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Sample records for a2 pla2 activity

  1. Inhibition of phospholipase A2 (PLA2) activity by nifedipine and nisoldipine is independent of their calcium-channel-blocking activity

    SciTech Connect

    Chang, J.; Blazek, E.; Carlson, R.P.

    1987-09-01

    The effects of several calcium antagonists on phospholipase A2 (PLA2) activity were examined. Nifedipine and nisoldipine inhibited a cell-free preparation of PLA2 in a dose-dependent manner with maximal inhibition of 71-77% observed at 100 microM. More potent or equipotent dihydropyridine calcium antagonists such as nitrendipine and felodipine did not inhibit PLA2 activity. In addition, nondihydropyridine calcium antagonists such as diltiazem, verapamil, and cinnarazine failed to reduce PLA2 activity markedly. Nifedipine and nisoldipine also reduced PLA2 activity in intact mouse peritoneal macrophages where PLA2 activity was monitored by free (/sup 14/C)arachidonic acid release from (/sup 14/C)arachidonic acid-prelabeled cells. When levels ofmore » PGE2 and LTC4 were measured by radioimmunoassay, it was found that the synthesis of these two metabolites was concomitantly inhibited by nifedipine and nisoldipine. In vivo, nifedipine and nisoldipine inhibited tetradecanoylphorbol acetate (TPA) induced ear edema. UV irradiation of nifedipine and nisoldipine (which destroys the slow calcium-channel-blocking activity of these compounds) did not result in a loss of PLA2 inhibitory activity. In fact, in both instances the UV-irradiated forms of nifedipine and nisoldipine were slightly more potent PLA2 inhibitors than the parent compound alone. We therefore conclude that the ability of nifedipine and nisoldipine to inhibit PLA2 was direct and unrelated to their actions on slow calcium channels.« less

  2. Antibacterial activity of an acidic phospholipase A2 (NN-XIb-PLA2) from the venom of Naja naja (Indian cobra).

    PubMed

    Sudarshan, S; Dhananjaya, B L

    2016-01-01

    The resistance of bacteria against the use of conventional antibiotics has become a serious threat to public health and considering the associated side effect with antibiotics; new strategies to find and develop new molecules with novel modes of action has received grate attention in recent years. In this study, when the antibacterial potential of an acidic protein-NN-XIb-PLA2 (Naja naja venom phospholipase A2 fraction-XIb) of Naja naja venom was evaluated, it showed significant bactericidal action against the human pathogenic strains tested. It inhibited more effectively the gram positive bacteria like Staphylococcus aureus and Bacillus subtilis, when compared to gram negative bacteria like Escherichia coli, Vibrio cholerae, Klebsiell pneumoniae and Salmonella paratyphi. It inhibited the bacterial growth, with a MIC values ranging from 17 to 20 µg/ml. It was interesting to observe that NN-XIb-PLA2 showed comparable antibacterial activity to the used standards antibiotics. It was found that their was a strong correlation between PLA2 activities, hemolytic and antibacterial activity. Furthermore, it is found that in the presence of p-bromophenacyl bromide (p-BPB), there is a significant decrease in enzymatic activity and associated antibacterial activities, suggesting that a strong association exists between catalytic activity and antimicrobial effects, which thereby destabilize the membrane bilayer. These studies encourage further in dept study on molecular mechanisms of bactericidal properties of NN-XIb-PLA2 and thereby help in development of this protein into a possible therapeutic lead molecule for treating bacterial infections.

  3. Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris.

    PubMed

    Karray, Aida; Bou Ali, Madiha; Kharrat, Nedia; Gargouri, Youssef; Bezzine, Sofiane

    2018-03-01

    Secretory class V phospholipase A2 (PLA2-V) has been shown to be involved in inflammatory processes in cellular studies, but the biochemical and physical properties of this important enzyme have been unclear. As a first step towards understanding the structure, function and regulation of this PLA2, we report the expression and characterization of PLA2-V from chicken (ChPLA2-V). The ChPLA2-V cDNA was synthesized from chicken heart polyA mRNA by RT-PCR, and an expression construct containing the PLA2 was established. After expression in Pichia pastoris cells, the active enzyme was purified. The purified ChPLA2-V protein was biochemically and physiologically characterized. The recombinant ChPLA2-V has an absolute requirement for Ca 2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with phosphatidylcholine as substrate. ChPLA2-V was found to display potent Gram-positive and Gram-negative bactericidal activity and antifungal activity in vitro. The purified enzyme ChPLA2-V with much stronger anticoagulant activity compared with the intestinal and pancreatic chicken PLA2-V was approximately 10 times more active. Chicken group V PLA2, like mammal one, may be considered as a future therapeutic agents against fungal and bacterial infections and as an anticoagulant agent. Copyright © 2017. Published by Elsevier B.V.

  4. Novel genetic approach to investigate the role of plasma secretory phospholipase A2 (sPLA2)-V isoenzyme in coronary heart disease: modified Mendelian randomization analysis using PLA2G5 expression levels.

