasing intracellular levels of Ca2+.

  9. Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA.

    PubMed

    Duchez, Anne-Claire; Boudreau, Luc H; Naika, Gajendra S; Bollinger, James; Belleannée, Clémence; Cloutier, Nathalie; Laffont, Benoit; Mendoza-Villarroel, Raifish E; Lévesque, Tania; Rollet-Labelle, Emmanuelle; Rousseau, Matthieu; Allaeys, Isabelle; Tremblay, Jacques J; Poubelle, Patrice E; Lambeau, Gérard; Pouliot, Marc; Provost, Patrick; Soulet, Denis; Gelb, Michael H; Boilard, Eric

    2015-07-07

    Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

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

    PubMed

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

    2010-05-01

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

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

    PubMed

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

    2003-06-01

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

  12. Group X Secretory Phospholipase A2 Negatively Regulates ABCA1 and ABCG1 expression and cholesterol efflux in macrophages

    PubMed Central

    Shridas, Preetha; Bailey, William M; Gizard, Florence; Oslund, Rob C; Gelb, Michael H; Bruemmer, Dennis; Webb, Nancy R

    2010-01-01

    Objectives Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids and has been implicated in inflammatory diseases including atherosclerosis. Here we identify a novel role for GX sPLA2 in modulating ABCA1 and ABCG1 expression and hence macrophage cholesterol efflux. Methods and Results Overexpression or exogenous addition of GX sPLA2 significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA2 deficiency in mouse peritoneal macrophages (MPMs) was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA2 overexpressing J774 cells, and increased efflux in GX sPLA2-deficient MPMs. Gene regulation was dependent on GX sPLA2 catalytic activity, mimicked by arachidonic acid, abrogated when LXRα/β expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA2 suppresses the ability of LXR to trans-activate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand binding domain. Conclusions GX sPLA2 modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA2 may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating genes critical for cellular cholesterol efflux. PMID:20844270

  13. Spectroscopic investigations on the binding of persimmon tannin to phospholipase A 2 from Chinese cobra ( Naja naja atra)

    NASA Astrophysics Data System (ADS)

    Yang, Jie; Zhong, Li; Zou, Bo; Tian, Yan; Xu, Shu-fen; Yao, Ping; Li, Chun-mei

    2012-01-01

    To understand the anti-venom mechanism of persimmon tannin, the interaction between persimmon tannin (PT) and phospholipase A 2 (PLA 2) under physiological conditions was investigated by fluorescence quenching technique in combination with Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra techniques. The results revealed that gradual fluorescence quenching was observed by titration of PLA 2 (2.0 μM) with increasing concentrations of PT (from 0 to 2.025 μM), and the type of quenching was found to be a static quenching process. Stern-Volmer plots were not linear but had an intersection at CPT ≈ 1.0 μM, indicating that PT binded to more than one class of sites on PLA 2. The binding sites calculated on basis of Scatchard plots were about 2, supporting this result. The enthalpy change (Δ H) and entropy change (Δ S) of the binding sites were -17.44 kJ/mol and 59.90 kJ/mol·, separately, suggesting that hydrophobic interaction played a main role in the binding. In addition, synchronous fluorescence, FT-IR and CD spectra showed that dramatic conformational changes in PLA 2 were induced by its interaction with PT.

  14. Regulation of the Ca(2+)-independent phospholipase A2 in liver mitochondria by changes in the energetic state.

    PubMed

    Rauckhorst, Adam J; Broekemeier, Kimberly M; Pfeiffer, Douglas R

    2014-05-01

    The effect of electron transport chain redox status on activity of the mitochondrial Ca(2+)-independent phospholipase A2 (iPLA2) has been examined. When oxidizing NAD-linked substrates, the enzyme is not active unless deenergization occurs. Uncoupler, rotenone, antimycin A, and cyanide are equally effective at upregulating the enzyme, while oligomycin is ineffective. Thenoyltrifluoroacetone causes deenergization and activates the enzyme, but only if succinate is the respiratory substrate. These findings show that the mitochondrial iPLA2 responds to the energetic state overall, rather than to the redox status of individual electron transport chain complexes. With NAD-linked substrates, and using rotenone to deenergize, iPLA2 activation can be reversed by adding succinate to reestablish a membrane potential. For this purpose, ascorbate plus N,N,N'N'-tetramethyl-phenylenediamine can be used instead of succinate and is equally effective. With succinate as substrate, the membrane potential can be reduced in a graded and stable fashion by adding increasing concentrations of malonate, which is a competitive inhibitor of succinate utilization. A partial and stable activation of the iPLA2 accompanies partial deenergization. These findings suggest that in addition to the several functions that have been proposed, the mitochondrial iPLA2 may help to coordinate local capillary blood flow with changing energy demands.

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

    PubMed Central

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

    2016-01-01

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

  16. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    PubMed Central

    West, Ewan; Osborne, Craig; Nolan, William; Bate, Clive

    2015-01-01

    Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage. PMID:26043272

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

    PubMed Central

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

    2015-01-01

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

  18. Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A 2 and synapse damage.

    PubMed

    Bate, Clive; Williams, Alun

    2012-01-01

    Precisely how the accumulation of PrP (Sc) causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP (Sc) oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP (C), either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A 2 (cPLA 2). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA 2 and synapse damage induced by cross-linked PrP (C). We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA 2 and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP (C), suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.

  19. Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates

    PubMed Central

    Mouchlis, Varnavas D.; Bucher, Denis; McCammon, J. Andrew; Dennis, Edward A.

    2015-01-01

    Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein–lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme–substrate–membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme’s interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface. PMID:25624474

  20. Suramin inhibits macrophage activation by human group IIA phospholipase A2, but does not affect bactericidal activity of the enzyme.

    PubMed

    Aragão, E A; Chioato, L; Ferreira, T L; de Medeiros, A I; Secatto, A; Faccioli, L H; Ward, R J

    2009-04-01

    Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A(2) (hsPLA(2)GIIA). The hsPLA(2)GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA(2) GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. The hydrolytic activity of the hsPLA(2) GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA(2) GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA(2) GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 microM. These results demonstrate that suramin selectively inhibits the activity of the hsPLA(2) GIIA against macrophages, whilst leaving the anti-bacterial function unchanged.

  1. Functions, structures and Triton X-100 effect for the catalytic subunits of heterodimeric phospholipases A2 from Vipera nikolskii venom.

    PubMed

    Gao, Wei; Starkov, Vladislav G; He, Zi-Xuan; Wang, Qi-hai; Tsetlin, Victor I; Utkin, Yuri N; Lin, Zheng-jiong; Bi, Ru-chang

    2009-11-01

    Phospholipases A(2) (PLA(2)s) from snake venoms have diverse pharmacological functions including neurotoxicity, and more studies are necessary to understand relevant mechanisms. Here we report the different crystal structures for two enzymatically active basic subunits (HDP-1P and HDP-2P) of heterodimeric neurotoxic PLA(2)s isolated from Vipera nikolskii venom. Structural comparisons with similar PLA(2)s clearly show some flexible regions which might be important for the catalytic function and neurotoxicity. Unexpectedly, Triton X-100 molecule bound in the hydrophobic channel of HDP-1P and HDP-2P was observed, and its binding induced conformational changes in the Ca(2+) binding loop. Enzymatic activity measurements indicated that Triton X-100 decreased the activity of PLA(2), although with comparatively low inhibitory activity. For the first time exocytosis experiments in pancreatic beta cells were used to confirm the presynaptic neurotoxicity of relevant snake PLA(2). These experiments also indicated that Triton X-100 inhibited the influence of HDP-1P on exocytosis, but the inhibition was smaller than that of MJ33, a phospholipid-analogue inhibitor of PLA(2). Our studies performed at a cellular level are in good agreement with earlier findings that enzymatic activity of the snake presynaptic PLA(2) neurotoxins is essential for effective block of nerve terminals.

  2. Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes.

    PubMed

    Canning, Paul; Kenny, Bridget-Ann; Prise, Vivien; Glenn, Josephine; Sarker, Mosharraf H; Hudson, Natalie; Brandt, Martin; Lopez, Francisco J; Gale, David; Luthert, Philip J; Adamson, Peter; Turowski, Patric; Stitt, Alan W

    2016-06-28

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood-retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.

  3. Lipoprotein-associated phospholipase A2 as a novel risk marker for cardiovascular disease: a systematic review of the literature.

    PubMed

    Madjid, Mohammad; Ali, Muzammil; Willerson, James T

    2010-01-01

    We sought to critically assess the role of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) in the prediction of cardiovascular events in primary and secondary prevention settings. The inclusion criteria for our study included population-based epidemiologic studies and the presence of clinical outcomes of interest, including atherosclerotic disease, coronary events, stroke, and cardiovascular death. Studies that lacked clinical outcomes or that involved animals were excluded. We included primary and secondary prevention studies of subjects in all ethnic groups and of either sex, with no age limitation. We searched MEDLINE, Google Scholar, and the Cochrane Library for studies with publication dates from January 1970 through July 2009, and we searched major cardiology meeting abstracts from 2000 through 2009. From each study, we used predictive ability-including relative risk, hazard ratio, odds ratio, and prevalence of high Lp-PLA(2) levels, with adjustment-along with baseline population characteristics.Of 33 studies that met our inclusion criteria, 30 showed a significant association between Lp-PLA(2) and cardiovascular events. Most of the studies had been adjusted for major Framingham risk factors and other variables that might influence the effect under question. After multivariate adjustments in cohort and nested case-control studies, increased levels of Lp-PLA(2) remained a significant predictor of cardiovascular events. The available body of evidence suggests that Lp-PLA(2) is a reliable marker of risk for cardiovascular events.

  4. The elevated lipoprotein-associated phospholipase A2 activity is associated with the occurrence and recurrence of acute cerebral infarction.

    PubMed

    Wei, Lingli; Ke, Zunyu; Zhao, Yu; Cai, Zhiyou

    2017-03-07

    There is a strong association between lipoprotein-associated phospholipase A2 (Lp-PLA2) levels and atherosclerosis-related diseases. The aim of this study was to investigate the role of Lp-PLA2 in the ischemic stroke and further offer clinical evidence that measuring Lp-PLA2 helps predict the risk of stroke occurrence and recurrence. A total of 328 hospitalized patients were recruited, including 179 cases of acute cerebral infarction (ACI) and 149 non-ACI controls. The serum level of Lp-PLA2 in ACI was significantly higher than non-ACI. The serum level of Lp-PLA2 in the recurrence of ACI was significantly higher than the nonrecurrence. The serum levels of Lp-PLA2 in large-artery atherosclerosis subtype were the highest among the subtypes of the Trial of Org 10172 in Acute Stroke Treatment and non-ACI controls. The level of Lp-PLA2 in large-artery atherosclerosis and the cardioembolism group was statistically significantly higher than that of the control cases. There was no statistically significant difference between the small-vessel occlusion group and the control cases. The present study confirmed that the elevated Lp-PLA2 level can be a risk factor for ischemic stroke in the Chinese population. The serum level of Lp-PLA2 may be a predictive factor for the recurrence of ACI.

  5. Distribution of secretory phospholipase A2 XIIA in the brain and its role in lipid metabolism and cognition.

    PubMed

    Ee, Sze-Min; Lo, Yew-Long; Shui, Guanghou; Wenk, Markus R; Shin, Eun-Joo; Kim, Hyoung-Chun; Ong, Wei-Yi

    2014-08-01

    Phospholipases A(2) (PLA(2)) catalyze the hydrolysis of membrane phospholipids to produce free fatty acids and lysophospholipids, which have important functions in cell signaling. The present study elucidated differential expression of PLA(2) isoforms in the rat cortex by quantitative reverse transcription PCR and demonstrated that sPLA(2)-XIIA had greater messenger RNA expression than iPLA(2)-VI or cPLA(2)-IVA in different brain regions, or compared to other sPLA(2) isoforms in the prefrontal cortex (PFC) and hippocampus. Western blots identified a 24-kDa band in different regions of the adult brain, and high levels of sPLA(2)-XIIA protein expression were detected in the PFC, striatum, and thalamus. Electron microscopy showed that sPLA(2)-XIIA is present in axon terminals and dendrites. Injection of antisense oligonucleotide to sPLA(2)-XIIA in the PFC and lipidomic analysis showed increase in phospholipid but decrease in lysophospholipid species consistent with decreased catalytic activity of the enzyme, changes in arachidonic acid release, and alterations in sphingolipids. sPLA(2)-XIIA knockdown also resulted in shorter latency timings in the passive avoidance test, and higher number of errors in the attention set-shifting task, indicating deficits in working memory and attention. Together, the results show an important role of sPLA(2)-XIIA in lipid metabolism, prefrontal cortical function, and cognition.