    PubMed

    Holmes, Michael V; Exeter, Holly J; Folkersen, Lasse; Nelson, Christopher P; Guardiola, Montse; Cooper, Jackie A; Sofat, Reecha; Boekholdt, S Matthijs; Khaw, Kay-Tee; Li, Ka-Wah; Smith, Andrew J P; Van't Hooft, Ferdinand; Eriksson, Per; Franco-Cereceda, Anders; Asselbergs, Folkert W; Boer, Jolanda M A; Onland-Moret, N Charlotte; Hofker, Marten; Erdmann, Jeanette; Kivimaki, Mika; Kumari, Meena; Reiner, Alex P; Keating, Brendan J; Humphries, Steve E; Hingorani, Aroon D; Mallat, Ziad; Samani, Nilesh J; Talmud, Philippa J

    2014-04-01

    Secretory phospholipase A2 (sPLA2) enzymes are considered to play a role in atherosclerosis. sPLA2 activity encompasses several sPLA2 isoenzymes, including sPLA2-V. Although observational studies show a strong association between elevated sPLA2 activity and CHD, no assay to measure sPLA2-V levels exists, and the only evidence linking the sPLA2-V isoform to atherosclerosis progression comes from animal studies. In the absence of an assay that directly quantifies sPLA2-V levels, we used PLA2G5 mRNA levels in a novel, modified Mendelian randomization approach to investigate the hypothesized causal role of sPLA2-V in coronary heart disease (CHD) pathogenesis. Using data from the Advanced Study of Aortic Pathology, we identified the single-nucleotide polymorphism in PLA2G5 showing the strongest association with PLA2G5 mRNA expression levels as a proxy for sPLA2-V levels. We tested the association of this SNP with sPLA2 activity and CHD events in 4 prospective and 14 case-control studies with 27 230 events and 70 500 controls. rs525380C>A showed the strongest association with PLA2G5 mRNA expression (P=5.1×10(-6)). There was no association of rs525380C>A with plasma sPLA2 activity (difference in geometric mean of sPLA2 activity per rs525380 A-allele 0.4% (95% confidence intervals [-0.9%, 1.6%]; P=0.56). In meta-analyses, the odds ratio for CHD per A-allele was 1.02 (95% confidence intervals [0.99, 1.04]; P=0.20). This novel approach for single-nucleotide polymorphism selection for this modified Mendelian randomization analysis showed no association between rs525380 (the lead single-nucleotide polymorphism for PLA2G5 expression, a surrogate for sPLA2-V levels) and CHD events. The evidence does not support a causal role for sPLA2-V in CHD.

  5. Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective.

    PubMed Central

    Williams, Scott D; Gottlieb, Roberta A

    2002-01-01

    Calcium-independent phospholipase A(2) (iPLA(2)) is the predominant phospholipase A(2) present in myocardium, and its pathophysiological role in acute myocardial infarction has been suggested by the rapid increase in membrane-associated iPLA(2) activity during myocardial ischaemia and reperfusion (I/R). We therefore examined iPLA(2) in mitochondrial fractions prepared from Langendorff-perfused adult rabbit hearts. Our studies indicate that iPLA(2)beta is present in rabbit heart mitochondrial inner membranes with no apparent translocation during ischaemia, I/R or preconditioning. Mitochondrion-associated iPLA(2) was catalytically competent and exhibited 2-, 3- and 2.5-fold increases in measured iPLA(2) activity following ischaemia, I/R and preconditioning, respectively, when compared with the activity of iPLA(2) measured in mitochondria from control hearts. Mitochondrial phospholipids are essential for maintaining the ordered structure and function of the organelle. I/R resulted in a rapid overall decrease in phosphatidylcholine and phosphatidylethanolamine glycerophospholipid species, as determined by electrospray ionization MS, that was partially alleviated by pretreatment of hearts with the iPLA(2)-specific inhibitor, bromoenol lactone (BEL). Pretreatment of I/R hearts with 10 microM BEL significantly reduced the infarct size almost to that of continuously perfused hearts and was cardioprotective only when administered prior to ischaemia. Cardioprotection by BEL was reversed by the simultaneous perfusion of 100 microM 5-hydroxydecanoate, implicating the mitochondrial K(ATP) channel in BEL-mediated protection from I/R. Preconditioning also significantly reduced the infarct size in response to I/R but protection was lost by concurrent perfusion of 10 microM arachidonic acid. Taken together, these data strongly implicate mitochondria-associated iPLA(2) in the signal transduction of myocardial I/R injury. PMID:11829736

  6. Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice

    PubMed Central

    Cheon, Yewon; Kim, Hyung-Wook; Igarashi, Miki; Modi, Hiren R.; Chang, Lisa; Ma, Kaizong; Greenstein, Deanna; Wohltmann, Mary; Turk, John; Rapoport, Stanley I; Taha, Ameer Y.

    2012-01-01

    Calcium-independent phospholipase A2 group VIA (iPLA2β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA2β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA2β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA2β+/+) and knockout (iPLA2β−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA2, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA2β+/+ mice, iPLA2β−/− mice showed decreased rates of incorporation of unesterified DHA in plasma into brain phospholipids, reduced concentration of several fatty acid residues (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover rates in brain phospholipids did not differ between genotypes. In iPLA2β−/− mice, brain levels of iPLA2β mRNA, protein, and activity were decreased, as was the iPLA2γ (Group VIB PLA2) mRNA level. Brain levels of secretory sPLA2-V mRNA, protein, and activity and cytosolic cPLA2-IVA mRNA were increased in iPLA2β−/− mice. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, genetic iPLA2β deficiency in mice is associated with profound reorganization of lipid-metabolizing enzyme expression and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to the neurologic abnormalities in humans with iPLA2β mutations. PMID:22349267

  7. Isolation and characterization of bioactive compounds of Clematis gouriana Roxb. ex DC against snake venom phospholipase A2 (PLA2) computational and in vitro insights.