  6. Phospholipase A2 Inhibitor from Crotalus durissus terrificus rattlesnake: Effects on human peripheral blood mononuclear cells and human neutrophils cells.

    PubMed

    Xavier, Caroline V; da S Setúbal, Sulamita; Lacouth-Silva, Fabianne; Pontes, Adriana S; Nery, Neriane M; de Castro, Onassis Boeri; Fernandes, Carla F C; Soares, Andreimar M; Fortes-Dias, Consuelo L; Zuliani, Juliana P

    2017-07-23

    Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2(-)) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2(-) release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2(-). However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Neuroaxonal dystrophy caused by group VIA phospholipase A2 deficiency in mice: a model of human neurodegenerative disease.

    PubMed

    Shinzawa, Koei; Sumi, Hisae; Ikawa, Masahito; Matsuoka, Yosuke; Okabe, Masaru; Sakoda, Saburo; Tsujimoto, Yoshihide

    2008-02-27

    Calcium-independent group VIA phospholipase A2 (iPLA2beta) is considered to play a role in signal transduction and maintenance of homeostasis or remodeling of membrane phospholipids. A role of iPLA2beta has been suggested in various physiological and pathological processes, including immunity, chemotaxis, and cell death, but the details remain unclear. Accordingly, we investigated mice with targeted disruption of the iPLA2beta gene. iPLA2beta-/- mice developed normally and grew to maturity, but all showed evidence of severe motor dysfunction, including a hindlimb clasping reflex during tail suspension, abnormal gait, and poor performance in the hanging wire grip test. Neuropathological examination of the nervous system revealed widespread degeneration of axons and/or synapses, accompanied by the presence of numerous spheroids (swollen axons) and vacuoles. These findings provide evidence that impairment of iPLA2beta causes neuroaxonal degeneration, and indicate that the iPLA2beta-/- mouse is an appropriate animal model of human neurodegenerative diseases associated with mutations of the iPLA2beta gene, such as infantile neuroaxonal dystrophy and neurodegeneration with brain iron accumulation.

  8. Physiological Roles of Group X-secreted Phospholipase A2 in Reproduction, Gastrointestinal Phospholipid Digestion, and Neuronal Function*

    PubMed Central

    Sato, Hiroyasu; Isogai, Yuki; Masuda, Seiko; Taketomi, Yoshitaka; Miki, Yoshimi; Kamei, Daisuke; Hara, Shuntaro; Kobayashi, Tetsuyuki; Ishikawa, Yukio; Ishii, Toshiharu; Ikeda, Kazutaka; Taguchi, Ryo; Ishimoto, Yoshikazu; Suzuki, Noriko; Yokota, Yasunori; Hanasaki, Kohji; Suzuki-Yamamoto, Toshiko; Yamamoto, Kei; Murakami, Makoto

    2011-01-01

    Although the secreted phospholipase A2 (sPLA2) family has been generally thought to participate in pathologic events such as inflammation and atherosclerosis, relatively high and constitutive expression of group X sPLA2 (sPLA2-X) in restricted sites such as reproductive organs, the gastrointestinal tract, and peripheral neurons raises a question as to the roles played by this enzyme in the physiology of reproduction, digestion, and the nervous system. Herein we used mice with gene disruption or transgenic overexpression of sPLA2-X to clarify the homeostatic functions of this enzyme at these locations. Our results suggest that sPLA2-X regulates 1) the fertility of spermatozoa, not oocytes, beyond the step of flagellar motility, 2) gastrointestinal phospholipid digestion, perturbation of which is eventually linked to delayed onset of a lean phenotype with reduced adiposity, decreased plasma leptin, and improved muscle insulin tolerance, and 3) neuritogenesis of dorsal root ganglia and the duration of peripheral pain nociception. Thus, besides its inflammatory action proposed previously, sPLA2-X participates in physiologic processes including male fertility, gastrointestinal phospholipid digestion linked to adiposity, and neuronal outgrowth and sensing. PMID:21266581

  9. Physiological roles of group X-secreted phospholipase A2 in reproduction, gastrointestinal phospholipid digestion, and neuronal function.

    PubMed

    Sato, Hiroyasu; Isogai, Yuki; Masuda, Seiko; Taketomi, Yoshitaka; Miki, Yoshimi; Kamei, Daisuke; Hara, Shuntaro; Kobayashi, Tetsuyuki; Ishikawa, Yukio; Ishii, Toshiharu; Ikeda, Kazutaka; Taguchi, Ryo; Ishimoto, Yoshikazu; Suzuki, Noriko; Yokota, Yasunori; Hanasaki, Kohji; Suzuki-Yamamoto, Toshiko; Yamamoto, Kei; Murakami, Makoto

    2011-04-01

    Although the secreted phospholipase A(2) (sPLA(2)) family has been generally thought to participate in pathologic events such as inflammation and atherosclerosis, relatively high and constitutive expression of group X sPLA(2) (sPLA(2)-X) in restricted sites such as reproductive organs, the gastrointestinal tract, and peripheral neurons raises a question as to the roles played by this enzyme in the physiology of reproduction, digestion, and the nervous system. Herein we used mice with gene disruption or transgenic overexpression of sPLA(2)-X to clarify the homeostatic functions of this enzyme at these locations. Our results suggest that sPLA(2)-X regulates 1) the fertility of spermatozoa, not oocytes, beyond the step of flagellar motility, 2) gastrointestinal phospholipid digestion, perturbation of which is eventually linked to delayed onset of a lean phenotype with reduced adiposity, decreased plasma leptin, and improved muscle insulin tolerance, and 3) neuritogenesis of dorsal root ganglia and the duration of peripheral pain nociception. Thus, besides its inflammatory action proposed previously, sPLA(2)-X participates in physiologic processes including male fertility, gastrointestinal phospholipid digestion linked to adiposity, and neuronal outgrowth and sensing.

  10. Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

    PubMed Central

    Canning, Paul; Kenny, Bridget-Ann; Prise, Vivien; Glenn, Josephine; Sarker, Mosharraf H.; Hudson, Natalie; Brandt, Martin; Lopez, Francisco J.; Gale, David; Luthert, Philip J.; Adamson, Peter; Turowski, Patric; Stitt, Alan W.

    2016-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood–retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents. PMID:27298369

  11. Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells.

    PubMed

    Blank, Simon; Michel, Yvonne; Seismann, Henning; Plum, Melanie; Greunke, Kerstin; Grunwald, Thomas; Bredehorst, Reinhard; Ollert, Markus; Braren, Ingke; Spillner, Edzard

    2011-04-01

    Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy.

  12. Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells.

    PubMed

    Moreira, Luciana S; Piva, Bruno; Gentile, Luciana B; Mesquita-Santos, Fabio P; D'Avila, Heloisa; Maya-Monteiro, Clarissa M; Bozza, Patricia T; Bandeira-Melo, Christianne; Diaz, Bruno L

    2009-03-01

    Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE(2), but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3 K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A(2)-alpha, while facilitated AA mobilization and synthesis of PGE(2) within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA (2)-alpha-driven enhanced AA mobilization and PGE(2)production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.

  13. Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain - evidence of increased phospholipase A(2) activity.

    PubMed

    Ma, M-T; Yeo, J-F; Shui, G; Wenk, M R; Ong, W-Y

    2012-01-01

    Recent studies suggest that CNS phospholipase A(2) (PLA(2) ) isoforms play a role in nociception, but until now, direct evidence of increased brain PLA(2) activity during allodynia or hyperalgesia is lacking. The present study was carried out, using lipidomics or systems wide analyses of lipids using tandem mass spectrometry, to elucidate possible changes in rat brain lipids after inflammatory pain induced by facial carrageenan injection. The caudal medulla oblongata showed decreases in phospholipids including phosphatidylethanolamine and phosphatidylinositol species, but increases in lysophospholipids, including lysophosphatidylethanolamine, lysophosphatidylinositol and lysophosphatidylserine, indicating increased PLA(2) activity and release of arachidonic acid after facial carrageenan injection. These changes likely occur in the spinal trigeminal nucleus which relays nociceptive input from the orofacial region. High levels of sPLA(2) -III, sPLA(2) -XIIA, cPLA(2) and iPLA(2) mRNA expression were detected in the medulla oblongata. Increase in sPLA(2) -III mRNA expression was found in the caudal medulla of carrageenan-injected rats, although no difference in sPLA(2) -III protein expression was detected. The changes in lipids as determined by lipidomics were therefore consistent with an increase in PLA(2) enzyme activity, but no change in enzyme protein expression. Together, these findings indicate enhanced PLA(2) activity in the caudal medulla oblongata after inflammatory orofacial pain. © 2011 European Federation of International Association for the Study of Pain Chapters.

  14. Group IVA phospholipase A2 is associated with the storage of lipids in adipose tissue and liver.

    PubMed

    Ii, Hiromi; Hatakeyama, Shinji; Tsutsumi, Kae; Sato, Takashi; Akiba, Satoshi

    2008-06-01

    Prostaglandin (PG) E(2) is considered to participate in the storage of fat in adipocytes and hepatocytes, but roles of group IVA phospholipase A(2) (PLA(2)), a key PLA(2) isozyme in the arachidonic acid cascade, remain unclear. The present study examined the possible involvement of the enzyme using group IVA PLA(2)-deficient mice (C57BL/6 background, 22 weeks of age) fed a normal diet (5.3% fat). The ratio of epididymal fat pad weight to body weight was significantly reduced in group IVA PLA(2)-deficient mice compared to wild-type mice. Histological analysis revealed that in group IVA PLA(2)-deficient mice, the adipocytes were smaller, and hepatocytes bearing cytoplasmic vacuolation were scarce. Hepatic triglyceride content and the serum levels of PGE(2) in the deficient mice were also lower. However, there was no difference in the serum levels of insulin, glucose, non-esterified free fatty acid, or total cholesterol between the deficient and wild-type mice. Our findings suggest that group IVA PLA(2) is involved in the storage of lipids in the adipose tissue and liver and in determining circulating PGE(2) levels.

  15. Structural Basis for the Inhibition of a Phospholipase A2-Like Toxin by Caffeic and Aristolochic Acids.

    PubMed

    Fernandes, Carlos A H; Cardoso, Fábio Florença; Cavalcante, Walter G L; Soares, Andreimar M; Dal-Pai, Maeli; Gallacci, Marcia; Fontes, Marcos R M

    2015-01-01

    One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from Bothropspirajai venom, complexed with two compounds present in several plants used in folk medicine against snakebites. These ligands partially neutralized the myotoxic activity of PrTX-I towards binding on the two independent sites of interaction between Lys49-PLA2 and muscle membrane. Our results corroborate the previously proposed mechanism of action of PLA2s-like and provide insights for the design of structure-based inhibitors that could prevent the permanent injuries caused by these proteins in snakebite victims.

  16. Structural Basis for the Inhibition of a Phospholipase A2-Like Toxin by Caffeic and Aristolochic Acids

    PubMed Central

    Fernandes, Carlos A. H.; Cardoso, Fábio Florença; Cavalcante, Walter G. L.; Soares, Andreimar M.; Dal-Pai, Maeli; Gallacci, Marcia; Fontes, Marcos R. M.