    PubMed

    Muthusamy, Karthikeyan; Chinnasamy, Sathishkumar; Nagarajan, Subbiah; Sivaraman, Thirunavukkarasu; Chinnasamy, Selvakumar

    2017-07-01

    Bioactive compounds were isolated from Clematis gouriana Roxb. ex DC. The compounds were separated, characterized, the structures elucidated and submitted to the PubChem Database. The PubChem Ids SID 249494134 and SID 249494135 were tested against phospholipases A 2 (PLA 2 ) of Naja naja (Indian cobra) venom for PLA 2 activity. Both the compounds showed promising inhibitory activity; computational data also substantiated the results. The two compounds underwent density functional theory calculation to observe the chemical stability and electrostatic potential profile. Molecular interactions between the compounds and PLA 2 were observed at the binding pocket of the PLA 2 protein. Further, this protein-ligand complexes were simulated for a timescale of 100 ns of molecular dynamics simulation. Experimental and computational results showed significant PLA 2 inhibition activity.

  8. Crystallization and preliminary X-ray diffraction studies of BmooPLA2-I, a platelet-aggregation inhibitor and hypotensive phospholipase A2 from Bothrops moojeni venom

    PubMed Central

    Salvador, Guilherme H. M.; Marchi-Salvador, Daniela P.; Silveira, Lucas B.; Soares, Andreimar M.; Fontes, Marcos R. M.

    2011-01-01

    Phospholipases A2 (PLA2s) are enzymes that cause the liberation of fatty acids and lysophospholipids by the hydrolysis of membrane phospholipids. In addition to their catalytic action, a wide variety of pharmacological activities have been described for snake-venom PLA2s. BmooPLA2-I is an acidic, nontoxic and catalytic PLA2 isolated from Bothrops moojeni snake venom which exhibits an inhibitory effect on platelet aggregation, an immediate decrease in blood pressure, inducing oedema at a low concentration, and an effective bactericidal effect. BmooPLA2-I has been crystallized and X-ray diffraction data have been collected to 1.6 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C2221, with unit-cell parameters a = 39.7, b = 53.2, c = 89.2 Å. The molecular-replacement solution of BmooPLA2-I indicated a monomeric conformation, which is in agreement with nondenaturing electrophoresis and dynamic light-scattering experiments. A comparative study of this enzyme with the acidic PLA2 from B. jararacussu (BthA-I) and other toxic and nontoxic PLA2s may provide important insights into the functional aspects of this class of proteins. PMID:21821890

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

    PubMed

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

    2016-08-01

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

  10. BmajPLA2-II, a basic Lys49-phospholipase A2 homologue from Bothrops marajoensis snake venom with parasiticidal potential.

    PubMed

    Grabner, Amy N; Alfonso, Jorge; Kayano, Anderson M; Moreira-Dill, Leandro S; Dos Santos, Ana Paula de A; Caldeira, Cleópatra A S; Sobrinho, Juliana C; Gómez, Ana; Grabner, Fernando P; Cardoso, Fabio F; Zuliani, Juliana Pavan; Fontes, Marcos R M; Pimenta, Daniel C; Gómez, Celeste Vega; Teles, Carolina B G; Soares, Andreimar M; Calderon, Leonardo A

    2017-09-01

    Snake venoms contain various proteins, especially phospholipases A 2 (PLA 2 s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA 2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA 2 -II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA 2 -II as a basic Lys49 PLA 2 homologue, compatible with other basic snake venom PLA 2 s (svPLA 2 ), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA 2 -II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100μg/mL. The venom and BmajPLA 2 -II presented IC 50 of 0.14±0.08 and 6.41±0.64μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC 50 cytotoxicity values against HepG2 cells of 43.64±7.94 and >150μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated. Copyright © 2017. Published by Elsevier B.V.

  11. Lp-PLA2 activity is associated with increased risk of diabetic retinopathy: a longitudinal disease progression study.

    PubMed

    Siddiqui, Moneeza K; Kennedy, Gwen; Carr, Fiona; Doney, Alexander S F; Pearson, Ewan R; Morris, Andrew D; Johnson, Toby; McLaughlin, Megan M; Williams, Rachel E; Palmer, Colin N A

    2018-04-06

    The aim of the study was to examine the association between lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) activity levels and incident diabetic retinopathy and change in retinopathy grade. This was a cohort study of diabetic participants with serum collected at baseline and routinely collected diabetic retinal screening data. Participants with type 2 diabetes from the GoDARTS (Genetics of Diabetes Audit and Research in Tayside Scotland) cohort were used. This cohort is composed of individuals of white Scottish ancestry from the Tayside region of Scotland. Survival analysis accounting for informative censoring by modelling death as a competing risk was performed for the development of incident diabetic retinopathy from a disease-free state in a 3 year follow-up period (n = 1364) by stratified Lp-PLA 2 activity levels (in quartiles). The same analysis was performed for transitions to more severe grades. The hazard of developing incident diabetic retinopathy was 2.08 times higher (95% CI 1.64, 2.63) for the highest quartile of Lp-PLA 2 activity compared with the lowest. Higher Lp-PLA 2 activity levels were associated with a significantly increased risk for transitions to all grades. The hazards of developing observable (or more severe) and referable (or more severe) retinopathy were 2.82 (95% CI 1.71, 4.65) and 1.87 (95% CI 1.26, 2.77) times higher for the highest quartile of Lp-PLA 2 activity compared with the lowest, respectively. Higher Lp-PLA 2 levels are associated with increased risk of death and the development of incident diabetic retinopathy, as well as transitions to more severe grades of diabetic retinopathy. These associations are independent of calculated LDL-cholesterol and other traditional risk factors. Further, this biomarker study shows that the association is temporally sensitive to the proximity of the event to measurement of Lp-PLA 2.

  12. Impact of 8-week linoleic acid intake in soy oil on Lp-PLA2 activity in healthy adults.

    PubMed

    Kim, Minkyung; Kim, Minjoo; Lee, Ayoung; Yoo, Hye Jin; Her, Jung Soo; Jee, Sun Ha; Lee, Jong Ho

    2017-01-01

    No intervention follow-up study has examined the association between plasma n-6 polyunsaturated fatty acids (PUFAs) and lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ), which is a risk factor for cardiovascular disease (CVD). We aimed to determine whether the administration of linoleic acid (LA, 18:2n-6) in soy oil affected Lp-PLA 2 activity in healthy adults. Self-reported healthy participants ( n  = 150) were randomly assigned to three groups: a low LA group, in which 10 mL soy oil was replaced with one apple; a medium LA group, in which the typical food intake was maintained; and a high LA group, in which 1/3 cup of cooked refined rice was replaced with 9.9 g of soy oil capsules daily. Plasma fatty acids and Lp-PLA 2 activity were measured along with other CVD risk factors. After 8 weeks of treatment, plasma LA levels decreased in the low LA group and increased in the high LA group. The high LA group showed greater increases in apolipoprotein B (apoB) and oxidized low-density lipoprotein (ox-LDL) than those in the low LA group. Plasma LA levels and Lp-PLA 2 activities demonstrated greater increases in the high LA group than those in the medium and low LA groups. Changes in plasma LA positively and independently correlated with changes in Lp-PLA 2 activity, which was negatively correlated with changes in collagen-epinephrine closure time (CEPI-CT). An increase in plasma LA following intake of soy oil was independently associated with Lp-PLA 2 activity, which was also related to apoB, ox-LDL and CEPI-CT. ClinicalTrail.gov Identifier: NCT02753907, registered 25 April 2016 (retrospectively registered).

  13. 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 A 2 receptor (PLA 2 R) is a landmark breakthrough for understanding adult idiopathic membranous nephropathy (iMN). However, potential roles for PLA 2 R 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 PLA 2 R 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 PLA 2 R staining procedure. Granular staining of PLA 2 R 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 PLA 2 R glomerular staining in these patients was weaker than that of a PLA 2 R-positive adult iMN patient. There were no PLA 2 R-positive patients among our cohort of children younger than 10 years. This preliminary study suggests PLA 2 R may play a role in some adolescent and preteen iMN patients but may be less frequently associated with iMN during childhood.

  14. Conservation analysis and decomposition of residue correlation networks in the phospholipase A2 superfamily (PLA2s): Insights into the structure-function relationships of snake venom toxins.

    PubMed

    Oliveira, Alberto; Bleicher, Lucas; Schrago, Carlos G; Silva Junior, Floriano Paes

    2018-05-01

    Phospholipases A2 (PLA 2 s) comprise a superfamily of glycerophospholipids hydrolyzing enzymes present in many organisms in nature, whose catalytic activity was majorly unveiled by analysis of snake venoms. The latter have pharmaceutical and biotechnological interests and can be divided into different functional sub-classes. Our goal was to identify important residues and their relation to the functional and class-specific characteristics in the PLA 2 s family with special emphasis on snake venom PLA 2 s (svPLA 2 s). We identified such residues by conservation analysis and decomposition of residue coevolution networks (DRCN), annotated the results based on the available literature on PLA 2 s, structural analysis and molecular dynamics simulations, and related the results to the phylogenetic distribution of these proteins. A filtered alignment of PLA 2 s revealed 14 highly conserved positions and 3 sets of coevolved residues, which were annotated according to their structural or functional role. These residues are mostly involved in ligand binding and catalysis, calcium-binding, the formation of disulfide bridges and a hydrophobic cluster close to the binding site. An independent validation of the inference of structure-function relationships from our co-evolution analysis on the svPLA2s family was obtained by the analysis of the pattern of selection acting on the Viperidae and Elapidae lineages. Additionally, a molecular dynamics simulation on the Lys49 PLA 2 from Agkistrodon contortrix laticinctus was carried out to further investigate the correlation of the Lys49-Glu69 pair. Our results suggest this configuration can result in a novel conformation where the binding cavity collapses due to the approximation of two loops caused by a strong salt bridge between Glu69 and Arg34. Finally, phylogenetic analysis indicated a correlation between the presence of residues in the coevolved sets found in this analysis and the clade localization. The results provide a guide for

  15. Interactions of pharmacologically active snake venom sPLA2 with different cell lines

    PubMed Central

    Doumanov, Jordan; Mladenova, Kirilka; Aleksandrov, Radoslav; Danovski, Georgi; Petrova, Svetla

    2014-01-01

    Secreted Phospholipases A2 (sPLA2s) represent a large family of structurally related enzymes, which target different tissues and organs and induce numerous pharmacological effects based on their catalytic specificity – hydrolysis of the sn-2 ester bond of glycerophospholipids. The neurotoxin vipoxin, isolated from the venom of Vipera ammodytes meriodionalis, is a heterodimeric postsynaptic ionic complex composed of two protein subunits – a basic and toxic His48 sPLA2 enzyme and an acidic, enzymatically inactive and non-toxic component. In this paper, for the first time, we demonstrate that vipoxin sPLA2 enzyme affects cell integrity and viability of four cell types and causes different cell responses. The most dramatic local tissue effects were observed with RPE-1 (retinal pigment epithelial) cells followed by A549 (adenocarcinomic human alveolar epithelial) cells and MDCK (Madin-Darby Canine Kidney epithelial) cells. Products of the enzymatic reaction, lysophospholipids and unsaturated free fatty acids, act as lipid mediators that can induce membrane damaging or can stimulate cell proliferation. Our preliminary results on the cytotoxic effect of vipoxin sPLA2 on A549 cells are promising in searching of its eventual anticancer potential. PMID:26019578