    2015-01-01

    One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from Bothropspirajai venom, complexed with two compounds present in several plants used in folk medicine against snakebites. These ligands partially neutralized the myotoxic activity of PrTX-I towards binding on the two independent sites of interaction between Lys49-PLA2 and muscle membrane. Our results corroborate the previously proposed mechanism of action of PLA2s-like and provide insights for the design of structure-based inhibitors that could prevent the permanent injuries caused by these proteins in snakebite victims. PMID:26192963

  17. Protective Effects of Intratracheally-Administered Bee Venom Phospholipase A2 on Ovalbumin-Induced Allergic Asthma in Mice

    PubMed Central

    Jung, Kyung-Hwa; Baek, Hyunjung; Shin, Dasom; Lee, Gihyun; Park, Sangwon; Lee, Sujin; Choi, Dabin; Kim, Woojin; Bae, Hyunsu

    2016-01-01

    Asthma is a common chronic disease characterized by bronchial inflammation, reversible airway obstruction, and airway hyperresponsiveness (AHR). Current therapeutic options for the management of asthma include inhaled corticosteroids and β2 agonists, which elicit harmful side effects. In the present study, we examined the capacity of phospholipase A2 (PLA2), one of the major components of bee venom (BV), to reduce airway inflammation and improve lung function in an experimental model of asthma. Allergic asthma was induced in female BALB/c mice by intraperitoneal administration of ovalbumin (OVA) on days 0 and 14, followed by intratracheal challenge with 1% OVA six times between days 22 and 30. The infiltration of immune cells, such as Th2 cytokines in the lungs, and the lung histology, were assessed in the OVA-challenged mice in the presence and absence of an intratracheal administration of bvPLA2. We showed that the intratracheal administration of bvPLA2 markedly suppressed the OVA-induced allergic airway inflammation by reducing AHR, overall area of inflammation, and goblet cell hyperplasia. Furthermore, the suppression was associated with a significant decrease in the production of Th2 cytokines, such as IL-4, IL-5, and IL-13, and a reduction in the number of total cells, including eosinophils, macrophages, and neutrophils in the airway. PMID:27669297

  18. Validation of a phospholipase A2 receptor antibody ELISA in an Australian cohort with membranous glomerulonephritis.

    PubMed

    Ong, Lawrence; Silvestrini, Roger; Chapman, Jeremy; Fulcher, David A; Lin, Ming Wei

    2016-04-01

    A commercial PLA2R Ab ELISA was validated by examining its ability to distinguish primary from secondary membranous nephropathy, correlating results with clinical markers of disease activity, and comparing its performance with an indirect immunofluorescence test (IIFT). PLA2R Ab levels were measured in 77 patients with biopsy proven membranous nephropathy, divided into either idiopathic (n = 61) or secondary groups (n = 6). In the idiopathic group, measures of contemporaneous disease activity (proteinuria, serum creatinine) were compared between seropositive and seronegative subjects. ELISA values were then compared with semi-quantitative results from an IIFT using PLA2R transfected HEK293 cells as substrate. The PLA2R Ab ELISA was positive in only 15 of 61 (25%) patients with idiopathic membranous nephropathy (IMN), but there was a significant negative relationship with time since diagnosis. Thus, in a subgroup of patients diagnosed within 6 months of analysis, the sensitivity was 6/15 (55%), rising to 6/8 (75%) in those recently-diagnosed patients who had not been treated. In the entire cohort, there was a significant positive correlation between ELISA values and degree of proteinuria, but our analysis did not control for variation of both variables with time. The PLA2R Ab ELISA also showed very high agreement with IIFT (96%). Therefore, the PLA2R Ab ELISA is a highly specific test for distinguishing primary from secondary membranous nephropathy that is most sensitive in newly diagnosed patients who have not received immunosuppression. Antibody levels correlated with degree of proteinuria, but this relationship was not shown to be independent of time. Both IIFT and ELISA platforms performed comparably.

  19. Membranous nephropathy as a manifestation of graft-versus-host disease: association with HLA antigen typing, phospholipase A2 receptor, and C4d.

    PubMed

    Byrne-Dugan, Cathryn J; Collins, A Bernard; Lam, Albert Q; Batal, Ibrahim

    2014-12-01

    Glomerulopathy is an uncommon but increasingly recognized complication of hematopoietic cell transplantation. It typically manifests as membranous nephropathy, less commonly as minimal change disease, and rarely as proliferative glomerulonephritis. There is evidence to suggest that these glomerulopathies might represent manifestations of chronic graft-versus-host disease. In this report, we focus on membranous nephropathy as the most common form of glomerulopathy after hematopoietic cell transplantation. We present a case of membranous nephropathy that developed 483 days post-allogeneic hematopoietic stem cell transplantation in a patient with a history of acute graft-versus-host disease. We also share our experience with 4 other cases of membranous nephropathy occurring after allogeneic hematopoietic stem cell transplantation. Clinicopathologic correlates, including the association with graft-versus-host-disease, HLA antigen typing, glomerular deposition of immunoglobulin G (IgG) subclasses, subepithelial colocalization of IgG deposits with phospholipase A2 receptor staining, C4d deposition along the peritubular capillaries, and treatment, are discussed with references to the literature.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  2. Non-steroidal anti-inflammatory drugs as potent inhibitors of phospholipase A2: structure of the complex of phospholipase A2 with niflumic acid at 2.5 Angstroms resolution.

    PubMed

    Jabeen, Talat; Singh, Nagendra; Singh, Rajendra K; Sharma, Sujata; Somvanshi, Rishi K; Dey, Sharmistha; Singh, Tej P

    2005-12-01

    Phospholipase A(2) (PLA(2); EC 3.1.3.4) catalyzes the first step of the production of proinflammatory compounds collectively known as eicosanoids. The binding of phospholipid substrates to PLA(2) occurs through a well formed hydrophobic channel. Surface plasmon resonance studies have shown that niflumic acid binds to Naja naja sagittifera PLA(2) with an affinity that corresponds to a dissociation constant (K(d)) of 4.3 x 10(-5) M. Binding studies of PLA(2) with niflumic acid were also carried out using a standard PLA(2) kit that gave an approximate binding constant, K(i), of 1.26 +/- 0.05 x 10(-6) M. Therefore, in order to establish the viability of PLA(2) as a potential target molecule for drug design against inflammation, arthritis and rheumatism, the three-dimensional structure of the complex of PLA(2) with the known anti-inflammatory agent niflumic acid [2-[3-(trifluoromethyl)anilino]nicotinic acid] has been determined at 2.5 Angstroms resolution. The structure of the complex has been refined to an R factor of 0.187. The structure determination reveals the presence of one niflumic acid molecule at the substrate-binding site of PLA(2). It shows that niflumic acid interacts with the important active-site residues His48 and Asp49 through two water molecules. It is observed that the niflumic acid molecule is completely buried in the substrate-binding hydrophobic channel. The conformations of the binding site in PLA(2) as well as that of niflumic acid are not altered upon binding. However, the orientation of the side chain of Trp19, which is located at the entry of the substrate-binding site, has changed from that found in the native PLA(2), indicating its familiar role.

  3. Lipoprotein-associated phospholipase A2, myeloperoxidase and vascular endothelial growth factor - predictors of high vascular risk in respiratory bacterial infections.

    PubMed

    Seri, A; Marta, D S; Madalan, A; Popescu, M; Tiglea, A I; Moldoveanu, E

    2016-01-01

    Objective. Respiratory bacterial infections are associated with important coagulation disturbances that amplify the pulmonary lesions and determine a more severe course of the disease. The aim of our study was to investigate the correlation between the evolution of the general clinical parameters and the occurrence of thrombotic events on one side, and plasma levels of selected proteins involved in inflammation and coagulation on the other side, with the intent to establish and to validate a laboratory test panel for the assessment of the vascular risk in patients with bacterial respiratory infections. Methods. The study included 111 patients (divided into two groups, 61 without thrombosis and 50 with thrombosis) with bacterial respiratory infections and 30 healthy controls, age and gender-matched. The baseline evaluation of the patients included clinical, biological, and respiratory examination. LpPLA2 and MPO activities were measured by the spectrophotometric method. VEGF was quantified with an ELISA kit. Results. The collected data showed a correlation between the occurrence of superimposed thrombosis in respiratory infection patients, and the intensity of the inflammatory process, reflected by the increased MPO activity, and the dynamics of LpPLA2 and VEGF. Conclusion. Bacterial respiratory infections associate thrombotic vascular events of various degrees of severity, which correlate with the intensity of the inflammatory process, and the severity of endothelium dysfunction at the level of microcirculation. Starting from the recorded data, and based on the established severity scales in use, it is possible to compute a vascular risk score that takes into consideration the values of the three biomarkers under investigation. Abbreviations: COPD = chronic obstructive pulmonary disease,hsCRP = high sensitivity C reactive protein,EC = endothelial cells, ICAM-1 = intercellular adhesion molecule1, LpPLA2 = lipoprotein-associated phospholipase A2, MPO

  4. Lipoprotein-associated phospholipase A2, myeloperoxidase and vascular endothelial growth factor - predictors of high vascular risk in respiratory bacterial infections

    PubMed Central

    Seri, A; Marta, DS; Madalan, A; Popescu, M; Tiglea, AI; Moldoveanu, E

    2016-01-01

    Objective. Respiratory bacterial infections are associated with important coagulation disturbances that amplify the pulmonary lesions and determine a more severe course of the disease. The aim of our study was to investigate the correlation between the evolution of the general clinical parameters and the occurrence of thrombotic events on one side, and plasma levels of selected proteins involved in inflammation and coagulation on the other side, with the intent to establish and to validate a laboratory test panel for the assessment of the vascular risk in patients with bacterial respiratory infections. Methods. The study included 111 patients (divided into two groups, 61 without thrombosis and 50 with thrombosis) with bacterial respiratory infections and 30 healthy controls, age and gender-matched. The baseline evaluation of the patients included clinical, biological, and respiratory examination. LpPLA2 and MPO activities were measured by the spectrophotometric method. VEGF was quantified with an ELISA kit. Results. The collected data showed a correlation between the occurrence of superimposed thrombosis in respiratory infection patients, and the intensity of the inflammatory process, reflected by the increased MPO activity, and the dynamics of LpPLA2 and VEGF. Conclusion. Bacterial respiratory infections associate thrombotic vascular events of various degrees of severity, which correlate with the intensity of the inflammatory process, and the severity of endothelium dysfunction at the level of microcirculation. Starting from the recorded data, and based on the established severity scales in use, it is possible to compute a vascular risk score that takes into consideration the values of the three biomarkers under investigation. Abbreviations: COPD = chronic obstructive pulmonary disease,hsCRP = high sensitivity C reactive protein,EC = endothelial cells, ICAM-1 = intercellular adhesion molecule1, LpPLA2 = lipoprotein-associated phospholipase A2, MPO

  5. Quantitative proteomic analysis of Vietnamese krait venoms: Neurotoxins are the major components in Bungarus multicinctus and phospholipases A2 in Bungarus fasciatus.

    PubMed

    Ziganshin, Rustam H; Kovalchuk, Sergey I; Arapidi, Georgij P; Starkov, Vladislav G; Hoang, Anh Ngoc; Thi Nguyen, Thao Thanh; Nguyen, Khoa Cuu; Shoibonov, Batozhab B; Tsetlin, Victor I; Utkin, Yuri N

    2015-12-01

    Kraits are venomous snakes of genus Bungarus from family Elapidae. Krait venoms are generally neurotoxic, but toxicity strongly depends on the particular species and regional origin of snakes. We analyzed the proteomes of Vietnamese Bungarus multicinctus and Bungarus fasciatus venoms both qualitatively and quantitatively. It should be noted that no proteomic data for B. multicinctus venom existed so far. We have found that in this venom, almost half (45%) of the proteins by weight was represented by β-bungarotoxins, followed by three finger toxins (28%) and phospholipases A2 (16%), other proteins being present at the level of 1-3%. In B. fasciatus venom, phospholipase A2 was the main component (71%), followed by oxidase of l-amino acids (8%), acetylcholinesterase (5%) and metalloproteinases (4%). Unexpectedly, extremely low amount of three finger toxins (1%) was found in this venom. Interestingly, the presence of complement depleting factor was observed in both venoms. Although our data showed the presence of the same toxin families in Vietnamese krait venoms as those found earlier in the venoms of kraits from other geographic regions, their relative ratio is completely different. This concerns especially B. fasciatus venom with predominant content of phospholipases A2 and very low amount of three finger toxins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Isolation, structural and functional characterization of a new Lys49 phospholipase A2 homologue from Bothrops neuwiedi urutu with bactericidal potential.

    PubMed

    Corrêa, Edailson A; Kayano, Anderson M; Diniz-Sousa, Rafaela; Setúbal, Sulamita S; Zanchi, Fernando B; Zuliani, Juliana P; Matos, Najla B; Almeida, José R; Resende, Letícia M; Marangoni, Sérgio; da Silva, Saulo L; Soares, Andreimar M; Calderon, Leonardo A

    2016-06-01

    Snake venom is a complex mixture of active compounds consisting of 80-90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A2 homologue from the venom of Bothrops neuwiedi urutu. The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA2s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A2 from Bothrops snake venoms, and identified this molecule as a novel PLA2 isoform from B. neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 μg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa. The results allowed for the identification of a new myotoxin isoform with PLA2 structure with promising biotechnological applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. M-type phospholipase A2 receptor (PLA2R) glomerular staining in pediatric idiopathic membranous nephropathy.

    PubMed

    Kanda, Shoichiro; Horita, Shigeru; Yanagihara, Takeshi; Shimizu, Akira; Hattori, Motoshi

    2017-04-01

    Identifying M-type phospholipase A2 receptor (PLA2R) is a landmark breakthrough for understanding adult idiopathic membranous nephropathy (iMN). However, potential roles for PLA2R in pediatric iMN have not been well investigated. A total of 34 pediatric iMN patients who underwent kidney biopsy between 1972 and 2015 were enrolled in this study. The study cohort consisted of 15 children aged from 3 to 9 years and 19 aged from 10 to 15 years. In all cases, secondary causes of MN, including infections, autoimmune diseases, and others, were ruled out. We examined PLA2R glomerular staining in stored, formalin-fixed, paraffin-embedded kidney biopsy samples. Kidney biopsy specimens obtained from an adult patient with iMN and an adult patient with lupus-associated MN were also examined to assess our PLA2R staining procedure. Granular staining of PLA2R along glomerular capillary loops was present in two patients: an 11-year-old girl and 12-year-old boy identified during a school urine screening test and who presented with mild proteinuria at the time of biopsy. Interestingly, the intensity of PLA2R glomerular staining in these patients was weaker than that of a PLA2R-positive adult iMN patient. There were no PLA2R-positive patients among our cohort of children younger than 10 years. This preliminary study suggests PLA2R may play a role in some adolescent and preteen iMN patients but may be less frequently associated with iMN during childhood.

  8. [Molecular cloning and characterization in silico of phospholipase A(2) transcript isolated from Lachesis muta peruvian snake venom].

    PubMed

    Jimenez, Karim L; Zavaleta, Amparo I; Izaguirre, Victor; Yarleque, Armando; Inga, Rosio R

    2010-01-01

    Isolate and characterize in silico gene phospholipase A(2) (PLA(2)) isolated from Lachesis muta venom of the Peruvian Amazon. Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13,976 kDa and 5.66 respectively. The aminoacid sequence was called Lm-PLA(2)-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA(2) from Lachesis stenophrys (93%) and other PLA(2) snake venoms and over 80% of other sPLA(2) family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA(2)-Peru grouped with other acidic [Asp(49)] sPLA(2) previously isolated from Bothriechis schlegelii venom showing 89 % nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA(2) group II that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. The nucleotide sequence corresponding to the first transcript of gene from PLA(2) cloned of Lachesis muta venom, snake from the Peruvian rainforest.

  9. Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries.

    PubMed

    Tsai, Inn-Ho; Wang, Ying-Ming; Chen, Yi-Hsuan; Tsai, Tein-Shun; Tu, Ming-Chung

    2004-01-01

    Phospholipases A2 (PLA2s) were purified from the Trimeresurus stejnegeri venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLA2 were determined. In addition to the six previously documented PLA2 isoforms of this species, we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLA2s. The molecular masses and partial sequences of most of the purified PLA2s matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLA2s of crotalid venoms, a novel type of PLA2 with Asn-49 substitution at the Ca2+-binding site was discovered. This type of PLA2 is non-catalytic, but may cause local oedema and appears to be a venom marker of many tree vipers. In particular, we showed that T. stejnegeri displayed high geographic variations of the PLA2s within and between their Taiwanese and Chinese populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLA2s of this species and other related Asian vipers reveals that T. stejnegeri contains venom genes related to those from several sympatric pit vipers, including the genera Tropedolaemus and Gloydius besides the Trimeresurus itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species.

  10. Anti-platelet aggregation activity of two novel acidic Asp49-phospholipases A2 from Bothrops brazili snake venom.

    PubMed

    Sobrinho, Juliana C; Kayano, Anderson M; Simões-Silva, Rodrigo; Alfonso, Jorge J; Gomez, Ana F; Gomez, Maria C Vega; Zanchi, Fernando B; Moura, Laura A; Souza, Vivian R; Fuly, André L; de Oliveira, Eliandre; da Silva, Saulo L; Almeida, José R; Zuliani, Juliana P; Soares, Andreimar M

    2017-09-23

    Phospholipases A2 (PLA2s) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLA2s from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLA2s was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ​​of 13,894 and 13,869Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLA2s using acid PLA2BthA-I-PLA2 from B. jararacussu template showed high quality. Both acidic PLA2s showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100μg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10μg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLA2s biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads. Copyright © 2017. Published by Elsevier B.V.

  11. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    PubMed

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-07

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  12. Nanoassemblies containing a fluorouracil/zidovudine glyceryl prodrug with phospholipase A2-triggered drug release for cancer treatment.

    PubMed

    Jin, Yiguang; Yang, Fang; Du, Lina

    2013-12-01

    Secretory phospholipase A2 (sPLA2), which is overexpressed in many tumors, cleaves ester bonds at the sn-2 position of phospholipids. A PLA2-sensitive amphiphilic prodrug, 1-O-octadecyl-2-(5-fluorouracil)-N-acetyl-3-zidovudine-phosphorylglycerol (OFZG), was synthesized and used to prepare nanoassemblies through the injection of a mixture of OFZG/cholesterol/Tween 80 (2:1:0.1, mol:mol:mol) into water. Cholesterol and Tween 80 was incorporated into the OFZG monolayers at the air/water interface to yield nanoassemblies. The resulting nanoassemblies exhibited a narrow size distribution with a mean size of 77.8nm and were stable due to their high surface charges. The in vitro experiments showed that PLA2 degraded OFZG. The nanoassemblies exhibited higher anticancer activity than the parent drug 5-fluorouracil (5-FU) in COLO205, HT-28, and HCT-116 cells. The intravenous (i.v.) administration of the nanoassemblies into mice resulted in the rapid elimination of OFZG from the circulation and its distribution mainly in the liver, lung, spleen, and kidney. After their injection into tumor-bearing mice, the nanoassemblies exhibited anticancer efficiency comparable to that of 5-FU, even though the nanoassemblies contained concentrations of only 1/10 of the molar amount of 5-FU. The lessons learned from the study and methods for the design of PLA2-sensitive amphiphilic prodrugs are also discussed. Enzyme-sensitive amphiphilic combinatorial prodrugs and prodrug-loaded nanoassemblies may represent a new strategy for anticancer drug design. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

    PubMed Central

    Duchez, Anne-Claire; Boudreau, Luc H.; Naika, Gajendra S.; Bollinger, James; Belleannée, Clémence; Cloutier, Nathalie; Laffont, Benoit; Mendoza-Villarroel, Raifish E.; Lévesque, Tania; Rollet-Labelle, Emmanuelle; Rousseau, Matthieu; Allaeys, Isabelle; Tremblay, Jacques J.; Poubelle, Patrice E.; Lambeau, Gérard; Pouliot, Marc; Provost, Patrick; Soulet, Denis; Gelb, Michael H.; Boilard, Eric

    2015-01-01

    Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms. PMID:26106157

  14. Crystal structure of a phospholipase A2 from Bothrops asper venom: Insights into a new putative "myotoxic cluster".

    PubMed

    Salvador, Guilherme H M; Dos Santos, Juliana I; Lomonte, Bruno; Fontes, Marcos R M

    2017-02-01

    Snake venoms from the Viperidae and Elapidae families often have several phospholipases A2 (PLA2s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA2s from these venoms are generally divided into two classes: (i) catalytic PLA2s (or Asp49-PLA2s) and (ii) non-catalytic PLA2-like toxins (or Lys49-PLA2s). In many Viperidae venoms, a subset of the basic Asp49-PLA2s displays some functional and structural characteristics of PLA2-like proteins and group within the same phylogenetic clade, but their myotoxic mechanism is still largely unknown. In the present study, we have crystallized and solved the structure of myotoxin I (MT-I), a basic myotoxic Asp49-PLA2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA2s and Lys49-PLA2s and has been confirmed by other biophysical and bioinformatics techniques. This arrangement suggests a possible cooperative action between both monomers to exert myotoxicity via two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS). This mechanism would resemble that proposed for Lys49-PLA2s, but the sites involved appear to be situated in a different region. Thus, as both sites are close to one another, they form a "myotoxic cluster", which is also found in two other basic myotoxic Asp49-PLA2s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA2s found in viperid snake venoms.

  15. Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries.

    PubMed Central

    Tsai, Inn-Ho; Wang, Ying-Ming; Chen, Yi-Hsuan; Tsai, Tein-Shun; Tu, Ming-Chung

    2004-01-01

    Phospholipases A2 (PLA2s) were purified from the Trimeresurus stejnegeri venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLA2 were determined. In addition to the six previously documented PLA2 isoforms of this species, we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLA2s. The molecular masses and partial sequences of most of the purified PLA2s matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLA2s of crotalid venoms, a novel type of PLA2 with Asn-49 substitution at the Ca2+-binding site was discovered. This type of PLA2 is non-catalytic, but may cause local oedema and appears to be a venom marker of many tree vipers. In particular, we showed that T. stejnegeri displayed high geographic variations of the PLA2s within and between their Taiwanese and Chinese populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLA2s of this species and other related Asian vipers reveals that T. stejnegeri contains venom genes related to those from several sympatric pit vipers, including the genera Tropedolaemus and Gloydius besides the Trimeresurus itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species. PMID:12959640

  16. Plasma secretory phospholipase A2-IIa as a potential biomarker for lung cancer in patients with solitary pulmonary nodules

    PubMed Central

    2011-01-01

    Background Five-year survival for lung cancer has remained at 16% over last several decades largely due to the fact that over 50% of patients are diagnosed with locally-advanced or metastatic disease. Diagnosis at an earlier and potentially curable stage is crucial. Solitary pulmonary nodules (SPNs) are common, but the difficulty lies in the determination of which SPN is malignant. Currently, there is no convenient and reliable biomarker effective for early diagnosis. Secretory phospholipase A2-IIa (sPLA2-IIa) is secreted into the circulation by cancer cells and may allow for an early detection of lung cancer. Methods Plasma samples from healthy donors, patients with only benign SPN, and patients with lung cancer were analyzed. Expression of sPLA2-IIa protein in lung cancer tissues was also determined. Results We found that the levels of plasma sPLA2-IIa were significantly elevated in lung cancer patients. The receiver operating characteristic curve analysis, comparing lung cancer patients to patients with benign nodules, revealed an optimum cutoff value for plasma sPLA2-IIa of 2.4 ng/ml to predict an early stage cancer with 48% sensitivity and 86% specificity and up to 67% sensitivity for T2 stage lung cancer. Combined sPLA2-IIa, CEA, and Cyfra21.1 tests increased the sensitivity for lung cancer prediction. High level of plasma sPLA2-IIa was associated with a decreased overall cancer survival. sPLA2-IIa was overexpressed in almost all non-small cell lung cancer and in the majority of small cell lung cancer by immunohistochemistry analysis. Conclusion Our finding strongly suggests that plasma sPLA2-IIa is a potential lung biomarker to distinguish benign nodules from lung cancer and to aid lung cancer diagnosis in patients with SPNs. PMID:22151235