  16. Cardiac Myocyte-specific Knock-out of Calcium-independent Phospholipase A2γ (iPLA2γ) Decreases Oxidized Fatty Acids during Ischemia/Reperfusion and Reduces Infarct Size *

    PubMed Central

    Moon, Sung Ho; Mancuso, David J.; Sims, Harold F.; Liu, Xinping; Nguyen, Annie L.; Yang, Kui; Guan, Shaoping; Dilthey, Beverly Gibson; Jenkins, Christopher M.; Weinheimer, Carla J.; Kovacs, Attila; Abendschein, Dana; Gross, Richard W.

    2016-01-01

    Calcium-independent phospholipase A2γ (iPLA2γ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA2γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2γ knock-out (CMiPLA2γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2γKO mice demonstrated attenuated Ca2+-induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA2γKO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA2γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca2+ by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA2γ, these results are consistent with salvage of myocardium after I/R by iPLA2γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion. PMID:27453526

  17. An acidic phospholipase A(2) (RVVA-PLA(2)-I) purified from Daboia russelli venom exerts its anticoagulant activity by enzymatic hydrolysis of plasma phospholipids and by non-enzymatic inhibition of factor Xa in a phospholipids/Ca(2+) independent manner.

    PubMed

    Saikia, Debashree; Thakur, Rupamoni; Mukherjee, Ashis K

    2011-05-01

    A homodimeric acidic PLA(2) (RVVA-PLA(2)-I) of 58.0 kDa molecular weight purified from Russell's viper (Daboia russelli) venom demonstrated dose-dependent catalytic, strong anticoagulant and indirect hemolytic activities and inhibited blood coagulation cascade in both enzymatic and non-enzymatic mechanisms. In in vitro condition, RVVA-PLA(2)-I showed preferential hydrolysis of phosphatidylcholine with a K(m) and V(max) values of 0.65 mM and 28.9 μmol min(-1), respectively. Biochemical study and GC-analysis of plasma phospholipids hydrolysis by PLA(2) revealed that anticoagulant activity of RVVA-PLA(2)-I was partly attributed by the enzymatic hydrolysis of pro-coagulant phospholipids PC, followed by PS. The spectrofluorometric and gel-filtration analyses documented binding of RVVA-PLA(2)-I with activated factor X and PC; however, it does not bind with factor Va, prothrombin and thrombin. Therefore, this anticoagulant PLA(2) inhibits the blood coagulation cascade non-enzymatically by binding with coagulation factor Xa, even in the absence of phospholipids and Ca(2+) and thus slows down the blood coagulation by partially inhibiting the prothrombin activation. Chemical modification of essential amino acids present in the active site, neutralization with Azadirachta indica leaves extract (AIPLAI) and heat-inactivation study reinforce the association of catalytic and anticoagulant activity of RVVA-PLA(2)-I and also throw a light on its non-enzymatic mechanism of anticoagulant action. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Increased iPLA2 activity and levels of phosphorylated GSK3B in platelets are associated with donepezil treatment in Alzheimer's disease patients.

    PubMed

    Talib, L L; Hototian, S R; Joaquim, H P G; Forlenza, O V; Gattaz, W F

    2015-12-01

    Reduced phospholipase A2 (PLA2) activity and increased phosphorylation of glycogen synthase kinase 3B (GSK3B) participate in the production of beta-amyloid plaques and of neurofibrillary tangles, which are two neuropathological hallmarks of Alzheimer's disease (AD). Experimental evidences suggest a neuroprotective effect of the cholinesterase inhibitor donepezil in the treatment the disease. The aims of the present study were to evaluate in AD patients the effects of treatment with donepezil on PLA2 activity and GSK3B level. Thirty patients with AD were treated during 6 months with 10 mg daily of donepezil. Radio-enzymatic assays were used to measure PLA2 activity and Elisa assays for GSK3B level, both in platelets. Before treatment and after 3 and 6 months on donepezil, AD patients underwent a cognitive assessment and platelet samples were collected. Values were compared to a healthy control group of 42 sex- and age-matched elderly individuals. Before treatment, iPLA2 activity was lower in patients with AD as compared to controls (p < 0.001). At baseline, no differences were found in GSK3B level between both groups. After 3 and 6 months of treatment, we found a significant increase in iPLA2 activity (p = 0.015 and p < 0.001, respectively). iPLA2 increment was related to the cognitive improvement during treatment (p = 0.037). After 6 months, we found an increase in phosphorylated GSK3B (p = 0.02). The present findings suggest two possible mechanisms by which donepezil delays the progression of AD. The increment of iPLA2 activity may reduce the production of beta-amyloid plaques, whereas the phosphorylation of GSK3B inactivates the enzyme, reducing thus the phosphorylation of tau protein.

  19. Replacing with whole grains and legumes reduces Lp-PLA2 activities in plasma and PBMCs in patients with prediabetes or T2D.

    PubMed

    Kim, Minjoo; Jeung, Se Ri; Jeong, Tae-Sook; Lee, Sang-Hyun; Lee, Jong Ho

    2014-08-01

    To determine dietary effects on circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and enzyme activity in peripheral blood mononuclear cells (PBMCs), 99 patients with impaired fasting glucose, impaired glucose tolerance, or newly-diagnosed T2D were randomly assigned to either a control group (usual diet with refined rice) or the whole grain and legume group. Substitution of whole grains and legumes for refined rice was associated with the replacement of 7% of energy from carbohydrates with energy from protein (about 4%) and fat. After 12 weeks, the whole grain and legume group showed a significant decrease in fasting glucose, insulin, homeostasis model assessment-insulin resistance, hemoglobin A1c, malondialdehyde, plasma Lp-PLA2 activity, and oxidized LDL (ox-LDL), and an increase in LDL particle size. The changes (Δs) in these variables in the whole grain and legume group were significantly different from those in controls after adjustment for the baseline levels. When all subjects were considered, Δ plasma Lp-PLA2 positively correlated with Δ glucose, Δ PBMC Lp-PLA2, Δ ox-LDL, and Δ urinary 8-epi-prostaglandin F2α after being adjusted for confounding factors. The Δ PBMC Lp-PLA2 correlated positively with Δ glucose and Δ ox-LDL, and negatively with Δ LDL particle size and baseline PBMC Lp-PLA2 The substitution of whole grains and legumes for refined rice resulted in a reduction in Lp-PLA2 activities in plasma and PBMCs partly through improved glycemic control, increased consumption of protein relative to carbohydrate, and reduced lipid peroxides. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  20. Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis

    PubMed Central

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-01-01

    The aim of this study is to determine if the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites induces apoptosis of corneal epithelial cells through a cytosolic phospholipase A2α (cPLA2α)-mediated pathway. The efficacy of cPLA2α inhibitors to provide protection against Acanthamoeba keratitis was examined in vivo. Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133. MIP-133 induces significant increase in cPLA2α and macrophage inflammatory protein-2 (MIP-2/CXCL2) levels from corneal cells. Moreover, cPLA2α inhibitors, MAFP (Methyl-arachidonyl fluorophosphonate) and AACOCF3 (Arachidonyl trifluoromethyl ketone), significantly reduce cPLA2α and CXCL2 from these cells (P< 0.05). Additionally, cPLA2α inhibitors significantly inhibit MIP-133-induced apoptosis in HCORN cells (P< 0.05). Subconjunctival injection of purified MIP-133 in Chinese hamster eyes induced cytopathic effects resulting in corneal ulceration. Animals infected with A. castellanii-laden contact lenses and treated with AACOCF3 and CAY10650, showed significantly less severe keratitis as compared with control animals. Collectively, the results indicate that cPLA2α is involved in MIP-133 induced apoptosis of corneal epithelial cells, polymorphonuclear neutrophil infiltration, and production of CXCL2. Moreover, cPLA2α inhibitors can be used as a therapeutic target in Acanthamoeba keratitis. PMID:23792108

  1. PLA2G6, encoding a phospholipase A2, is mutated in neurodegenerative disorders with high brain iron

    PubMed Central

    Morgan, Neil V; Westaway, Shawn K; Morton, Jenny E V; Gregory, Allison; Gissen, Paul; Sonek, Scott; Cangul, Hakan; Coryell, Jason; Canham, Natalie; Nardocci, Nardo; Zorzi, Giovanna; Pasha, Shanaz; Rodriguez, Diana; Desguerre, Isabelle; Mubaidin, Amar; Bertini, Enrico; Trembath, Richard C; Simonati, Alessandro; Schanen, Carolyn; Johnson, Colin A; Levinson, Barbara; Woods, C Geoffrey; Wilmot, Beth; Kramer, Patricia; Gitschier, Jane; Maher, Eamonn R; Hayflick, Susan J

    2007-01-01

    Neurodegenerative disorders with high brain iron include Parkinson disease, Alzheimer disease and several childhood genetic disorders categorized as neuroaxonal dystrophies. We mapped a locus for infantile neuroaxonal dystrophy (INAD) and neurodegeneration with brain iron accumulation (NBIA) to chromosome 22q12-q13 and identified mutations in PLA2G6, encoding a calcium-independent group VI phospholipase A2, in NBIA, INAD and the related Karak syndrome. This discovery implicates phospholipases in the pathogenesis of neurodegenerative disorders with iron dyshomeostasis. PMID:16783378

  2. cPLA2α Gene Activation by IL-1β is Dependent on an Upstream Kinase pathway, Enzymatic Activation and Downstream 15-lipoxygenase Activity: A Positive Feedback Loop

    PubMed Central

    Walters, Jewell N.; Bickford, Justin S.; Beachy, Dawn E.; Newsom, Kimberly J.; Herlihy, John-David H.; Peck, Molly V.; Qiu, Xiaolei; Nick, Harry S.

    2011-01-01

    Cytosolic phospholipase A2α (cPLA2α) is the most widely studied member of the Group IV PLA2 family. The enzyme is Ca2+-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA2α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA2α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca2+ levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA2α is phosphorylated within 10 min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA2α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA2α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA2α→15-LOX2-dependent, positive feedback loop where a protein’s enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus. PMID:21771656

  3. CD64 and Group II Secretory Phospholipase A2 (sPLA2-IIA) as Biomarkers for Distinguishing Adult Sepsis and Bacterial Infections in the Emergency Department.