  17. PEDF Inhibits the Activation of NLRP3 Inflammasome in Hypoxia Cardiomyocytes through PEDF Receptor/Phospholipase A2.

    PubMed

    Zhou, Zhongxin; Wang, Zhu; Guan, Qiuhua; Qiu, Fan; Li, Yufeng; Liu, Zhiwei; Zhang, Hao; Dong, Hongyan; Zhang, Zhongming

    2016-12-12

    The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome has been linked to sterile inflammation, which is involved in ischemic injury in myocardial cells. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein with many biological activities, such as anti-inflammatory, antioxidant and anti-angiogenic properties. However, it is not known whether and how PEDF acts to regulate the activation of the NLRP3 inflammasome in cardiomyocytes. In the present study, we used the neonatal cardiomyocytes models of ischemia-like conditions to evaluate the mitochondrial fission and the activation of the NLRP3 inflammasome. We also determined the mechanism by which PEDF inhibits hypoxia-induced activation of the NLRP3 inflammasome. We found that PEDF decreased the activation of the NLRP3 inflammasome in neonatal cardiomyocytes through pigment epithelial-derived factor receptor/calcium-independent phospholipase A2 (PEDFR/iPLA2). Meanwhile, PEDF reduced Drp1-induced mitochondrial fission and mitochondrial fission-induced mitochondrial DNA (mtDNA), as well as mitochondrial reactive oxygen species (mtROS) release into cytosol through PEDFR/iPLA2. We also found that PEDF inhibited mitochondrial fission-induced NLRP3 inflammasome activation. Furthermore, previous research has found that endogenous cytosolic mtDNA and mtROS can serve as activators of NLRP3 inflammasome activity. Therefore, we hypothesized that PEDF can protect against hypoxia-induced activation of the NLRP3 inflammasome by inhibiting mitochondrial fission though PEDFR/iPLA2.

  18. Biochemical and enzymatic characterization of two basic Asp49 phospholipase A2 isoforms from Lachesis muta muta (Surucucu) venom.

    PubMed

    Damico, Daniela C S; Lilla, Sérgio; de Nucci, Gilberto; Ponce-Soto, Luis A; Winck, Flávia V; Novello, José Camillo; Marangoni, Sérgio

    2005-10-30

    Two basic phospholipase A2 (PLA2) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 mu-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg65 (LmTX-I)-->Pro65 (LmTX-II). The amino acid sequence showed a high degree of homology between PLA2 isoforms from Lachesis muta muta and other PLA2 snake venoms. LmTX-I and LmTX-II had PLA2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35-45 degrees C. Full PLA2 activity required Ca2+ and was respectively inhibited by Cu2+ and Zn2+ in the presence and absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P<0.05) the enzymatic activity of LmTX-I, suggesting that the binding site for crotapotin in this PLA2 was similar to another in the basic PLA2 of the crotoxin complex from C. durissus cascavella venom.

  19. Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

    PubMed

    Park, Dae-Won; Kim, Jae-Ryong; Kim, Seong-Yong; Sonn, Jong-Kyung; Bang, Ok-Sun; Kang, Shin-Sung; Kim, Jung-Hye; Baek, Suk-Hwan

    2003-02-15

    The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

  20. Resveratrol suppresses lipoprotein-associated phospholipase A2 expression by reducing oxidative stress in macrophages and animal models.

    PubMed

    Sun, Shengnan; Zhang, Mingjun; Yang, Qiangbing; Shen, Ziying; Chen, Jiahuan; Yu, Biao; Wang, He; Qu, Jiali; Pang, Daxin; Ren, Wenzhi; Ouyang, Hongsheng; Tang, Xiaochun

    2017-10-01

    Resveratrol is a naturally occurring polyphenolic compound with known cardioprotective, anti-inflammatory, and antioxidant properties. Lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is associated with the risk of cardiovascular disease. Here, we investigated the effects of resveratrol on Lp-PLA2 expression in vitro and in vivo and explored the underlying mechanisms. Human monocytic cells (THP-1) were induced to differentiate into macrophages for an in vitro experimental model. Resveratrol suppressed Lp-PLA2 expression and reduced inflammation; lipopolysaccharide (LPS, 1 μg/mL), tumor necrosis factor-α (TNF-α, 10 ng/mL) and reactive oxygen species (ROS) were employed to stimulate an increase in Lp-PLA2 expression and ROS levels, and the stimulation was inhibited by resveratrol (50 μM) and other antioxidants. The inhibition of resveratrol was inversed partially by sirtuin 1 (SIRT1) inhibitors (Nicotinamide, 1-10 mM) (p<0.05). Next, a chronic inflammation mouse model induced by a HFD (high fat diet) supplemented with resveratrol 100 mg/kg/day orally for 12 weeks, resulted in resveratrol-induced decreases in the Lp-PLA2 levels in the plasma and liver and increases in the superoxide dismutase 2 (SOD2) expression in the liver (p<0.05). Based on our results, the protective effects of resveratrol on cardiovascular events may be related to its ability to suppress Lp-PLA2 expression. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A2 Pathways

    PubMed Central

    Seo, Jihui; Maeng, Jeehye; Kim, Hwa-Jung

    2016-01-01

    The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [3H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i), the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2) in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions. PMID:27783042

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

    PubMed

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

    2015-12-07

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

  3. Secretory phospholipase A2 in dromedary tears: a host defense against staphylococci and other gram-positive bacteria.

    PubMed

    Ben Bacha, Abir; Abid, Islem

    2013-03-01

    The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

  4. Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin.

    PubMed

    Chiou, Yi-Ling; Lin, Shinne-Ren; Hu, Wan-Ping; Chang, Long-Sen

    2014-12-15

    Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Alleviation of high-fat diet-induced fatty liver damage in group IVA phospholipase A2-knockout mice.

    PubMed

    Ii, Hiromi; Yokoyama, Naoki; Yoshida, Shintaro; Tsutsumi, Kae; Hatakeyama, Shinji; Sato, Takashi; Ishihara, Keiichi; Akiba, Satoshi

    2009-12-01

    Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A(2) (IVA-PLA(2)), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA(2)-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA(2)-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA(2)-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA(2)-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA(2)-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E(2), which has a fat storage effect, was lower in IVA-PLA(2)-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA(2) alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA(2) metabolites, such as prostaglandin E(2). IVA-PLA(2) could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.

  6. Group IVA phospholipase A2-associated production of MMP-9 in macrophages and formation of atherosclerotic lesions.

    PubMed

    Ii, Hiromi; Hontani, Naoya; Toshida, Issei; Oka, Mayuko; Sato, Takashi; Akiba, Satoshi

    2008-03-01

    Matrix metalloproteinase-9 (MMP-9) is involved in atherogenesis, and the production of MMP-9 in macrophages is considered to be mediated by the arachidonic acid cascade. The present study examined the possible involvement of group IVA phospholipase A2 (IVA-PLA2), a key enzyme in the arachidonic acid cascade, in the production of MMP-9 induced by oxidized low-density lipoprotein (oxLDL) in macrophages and high-fat diet-induced formation of atherosclerotic lesions using IVA-PLA2-deficient mice (C57BL/6 background). In wild-type mouse peritoneal macrophages, oxLDL induced an increase in MMP-9 in the culture medium. The oxLDL-promoted production of MMP-9 was markedly reduced in IVA-PLA2-deficient macrophages compared to wild-type macrophages. Feeding of wild-type mice with a high-fat diet caused the formation of early atherosclerotic lesions in the aortic root with increases in MMP-9 and macrophages in the lesions and with higher serum levels of total cholesterol. Such lesions were apparently less severe in IVA-PLA2-deficient mice fed a high-fat diet, despite higher total cholesterol levels. Under the conditions, a high-fat diet reduced the serum levels of high-density lipoprotein-cholesterol (HDL-C) in wild-type mice. However, IVA-PLA2-deficient mice fed a high-fat diet were protected against the decrease in HDL-C levels. The present results suggest that IVA-PLA2 is involved in the oxLDL-induced production of MMP-9 in macrophages and the high-fat diet-induced formation of early atherosclerotic lesions. The protection against the lesions in IVA-PLA2-deficient mice may be ascribable, in part, to the impaired production of MMP-9 and/or the maintained levels of HDL-C.

  7. Group X Phospholipase A2 Stimulates the Proliferation of Colon Cancer Cells by Producing Various Lipid Mediators

    PubMed Central

    Surrel, Fanny; Jemel, Ikram; Boilard, Eric; Bollinger, James G.; Payré, Christine; Mounier, Carine M.; Talvinen, Kati A.; Laine, Veli J. O.; Nevalainen, Timo J.; Gelb, Michael H.

    2009-01-01

    Among mammalian secreted phospholipases A2 (sPLA2s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA2 [mouse (m)GX] is one of the most highly expressed PLA2 in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA2s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA2 inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA2α and M-type sPLA2 receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA2 mitogenic effects. Together, our results indicate that group X sPLA2 may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression. PMID:19602573

  8. Group X phospholipase A2 stimulates the proliferation of colon cancer cells by producing various lipid mediators.

    PubMed

    Surrel, Fanny; Jemel, Ikram; Boilard, Eric; Bollinger, James G; Payré, Christine; Mounier, Carine M; Talvinen, Kati A; Laine, Veli J O; Nevalainen, Timo J; Gelb, Michael H; Lambeau, Gérard

    2009-10-01

    Among mammalian secreted phospholipases A2 (sPLA(2)s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA(2) [mouse (m)GX] is one of the most highly expressed PLA(2) in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA(2)s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA(2) inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA(2)alpha and M-type sPLA(2) receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA(2) mitogenic effects. Together, our results indicate that group X sPLA(2) may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression.

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

    PubMed

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

    2005-01-01

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

  10. Phospholipase A2 of Peroxiredoxin 6 Plays a Critical Role in Cerebral Ischemia/Reperfusion Inflammatory Injury.

    PubMed

    Shanshan, Yu; Beibei, Jiang; Li, Tan; Minna, Gao; Shipeng, Lei; Li, Peng; Yong, Zhao

    2017-01-01

    Microglia-mediated inflammation is an important step in the progression of cerebral ischemia/reperfusion injury and the associated production of receptors of immunomoudulation, including Toll-like receptors (TLRs). Peroxiredoxin 6 (Prdx6) has been demonstrated as the endogenous antioxidant protein for its peroxidase properties. However, the role of the independent phospholipase A2 (iPLA2) activity of Prdx6 in stroke has not been well studied. In this study, we evaluated whether blocking the calcium-iPLA2 activity of Prdx6 using siRNA and inhibitors (1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol, MJ33) would have a critical effect on inflammatory brain damage. We conducted oxygen-glucose deprivation (OGD)/recovery (R) in vitro and middle cerebral artery occlusion (MCAO) in vivo in a microglia/neuron co-culture system and in rats. In vitro, we found that Prdx6-iPLA2 activity was associated with the secretion of neurotoxic inflammatory mediators interleukin1β (IL-1β), interleukin-17 (IL-17) and interleukin-23 (IL-23) and elevated expression of Toll-like receptor 2/4 (TLR2/4), leading to the formation of nuclear factor-kappa B (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in microglial cells. In vivo, combined treatment with Prdx6-iPLA2 activity inhibitor MJ33 showed a greater diminution in neurologic deficits, cerebral infarction, brain water content and inflammatory molecules than Prdx6-siRNA treatment alone. Our findings provide new insight into Prdx6-iPLA2 function in the brain. Inhibition of Prdx6-iPLA2 activity by gene therapy and/or pharmacology may constitute a promising new therapeutic approach to the treatment of stroke.