    PubMed

    Tan, Toh Leong; Ahmad, Nurul Saadah; Nasuruddin, Dian Nasriana; Ithnin, Azlin; Tajul Arifin, Khaizurin; Zaini, Ida Zarina; Wan Ngah, Wan Zurinah

    2016-01-01

    Early diagnosis of sepsis and bacterial infection is imperative as treatment relies on early antibiotic administration. There is a need to develop new biomarkers to detect patients with sepsis and bacterial infection as early as possible, thereby enabling prompt antibiotic treatment and improving the survival rate. Fifty-one adult patients with suspected bacterial sepsis on admission to the Emergency Department (ED) of a teaching hospital were included into the study. All relevant cultures and serology tests were performed. Serum levels for Group II Secretory Phospholipase A2 (sPLA2-IIA) and CD64 were subsequently analyzed. Sepsis was confirmed in 42 patients from a total of 51 recruited subjects. Twenty-one patients had culture-confirmed bacterial infections. Both biomarkers were shown to be good in distinguishing sepsis from non-sepsis groups. CD64 and sPLA2-IIA also demonstrated a strong correlation with early sepsis diagnosis in adults. The area under the curve (AUC) of both Receiver Operating Characteristic curves showed that sPLA2-IIA was better than CD64 (AUC = 0.93, 95% confidence interval (CI) = 0.83-0.97 and AUC = 0.88, 95% CI = 0.82-0.99, respectively). The optimum cutoff value was 2.13μg/l for sPLA2-IIA (sensitivity = 91%, specificity = 78%) and 45 antigen bound cell (abc) for CD64 (sensitivity = 81%, specificity = 89%). In diagnosing bacterial infections, sPLA2-IIA showed superiority over CD64 (AUC = 0.97, 95% CI = 0.85-0.96, and AUC = 0.95, 95% CI = 0.93-1.00, respectively). The optimum cutoff value for bacterial infection was 5.63μg/l for sPLA2-IIA (sensitivity = 94%, specificity = 94%) and 46abc for CD64 (sensitivity = 94%, specificity = 83%). sPLA2-IIA showed superior performance in sepsis and bacterial infection diagnosis compared to CD64. sPLA2-IIA appears to be an excellent biomarker for sepsis screening and for diagnosing bacterial infections, whereas CD64 could be used for screening bacterial infections. Both biomarkers either alone or in

  4. Helicobacter pylori-elicited induction in gastric mucosal matrix metalloproteinase-9 (MMP-9) release involves ERK-dependent cPLA2 activation and its recruitment to the membrane-localized Rac1/p38 complex.

    PubMed

    Slomiany, B L; Slomiany, A

    2016-06-01

    Matrix metalloproteinases (MMPs) are a family of endopeptidases implicated in a wide rage of degenerative and inflammatory diseases, including Helicobacter pylori-associated gastritis, and gastric and duodenal ulcer. As gastric mucosal inflammatory responses to H. pylori are characterized by the rise in MMP-9 production, as well as the induction in mitogen-activated protein kinase (MAPK) and Rac1 activation, we investigated the role of Rac1/MAPK in the processes associated with the release of MMP-9. We show that H. pylori LPS-elicited induction in gastric mucosal MMP-9 release is associated with MAPK, ERK and p38 activation, and occurs with the involvement of Rac1 and cytosolic phospholipase A2 (cPLA2). Further, we demonstrate that the LPS-induced MMP-9 release requires ERK-mediated phosphorylation of cPLA2 on Ser(505) that is essential for its membrane localization with Rac1, and that this process necessitates p38 participation. Moreover, we reveal that the activation and membrane translocation of p38 to the Rac1-GTP complex plays a pivotal role in cPLA2-dependent enhancement in MMP-9 release. Hence, our findings provide a strong evidence for the role of ERK/cPLA2 and Rac1/p38/cPLA2 cascade in H. pylori LPS-induced up-regulation in gastric mucosal MMP-9 release.

  5. The anti-inflammatory activity of standard aqueous stem bark extract of Mangifera indica L. as evident in inhibition of Group IA sPLA2.

    PubMed

    Dhananjaya, Bhadrapura Lakkappa; Shivalingaiah, Sudharshan

    2016-03-01

    The standard aqueous stem bark extract is consumed as herbal drink and used in the pharmaceutical formulations to treat patients suffering from various disease conditions in Cuba. This study was carried out to evaluate the modulatory effect of standard aqueous bark extract of M. indica on Group IA sPLA2. M. indica extract, dose dependently inhibited the GIA sPLA2 (NN-XIa-PLA2) activity with an IC50 value 8.1 µg/ml. M. indica extract effectively inhibited the indirect hemolytic activity up to 98% at ~40 µg/ml concentration and at various concentrations (0-50 µg/ml), it dose dependently inhibited the edema formation. When examined as a function of increased substrate and calcium concentration, there was no relieve of inhibitory effect on the GIA sPLA2. Furthermore, the inhibition was irreversible as evidenced from binding studies. It is observed that the aqueous extract ofM. indica effectively inhibits sPLA2 and it is associated inflammatory activities, which substantiate their anti-inflammatory properties. The mode of inhibition could be due to direct interaction of components present in the extract, with sPLA2 enzyme. Further studies on understanding the principal constituents, responsible for the anti-inflammatory activity would be interesting to develop this into potent anti-inflammatory agent.

  6. Early-onset L-dopa-responsive parkinsonism with pyramidal signs due to ATP13A2, PLA2G6, FBXO7 and spatacsin mutations.