  11. PEDF Inhibits the Activation of NLRP3 Inflammasome in Hypoxia Cardiomyocytes through PEDF Receptor/Phospholipase A2

    PubMed Central

    Zhou, Zhongxin; Wang, Zhu; Guan, Qiuhua; Qiu, Fan; Li, Yufeng; Liu, Zhiwei; Zhang, Hao; Dong, Hongyan; Zhang, Zhongming

    2016-01-01

    The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome has been linked to sterile inflammation, which is involved in ischemic injury in myocardial cells. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein with many biological activities, such as anti-inflammatory, antioxidant and anti-angiogenic properties. However, it is not known whether and how PEDF acts to regulate the activation of the NLRP3 inflammasome in cardiomyocytes. In the present study, we used the neonatal cardiomyocytes models of ischemia-like conditions to evaluate the mitochondrial fission and the activation of the NLRP3 inflammasome. We also determined the mechanism by which PEDF inhibits hypoxia-induced activation of the NLRP3 inflammasome. We found that PEDF decreased the activation of the NLRP3 inflammasome in neonatal cardiomyocytes through pigment epithelial-derived factor receptor/calcium-independent phospholipase A2 (PEDFR/iPLA2). Meanwhile, PEDF reduced Drp1-induced mitochondrial fission and mitochondrial fission-induced mitochondrial DNA (mtDNA), as well as mitochondrial reactive oxygen species (mtROS) release into cytosol through PEDFR/iPLA2. We also found that PEDF inhibited mitochondrial fission-induced NLRP3 inflammasome activation. Furthermore, previous research has found that endogenous cytosolic mtDNA and mtROS can serve as activators of NLRP3 inflammasome activity. Therefore, we hypothesized that PEDF can protect against hypoxia-induced activation of the NLRP3 inflammasome by inhibiting mitochondrial fission though PEDFR/iPLA2. PMID:27973457

  12. Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

    PubMed Central

    Escoffier, Jessica; Jemel, Ikram; Tanemoto, Akemi; Taketomi, Yoshitaka; Payre, Christine; Coatrieux, Christelle; Sato, Hiroyasu; Yamamoto, Kei; Masuda, Seiko; Pernet-Gallay, Karin; Pierre, Virginie; Hara, Shuntaro; Murakami, Makoto; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

    2010-01-01

    Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization. PMID:20424324

  13. The role of lipoprotein-associated phospholipase A2 in a murine model of experimental autoimmune uveoretinitis.

    PubMed

    Crawford, G L; Boldison, J; Copland, D A; Adamson, P; Gale, D; Brandt, M; Nicholson, L B; Dick, A D

    2015-01-01

    Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp-PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity.

  14. Biosynthesis of oxidized lipid mediators via lipoprotein-associated phospholipase A2 hydrolysis of extracellular cardiolipin induces endothelial toxicity.

    PubMed

    Buland, Justin R; Wasserloos, Karla J; Tyurin, Vladimir A; Tyurina, Yulia Y; Amoscato, Andrew A; Mallampalli, Rama K; Chen, Bill B; Zhao, Jing; Zhao, Yutong; Ofori-Acquah, Solomon; Kagan, Valerian E; Pitt, Bruce R

    2016-08-01

    We (66) have previously described an NSAID-insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis [by calcium-independent mitochondrial calcium-independent phospholipase A2-γ (iPLA2γ)] of oxidized CL (CLox), leading to the formation of lysoCL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAECs) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by lipoprotein-associated PLA2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolysoCL and dilysoCL and oxidized octadecadienoic metabolites including 9- and 13-hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2 Lower doses of nonlethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolysoCL or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa (P. aeruginosa) infection, 34 ± 8 mol% (n = 6; P < 0.02) of circulating CL was oxidized. In addition, molar percentage of monolysoCL increased twofold after P. aeruginosa in a subgroup analyzed for these changes. Collectively, these studies suggest an important role for 1) oxidation of CL in proinflammatory environments and 2) possible hydrolysis of CLox in extracellular spaces producing lysoCL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.

  15. Oxidized phospholipids and lipoprotein-associated phospholipase A2 as important determinants of Lp(a) functionality and pathophysiological role.

    PubMed

    Tselepis, Alexandros D

    2016-04-02

    Lipoprotein(a) [Lp(a)] is composed of a low density lipoprotein (LDL)-like particle to which apolipoprotein (a) [apo(a)] is linked by a single disulfide bridge. Lp(a) is considered a causal risk factor for ischemic cardiovascular disease (CVD) and calcific aortic valve stenosis (CAVS). The evidence for a causal role of Lp(a) in CVD and CAVS is based on data from large epidemiological databases, mendelian randomization studies, and genomewide association studies. Despite the well-established role of Lp(a) as a causal risk factor for CVD and CAVS, the underlying mechanisms are not well understood. A key role in the Lp(a) functionality may be played by its oxidized phospholipids (OxPL) content. Importantly, most of circulating OxPL are associated with Lp(a); however, the underlying mechanisms leading to this preferential sequestration of OxPL on Lp(a) over the other lipoproteins, are mostly unknown. Several studies support the hypothesis that the risk of Lp(a) is primarily driven by its OxPL content. An important role in Lp(a) functionality may be played by the lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme that catalyzes the degradation of OxPL and is bound to plasma lipoproteins including Lp(a). The present review article discusses new data on the pathophysiological role of Lp(a) and particularly focuses on the functional role of OxPL and Lp-PLA2 associated with Lp(a).

  16. Role of prefrontal cortical calcium-independent phospholipase A2 in antinociceptive effect of the norepinephrine reuptake inhibitor antidepresssant maprotiline.

    PubMed

    Chew, Wee-Siong; Shalini, Suku-Maran; Torta, Federico; Wenk, Markus R; Stohler, Christian; Yeo, Jin-Fei; Herr, Deron R; Ong, Wei-Yi

    2017-01-06

    The prefrontal cortex is essential for executive functions such as decision-making and planning. There is also accumulating evidence that it is important for the modulation of pain. In this study, we investigated a possible role of prefrontal cortical calcium-independent phospholipase A2 (iPLA2) in antinociception induced by the norepinephrine reuptake inhibitor (NRI) and tetracyclic (tricyclic) antidepressant, maprotiline. Intraperitoneal injections of maprotiline increased iPLA2 mRNA and protein expression in the prefrontal cortex. This treatment also reduced grooming responses to von-Frey hair stimulation of the face after facial carrageenan injection, indicating decreased sensitivity to pain. The antinociceptive effect of maprotiline was abrogated by iPLA2 antisense oligonucleotide injection to the prefrontal cortex, indicating a role of this enzyme in antinociception. In contrast, injection of iPLA2 antisense oligonucleotide to the somatosensory cortex did not reduce the antinociceptive effect of maprotiline. Lipidomic analysis of the prefrontal cortex showed decrease in phosphatidylcholine species, but increase in lysophosphatidylcholine species, indicating increased PLA2 activity, and release of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) after maprotiline treatment. Differences in sphingomyelin/ceramide were also detected. These changes were not observed in maprotiline-treated mice that received iPLA2 antisense oligonucleotide to the prefrontal cortex. Metabolites of DHA and EPA may help to strengthen a known supraspinal antinociceptive pathway from the prefrontal cortex to the periaqueductal gray. Together, results indicate a role of prefrontal cortical iPLA2 and its enzymatic products in the antinociceptive effect of maprotiline.

  17. Hemilipin, a novel Hemiscorpius lepturus venom heterodimeric phospholipase A2, which inhibits angiogenesis in vitro and in vivo.

    PubMed

    Jridi, Imen; Catacchio, Ivana; Majdoub, Hafed; Shahbazeddah, Delavar; El Ayeb, Mohamed; Frassanito, Maria Antonia; Ribatti, Domenico; Vacca, Angelo; Borchani, Lamia

    2015-10-01

    Phospholipases A2 (PLA2) are enzymes which specifically hydrolyze the sn-2 acyl ester bond of phospholipids producing free fatty acids and lysophospholipids. The secreted PLA2 (sPLA2) are the most common types of PLA2 purified from the snake venom, mammalian pancreatic juice and other sources. They display a variety of toxic actions and biological activities, including antitumoral and antiangiogenic effects. In this study, we report the isolation, characterization and the antiangiogenic activity of Hemilipin, a novel sPLA2 extracted from Hemiscorpius lepturus venom, the most dangerous scorpion in Iran. Hemilipin was purified by HPLC and analyzed by MALDI TOF/MS. The primary structure was determined by EDMAN degradation method and the PLA2 activity by titration of fatty acids released from the egg yolk phospholipids. Its antiangiogenic activity was studied in vitro by evaluating effects on apoptosis, Matrigel angiogenesis, migration and adhesion of human umbilical vein endothelial cells (HUVECs) and human pulmonary artery endothelial cells (HPAECs) and in vivo by the chorioallantoic membrane (CAM) assay. Mass spectrometry profile showed that Hemilipin is heterodimeric and the PLA2 test demonstrated its strong hydrolytic activity. N-terminal aminoacid sequence highlighted a significant homology of Hemilipin's small and large subunits with other sPLA2 group III. Hemilipin had no effect on apoptosis, but strongly impacted angiogenesis both in vitro and in vivo. Our results demonstrate that this novel non toxic sPLA2 could be a new tool to disrupt at different steps human angiogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Regional protein levels of cytosolic phospholipase A2 and cyclooxygenase-2 in Rhesus monkey brain as a function of age

    PubMed Central

    Weerasinghe, Gayani R.; Coon, Steven L.; Bhattacharjee, Abesh Kumar; Harry, G. Jean; Bosetti, Francesca

    2006-01-01

    Limited evidence suggests that brain cytosolic phospholipase A2 (cPLA2), which selectively releases arachidonic acid (AA) from membrane phospholipids, and cyclooxygenase-2 (COX-2), the rate-limiting enzyme for AA metabolism to prostanoids, change as a function of normal aging. In this study, we examined the protein levels of cPLA2 and COX-2 enzymes in hippocampus, frontal pole and cerebellum from young (2–5 year-old), middle-aged (8–11 year-old) and old (23 year-old) male and female Rhesus monkeys. In the cerebellum, cPLA2 protein level was higher in the young brain as compared to levels seen at both middle-aged and old. Similarly, in the frontal pole, the young brain showed a higher level of COX-2 protein as compared to the levels seen at both older ages. For both, once an animal reached 8–11 years of age the levels appeared to remain relatively constant over the next decade. Immunohistochemistry of COX-2 protein within the brain demonstrated no significant change in the localization to neurons within the frontal pole. In the young brain, the distribution of a low level of COX-2 protein within numerous neurons was different than the decreased number of neurons stained at a greater intensity in the adult brain. Based on the previous reports of localization of cPLA2 and COX-2 at post-synaptic sites in neurons results from the current study suggest that the elevated protein levels of the two enzymes seen in the younger brain is related to the greater potential for synaptic plasticity across multiple neurons as a function of age and that cPLA2 and COX-2 may be considered as post-synaptic markers. PMID:16716827

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

    SciTech Connect

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

    2009-09-02

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

  20. Biological and biochemical characterization of new basic phospholipase A(2) BmTX-I isolated from Bothrops moojeni snake venom.

    PubMed

    Calgarotto, Andrana K; Damico, Daniela C S; Ponce-Soto, L A; Baldasso, Paulo A; Da Silva, Saulo L; Souza, Gustavo H M F; Eberlin, Marcos N; Marangoni, Sergio

    2008-06-15

    BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+) and in the presence of Mg(2+), Cd(2+) and Mn(2+) it was reduced in presence or absence of Ca(2+). Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.

  1. Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.

    PubMed

    Heinze, Michael; Steighardt, Jörg; Gesell, Andreas; Schwartze, Wieland; Roos, Werner

    2007-12-01

    Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).

  2. Analgesic Effects of Bee Venom Derived Phospholipase A2 in a Mouse Model of Oxaliplatin-Induced Neuropathic Pain

    PubMed Central

    Li, Dongxing; Lee, Younju; Kim, Woojin; Lee, Kyungjin; Bae, Hyunsu; Kim, Sun Kwang

    2015-01-01

    A single infusion of oxaliplatin, which is widely used to treat metastatic colorectal cancer, induces specific sensory neurotoxicity signs that are triggered or aggravated when exposed to cold or mechanical stimuli. Bee Venom (BV) has been traditionally used in Korea to treat various pain symptoms. Our recent study demonstrated that BV alleviates oxaliplatin-induced cold allodynia in rats, via noradrenergic and serotonergic analgesic pathways. In this study, we have further investigated whether BV derived phospholipase A2 (bvPLA2) attenuates oxaliplatin-induced cold and mechanical allodynia in mice and its mechanism. The behavioral signs of cold and mechanical allodynia were evaluated by acetone and a von Frey hair test on the hind paw, respectively. The significant allodynia signs were observed from one day after an oxaliplatin injection (6 mg/kg, i.p.). Daily administration of bvPLA2 (0.2 mg/kg, i.p.) for five consecutive days markedly attenuated cold and mechanical allodynia, which was more potent than the effect of BV (1 mg/kg, i.p.). The depletion of noradrenaline by an injection of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4, 50 mg/kg, i.p.) blocked the analgesic effect of bvPLA2, whereas the depletion of serotonin by injecting DL-p-chlorophenylalanine (PCPA, 150 mg/kg, i.p.) for three successive days did not. Furthermore, idazoxan (α2-adrenegic receptor antagonist, 1 mg/kg, i.p.) completely blocked bvPLA2-induced anti-allodynic action, whereas prazosin (α1-adrenegic antagonist, 10 mg/kg, i.p.) did not. These results suggest that bvPLA2 treatment strongly alleviates oxaliplatin-induced acute cold and mechanical allodynia in mice through the activation of the noradrenergic system, via α2-adrenegic receptors, but not via the serotonergic system. PMID:26131771

  3. Interactions of two structurally related anionic phospholipids cardiolipin and phosphatidylglycerol with phospholipase A2. Langmuir monolayer studies.

    PubMed

    Broniatowski, Marcin; Urbaś, Monika

    2017-02-01

    Anionic phospholipids cardiolipins (CL) and phosphatidylglycerols (PG) dominate in the biomembranes of the majority of soil bacteria. CL to PG ratio differs between the species and is also dependent on the external conditions. CL/PG ratio is different in polluted than in unspoiled soils and it was hypothesized that it is connected with the activity of the membranelytic enzymes from the phospholipase A2 class (PLA2) as it was proved that persistent soil pollutants can activate PLA2. In our studies we applied the Langmuir monolayer technique and Brewster angle microscopy to elucidate the mechanism of the interactions of PLA2 with the model membranes formed by anionic phospholipids. It turned out that there are significant differences between CL and PG. The monolayer of PG is hydrolyzed readily and entirely, whereas for CL approximately 30% of the phospholipid molecules are hydrolyzed after which the enzyme is inhibited. The observed differences between PG and CL are strictly connected with the hydrophobicity of the generated lysolipids: lyso-PG and lyso-CL. Lyso-PG is water soluble and leaves the interface whereas lyso-CL is water-insoluble remains at the interface and modifies the monolayer properties. The second hydrolysis product - myristic acid (MA) forms crystallites of calcium myristate when generated from PG, whereas when generated from CL it is shielded by the lysolipid and does not interact with calcium. Therefore, on the basis of our study it can be concluded that the increase in CL content protects the soil bacteria from PLA2 activity and from the loss of calcium homeostasis.

  4. Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells.

    PubMed

    Yao, Mu; Xie, Chanlu; Kiang, Mei-Yee; Teng, Ying; Harman, David; Tiffen, Jessamy; Wang, Qian; Sved, Paul; Bao, Shisan; Witting, Paul; Holst, Jeff; Dong, Qihan

    2015-10-27

    Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A2α (cPLA2α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA2α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA2α with a phosphorylation at Ser505 was lower compared to their proliferating state. Conversely, the phospho-cPLA2α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA2α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G0/G1 and low percentage of cells in S and G2/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA2α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA2α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.

  5. Atomic resolution structure of prokaryotic phospholipase A2: analysis of internal motion and implication for a catalytic mechanism.

    PubMed

    Matoba, Yasuyuki; Sugiyama, Masanori

    2003-05-15

    We have found a secreted phospholipase A(2) (PLA(2), EC 3.1.1.4) from Streptomyces violaceoruber A-2688, which is the first PLA(2) identified in prokaryote, and determined its tertiary structure by NMR and X-ray analyses. In this study, we collected the X-ray diffraction data of the bacterial PLA(2) at room temperature (297 K) using conventional MoK(alpha) radiation and refined the structure at a 1.05 A resolution. The atomic resolution analysis led us to introduce disordered conformations and hydrogen atoms into a full anisotropic model. The molecular motion, which is expressed as the sum of rigid-body motion and internal motion of protein, is roughly estimated as the thermal motion when the X-ray diffraction data are collected at room temperature. In this study, we applied a TLS (rigid-body motion in terms of translation, libration, and screw motions) model to analyze the rigid-body motion of the bacterial PLA(2) and calculated the internal motion by subtracting the estimate of the rigid-body motion from the observed anisotropic temperature factor. We also subjected the TLS model to estimate the internal motion of the bovine pancreatic PLA(2) using the anisotropic temperature factor deposited in the Protein Data Bank. Both results indicate that the localization of regions exhibiting larger internal motion in the bacterial PLA(2) is almost the same as that in the bovine pancreatic PLA(2), suggesting that although the tertiary structure of the bacterial PLA(2) is strikingly different from that of the bovine pancreatic PLA(2), the internal motion, which is associated with the calcium(II) ion-binding, phospholipid-binding, and allosteric interfacial activation, is commonly observed in both PLA(2)s.

  6. Influence of Race and Sex on Lipoprotein-Associated Phospholipase A2 Levels: Observations from the Dallas Heart Study

    PubMed Central

    Brilakis, Emmanouil S.; Khera, Amit; McGuire, Darren K.; See, Raphael; Banerjee, Subhash; Murphy, Sabina A.; de Lemos, James A.

    2008-01-01

    Aims Most lipoprotein-associated phospholipase A2 (Lp-PLA2) studies included mainly white men. We sought to determine whether Lp-PLA2 levels differ according to race and sex. Methods Lp-PLA2 mass and activity were measured in 3,332 subjects age 30 to 65 participating in the Dallas Heart Study, a multiethnic, population-based, probability sample. Lp-PLA2 levels were compared between different race and sex groups. Results Mean age was 45 ± 9 years and 44% were men; 30% were white, 17% hispanic, and 53% black. Mean Lp-PLA2 activity and mass were 146 ± 40 nmol/min/mL and 191 ± 60 ng/mL, respectively. Lp-PLA2 activity was lower in women compared with men (134 ± 35 vs. 161 ± 40, p=0.001) and was lowest in black (136 ± 38), intermediate in hispanic (151 ± 36), and highest in white subjects (161 ± 39) (trend p=0.0001). In multivariable linear regression models, after adjusting for age, body mass index (BMI), smoking, total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, triglycerides and high sensitivity C-reactive protein (hsCRP), Lp-PLA2 activity was 19 nmol/min/mL higher in men vs. women (p<0.001); compared with black subjects, adjusted Lp-PLA2 activity was 11 and 20 nmol/min/mL higher in white and hispanic subjects, respectively (both p<0.001). Similar race and sex differences were observed for Lp-PLA2 mass. Conclusion Race and sex independently influence Lp-PLA2 activity and mass. Thresholds to define Lp-PLA2 elevation may need to be sex and race specific. PMID:18061193

  7. Influence of race and sex on lipoprotein-associated phospholipase A2 levels: observations from the Dallas Heart Study.

    PubMed

    Brilakis, Emmanouil S; Khera, Amit; McGuire, Darren K; See, Raphael; Banerjee, Subhash; Murphy, Sabina A; de Lemos, James A

    2008-07-01

    Most lipoprotein-associated phospholipase A2 (Lp-PLA2) studies included mainly white men. We sought to determine whether Lp-PLA2 levels differ according to race and sex. Lp-PLA2 mass and activity were measured in 3332 subjects age 30-65 participating in the Dallas Heart Study, a multiethnic, population-based, probability sample. Lp-PLA2 levels were compared between different race and sex groups. Mean age was 45+/-9 years and 44% were men; 30% were white, 17% hispanic, and 53% black. Mean Lp-PLA2 activity and mass were 146+/-40 nmol/min/mL and 191+/-60 ng/mL, respectively. Lp-PLA2 activity was lower in women compared with men (134+/-35 vs. 161+/-40, p=0.001) and was lowest in black (136+/-38), intermediate in hispanic (151+/-36), and highest in white subjects (161+/-39) (trend p=0.0001). In multivariable linear regression models, after adjusting for age, body mass index (BMI), smoking, total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, triglycerides and high sensitivity C-reactive protein (hsCRP), Lp-PLA2 activity was 19 nmol/min/mL higher in men vs. women (p<0.001); compared with black subjects, adjusted Lp-PLA2 activity was 11 and 20 nmol/min/mL higher in white and hispanic subjects, respectively (both p<0.001). Similar race and sex differences were observed for Lp-PLA2 mass. Race and sex independently influence Lp-PLA2 activity and mass. Thresholds to define Lp-PLA2 elevation may need to be sex and race specific.

  8. Secretory phospholipase A2 pathway in various types of lung injury in neonates and infants: a multicentre translational study

    PubMed Central

    2011-01-01

    Background Secretory phospholipase A2 (sPLA2) is a group of enzymes involved in lung tissue inflammation and surfactant catabolism. sPLA2 plays a role in adults affected by acute lung injury and seems a promising therapeutic target. Preliminary data allow foreseeing the importance of such enzyme in some critical respiratory diseases in neonates and infants, as well. Our study aim is to clarify the role of sPLA2 and its modulators in the pathogenesis and clinical severity of hyaline membrane disease, infection related respiratory failure, meconium aspiration syndrome and acute respiratory distress syndrome. sPLA2 genes will also be sequenced and possible genetic involvement will be analysed. Methods/Design Multicentre, international, translational study, including several paediatric and neonatal intensive care units and one coordinating laboratory. Babies affected by the above mentioned conditions will be enrolled: broncho-alveolar lavage fluid, serum and whole blood will be obtained at definite time-points during the disease course. Several clinical, respiratory and outcome data will be recorded. Laboratory researchers who perform the bench part of the study will be blinded to the clinical data. Discussion This study, thanks to its multicenter design, will clarify the role(s) of sPLA2 and its pathway in these diseases: sPLA2 might be the crossroad between inflammation and surfactant dysfunction. This may represent a crucial target for new anti-inflammatory therapies but also a novel approach to protect surfactant or spare it, improving alveolar stability, lung mechanics and gas exchange. PMID:22067747

  9. Expression of group IIA phospholipase A2 is an independent predictor of favorable outcome for patients with gastric cancer.

    PubMed

    Wang, Xi; Huang, Chun-Jin; Yu, Guan-Zhen; Wang, Jie-Jun; Wang, Rui; Li, Yu-Mei; Wu, Qiong

    2013-10-01

    Growing evidence suggests that phospholipase A2 (PLA2) plays a pivotal role in tumorigenesis in human gastrointestinal cancer. One of the well-studied isoforms of PLA2, group IIA PLA2 (PLA2G2A), appears to exert its protumorigenic or antitumorigenic effects in a tissue-specific manner. The present study was designed to determine the expression profile and prognostic value of PLA2G2A in gastric cancer in a large Chinese cohort. By using real-time polymerase chain reaction, the amount of PLA2G2A messenger RNA in 60 pairs of fresh gastric tumors and adjacent noncancerous mucosa was measured. The immunostaining of PLA2G2A in 866 gastric cancers with paired noncancerous tissues was assayed. No expression of PLA2G2A was found in normal gastric mucosa, and focal expression of PLA2G2A was noticed in intestinal metaplasia, whereas significantly increased expression of PLA2G2A was observed in the cytoplasm of gastric cancer cells. Furthermore, the extent of PLA2G2A expression was associated with tumor size (P < .001), tumor differentiation (P = .001), T class (P < .001), N class (P < .001), and TNM stage (P < .001) of gastric cancer. Multivariate analysis showed that PLA2G2A expression was an independent predictor of survival for patients with gastric cancer (P = .024). Expression of PLA2G2A seems to be protective for patients with gastric cancer (hazard ratio, 1.423; 95% confidence interval, 1.047-1.935), and it may be a target for achieving better treatment outcomes.

  10. Extracellular phospholipase A2 secretion is a common effector pathway of interleukin-1 and tumour necrosis factor action.

    PubMed

    Vadas, P; Pruzanski, W; Stefanski, E; Ellies, L G; Aubin, J E; Sos, A; Melcher, A

    1991-06-01

    Inflammatory processes are characterized by increased levels of extracellular phospholipase A2 (PLA2) and cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF). IL-1, TNF and PLA2 share a number of proinflammatory, arthritogenic effects. The sequential induction, first of the cytokines followed by PLA2, suggests that these cytokines may regulate synthesis and secretion of PLA2. To test this postulate, foetal rat calvarial bone-forming cells (FRCC) were treated with recombinant human IL-1 and TNF and extracellular PLA2 release was quantitated. Both IL-1 and TNF induced the de novo synthesis of PLA2 in a concentration-dependent manner. Continuous exposure of FRCC in primary culture to IL-1 (50 units/ml) over 15 days resulted in as much as 100-fold increase in PLA2 secretion. IL-1 (50 units/ml) added to post-confluent cultures for a 48-h pulse increased PLA2 activity 9.4-fold. The combination of IL-1 (50 units/ml) and TNF (500 units/ml) was synergistic with an observed increase in extracellular PLA2 secretion of 146-fold following a 48-h pulse. Interleukin-6, alone or in combination with IL-1 or TNF, did not further enhance PLA2 synthesis of secretion. Cytokine-induced synthesis of PLA2 was inhibited 80% by 10 microM cycloheximide but not by dexamethasone over the range of 10(-6) to 10(-8) M. FRCC-derived PLA2 was neutral-active with a pH optimum of 6-7.5 and was calcium-dependent with optimal activity in the presence of 2-7 mM calcium. It had absolute 2-acyl specificity using micellar phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

    PubMed Central

    Beck, Goichi; Shinzawa, Koei; Hayakawa, Hideki; Baba, Kousuke; Yasuda, Toru; Sumi-Akamaru, Hisae; Tsujimoto, Yoshihide; Mochizuki, Hideki

    2015-01-01

    Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA2β), which is encoded by the PLA2G6 gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA2β-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA2β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA2β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA2β-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA2β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction. PMID:26506412

  12. Primary structures and partial toxicological characterization of two phospholipases A2 from Micrurus mipartitus and Micrurus dumerilii coral snake venoms.

    PubMed

    Rey-Suárez, Paola; Núñez, Vitelbina; Saldarriaga-Córdoba, Mónica; Lomonte, Bruno

    2017-03-14

    Snake venom phospholipases A2 (PLA2) share high sequence identities and a conserved structural scaffold, but show important functional differences. Only a few PLA2s have been purified and characterized from coral snake (Micrurus spp.) venoms, and their role in envenomation remains largely unknown. In this report, we describe the isolation, sequencing and partial functional characterization of two Micrurus PLA2s: MmipPLA2 from Micrurus mipartitus and MdumPLA2 from Micrurus dumerilii, two species of clinical importance in Colombia. MmipPLA2 consisted of 119 amino acid residues with a predicted pI of 8.4, whereas MdumPLA2 consisted of 117 residues with a pI of 5.6. Both PLA2s showed the conserved 'group I' cysteine pattern and were enzymatically active, although MdumPLA2 had higher activity. The two enzymes differed notably in their toxicity, with MmipPLA2 being highly lethal to mice and mildly myotoxic, whereas MdumPLA2 was not lethal (up to 3 μg/g body weight) but strongly myotoxic. MdumPLA2 displayed higher anticoagulant activity than MmipPLA2in vitro and caused more sustained edema in the mouse footpad assay. Neither of these enzymes was cytolytic to cultured skeletal muscle C2C12 myotubes. Based on their structural differences, the two enzymes were placed in separate lineages in a partial phylogeny of Micrurus venom PLA2s and this classification agreed with their divergent biological activities. Overall, these findings highlight the structural and functional diversity of Micrurus venom PLA2.

  13. Nanomolar vitamin E alpha-tocotrienol inhibits glutamate-induced activation of phospholipase A2 and causes neuroprotection.

    PubMed

    Khanna, Savita; Parinandi, Narasimham L; Kotha, Sainath R; Roy, Sashwati; Rink, Cameron; Bibus, Douglas; Sen, Chandan K

    2010-03-01

    Our previous works have elucidated that the 12-lipoxygenase pathway is directly implicated in glutamate-induced neural cell death, and that such that toxicity is prevented by nM concentrations of the natural vitamin E alpha-tocotrienol (TCT). In the current study we tested the hypothesis that phospholipase A(2) (PLA(2)) activity is sensitive to glutamate and mobilizes arachidonic acid (AA), a substrate for 12-lipoxygenase. Furthermore, we examined whether TCT regulates glutamate-inducible PLA(2) activity in neural cells. Glutamate challenge induced the release of [(3)H]AA from HT4 neural cells. Such response was attenuated by calcium chelators (EGTA and BAPTA), cytosolic PLA(2) (cPLA(2))-specific inhibitor (AACOCF(3)) as well as TCT at 250 nM. Glutamate also caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of phospholipid-esterified AA in neural cells. Furthermore, glutamate induced a time-dependent translocation and enhanced serine phosphorylation of cPLA(2) in the cells. These effects of glutamate on fatty acid levels and on cPLA(2) were significantly attenuated by nM TCT. The observations that AACOCF(3), transient knock-down of cPLA(2) as well as TCT significantly protected against the glutamate-induced death of neural cells implicate cPLA(2) as a TCT-sensitive mediator of glutamate induced neural cell death. This work presents first evidence recognizing glutamate-induced changes in cPLA(2) as a novel mechanism responsible for neuroprotection observed in response to nanomolar concentrations of TCT.

  14. Lipoprotein-Associated Phospholipase A2 and Incident Peripheral Arterial Disease in Older Adults: The Cardiovascular Health Study.

    PubMed

    Garg, Parveen K; Arnold, Alice M; Hinckley Stukovsky, Karen D; Koro, Carol; Jenny, Nancy S; Mukamal, Kenneth J; Criqui, Michael H; Furberg, Curt D; Newman, Anne B; Cushman, Mary

    2016-04-01

    Although prior studies report a relationship between elevated lipoprotein-associated phospholipase A2 (Lp-PLA2) and incident cardiovascular disease, the prospective association of Lp-PLA2 with incident peripheral arterial disease (PAD) has not been studied. We investigated the association between Lp-PLA2 mass and activity and the risk of developing clinical PAD and low ankle-brachial index (ABI). Among Cardiovascular Health Study participants, a population-based cohort of 5888 adults aged ≥65 years enrolled in 1989 to 1990, Lp-PLA2 mass and activity were measured in 4537 individuals without baseline PAD. Clinical PAD, defined as leg artery revascularization or diagnosed claudication, was ascertained through 2011. Incident low ABI, defined as ABI <0.9 and decline of ≥0.15, was assessed among 3537 individuals who had an ABI >0.9 at baseline and a second ABI measurement 3 or 6 years later. Analyses were adjusted for demographics, cholesterol, smoking, comorbidities, and C-reactive protein. Each standard deviation increment in Lp-PLA2 mass (117 ng/mL) was associated with a higher risk of developing clinical PAD (hazard ratio 1.28; 95% confidence interval 1.13, 1.45) and incident low ABI (odds ratio 1.16; 95% confidence interval 1.00, 1.33). Results per standard deviation increment in Lp-PLA2 activity (13 nmol/min per mL) were similar for clinical PAD (hazard ratio 1.24; 95% confidence interval 1.07, 1.44) and low ABI (odds ratio 1.28; 95% confidence interval 1.09, 1.50). Higher Lp-PLA2 mass and activity were associated with development of both incident clinical PAD and low ABI. Future studies are needed to determine whether pharmacological inhibition of Lp-PLA2 reduces the incidence of PAD. © 2016 American Heart Association, Inc.

  15. Secreted phospholipase A2 inhibitor modulates fatty acid composition and reduces obesity-induced inflammation in Beagle dogs.

    PubMed

    Xu, J; Bourgeois, H; Vandermeulen, E; Vlaeminck, B; Meyer, E; Demeyere, K; Hesta, M

    2015-05-01

    Secreted phospholipase A2 inhibitor (sPLA2i) has been reported to have an anti-inflammatory function by blocking the production of inflammatory mediators. Obesity is characterized by low-grade inflammation and oxidative stress. The aim of this study was to investigate the effects of dietary supplementation of sPLA2i on inflammation, oxidative stress and serum fatty acid profile in dogs. Seven obese and seven lean Beagle dogs were used in a 28-day double blind cross-over design. Dogs were fed a control diet without supplemental sPLA2i or an sPLA2i supplemented diet. The sPLA2i diet decreased plasma fibrinogen levels and increased the protein:fibrinogen ratio in obese dogs to levels similar to those of lean dogs fed the same diet. Obese dogs had a higher plasma concentration of the lipophilic vitamin A with potential antioxidative capacity and a lower ratio of retinol binding protein 4:vitamin A compared to lean dogs, independent of the diets. A higher proportion of myristic acid (C14:0) and a lower proportion of linoleic acid (C18:2n-6) were observed in the dogs fed with the sPLA2i diet compared to dogs fed with the control diet. Furthermore, a higher ratio of n-6 to n-3, a lower proportion of n-3 polyunsaturated fatty acids and lower omega-3 index were observed in obese compared to lean dogs. The results indicate that obese dogs are characterized by a more 'proinflammatory' serum fatty acid profile and that diet inclusion of sPLA2i may reduce inflammation and alter fatty acid profile. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Neutralizing properties of Musa paradisiaca L. (Musaceae) juice on phospholipase A2, myotoxic, hemorrhagic and lethal activities of crotalidae venoms.

    PubMed

    Borges, M H; Alves, D L F; Raslan, D S; Piló-Veloso, D; Rodrigues, V M; Homsi-Brandeburgo, M I; de Lima, M E

    2005-04-08

    The use of plants as medicine has been referred to since ancient peoples, perhaps as early as Neanderthal man. Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. The study of how people of different culture use plants in particular ways has led to the discovery of important new medicines. In this work, we verify the possible activity of Musa paradisiaca L. (Musaceae) against the toxicity of snake venoms. Musa paradisiaca, an important source of food in the world, has also been reported to be popularly used as an anti-venom. Interaction of Musa paradisiaca extract (MsE) with snake venom proteins has been examined in this study. Phospholipase A2 (PLA2), myotoxic and hemorrhagic activities, including lethality in mice, induced by crotalidae venoms were significantly inhibited when different amounts of MsE were mixed with these venoms before assays. On the other hand, mice that received MsE and venoms without previous mixture or by separated routes were not protected against venom toxicity. Partial chemical characterization of MsE showed the presence of polyphenols and tannins and they are known to non-specifically inactivate proteins. We suggest that these compounds can be responsible for the in vitro inhibition of the toxic effects of snake venoms. In conclusion, according to our results, using mice as experimental model, MsE does not show protection against the toxic effects of snake venoms in vivo, but if was very effective when