    PubMed

    Paisán-Ruiz, Coro; Guevara, Rocio; Federoff, Monica; Hanagasi, Hasmet; Sina, Fardaz; Elahi, Elahe; Schneider, Susanne A; Schwingenschuh, Petra; Bajaj, Nin; Emre, Murat; Singleton, Andrew B; Hardy, John; Bhatia, Kailash P; Brandner, Sebastian; Lees, Andrew J; Houlden, Henry

    2010-09-15

    Seven autosomal recessive genes associated with juvenile and young-onset Levodopa-responsive parkinsonism have been identified. Mutations in PRKN, DJ-1, and PINK1 are associated with a rather pure parkinsonian phenotype, and have a more benign course with sustained treatment response and absence of dementia. On the other hand, Kufor-Rakeb syndrome has additional signs, which distinguish it clearly from Parkinson's disease including supranuclear vertical gaze palsy, myoclonic jerks, pyramidal signs, and cognitive impairment. Neurodegeneration with brain iron accumulation type I (Hallervorden-Spatz syndrome) due to mutations in PANK2 gene may share similar features with Kufor-Rakeb syndrome. Mutations in three other genes, PLA2G6 (PARK14), FBXO7 (PARK15), and Spatacsin (SPG11) also produce clinical similar phenotypes in that they presented with rapidly progressive parkinsonism, initially responsive to Levodopa treatment but later, developed additional features including cognitive decline and loss of Levodopa responsiveness. Here, using homozygosity mapping and sequence analysis in families with complex parkinsonisms, we identified genetic defects in the ATP13A2 (1 family), PLA2G6 (1 family) FBXO7 (2 families), and SPG11 (1 family). The genetic heterogeneity was surprising given their initially common clinical features. On careful review, we found the FBXO7 cases to have a phenotype more similar to PRKN gene associated parkinsonism. The ATP13A2 and PLA2G6 cases were more seriously disabled with additional swallowing problems, dystonic features, severe in some, and usually pyramidal involvement including pyramidal weakness. These data suggest that these four genes account for many cases of Levodopa responsive parkinsonism with pyramidal signs cases formerly categorized clinically as pallido-pyramidal syndrome. © 2010 Movement Disorder Society.

  7. Novel enzymatic method for assaying Lp-PLA2in serum.

    PubMed

    Yamaura, Saki; Sakasegawa, Shin-Ichi; Koguma, Emisa; Ueda, Shigeru; Kayamori, Yuzo; Sugimori, Daisuke; Karasawa, Ken

    2018-03-15

    Measurement of lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. This can be performed by quantification of the protein concentration using an ELISA platform or by measuring Lp-PLA 2 activity using platelet-activating factor (PAF) analog as substrate. Here, an enzymatic Lp-PLA 2 activity assay method using 1-O-Hexadecyl-2-acetyl-rac-glycero-3-phosphocholine (rac C 16 PAF) was developed. The newly revealed substrate specificity of lysoplasmalogen-specific phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA 2 hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C 16 PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase. Regression analysis of Lp-PLA 2 activity measured by the enzymatic Lp-PLA 2 activity assay vs. two chemical Lp-PLA 2 activity assays, i.e. LpPLA 2 FS and PLAC® test, and ELISA, gave the following correlation coefficients: 0.990, 0.893 and 0.785, respectively (n = 30). Advantages of this enzymatic Lp-PLA 2 activity assay compared with chemical Lp-PLA 2 methods include the following; (i) only requires two reagents enabling a simple two-point linear calibration method with one calibrator (ii) no need for inhibitors of esterase-like activity in serum. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Regulation of sPLA2-IID in Human Decidua

    PubMed Central

    Mosher, Andrea A.; Rainey, Kelly J.; Riley, Brigitta; Levinson, Hayley S.; Vinturache, Angela E.; Wood, Stephen L.

    2014-01-01

    Prostaglandins are implicated in the labor process, yet the precise role and regulation of the prostaglandin pathway remains to be elucidated. The first step in the pathway is cleavage of membrane phospholipids by phospholipase A2 (PLA2). Previous work demonstrated upregulation of secretory PLA2 (sPLA2)-IIA with labor in human myometrium, and recent evidence shows that there are numerous PLA2 isoforms. The present study investigates the potential of additional sPLA2 isoforms during pregnancy and labor. Real-time reverse transcriptase–polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to determine sPLA2 expression and localization. Results show the presence of sPLA2-IID in amnion, chorion, placenta, decidua, and myometrium. Expression of sPLA2-IID in decidua was significantly decreased in term labor compared to nonlabor patients, whereas no significant labor-associated changes were observed in other gestational tissues. Secretory PLA2-IID was localized within chorion fibroblasts, placenta trophoblasts, decidual cells, and in myometrial smooth muscle cells. In primary decidual cell cultures, interleukin (IL) 10 (IL-10) increased sPLA2-IID messenger RNA (mRNA) expression, while IL-1β had no effect on sPLA2-IID mRNA expression. In conclusion, decreased expression of sPLA2-IID in the decidua at labor indicates that it is unlikely to contribute to increased prostaglandin production during labor. However, increased expression of sPLA2-IID, induced by IL-10, suggests that sPLA2-IID may play an important anti-inflammatory role at the maternal–fetal interface. Nevertheless, precise functions of sPLA2-IID within the human uterus remain to be determined. PMID:24440994

  9. PPAR Activation Induces M1 Macrophage Polarization via cPLA2-COX-2 Inhibition, Activating ROS Production against Leishmania mexicana

    PubMed Central

    Díaz-Gandarilla, J. A.; Osorio-Trujillo, C.; Hernández-Ramírez, V. I.; Talamás-Rohana, P.

    2013-01-01

    Defence against Leishmania depends upon Th1 inflammatory response and, a major problem in susceptible models, is the turnoff of the leishmanicidal activity of macrophages with IL-10, IL-4, and COX-2 upregulation, as well as immunosuppressive PGE2, all together inhibiting the respiratory burst. Peroxisome proliferator-activated receptors (PPAR) activation is responsible for macrophages polarization on Leishmania susceptible models where microbicide functions are deactivated. In this paper, we demonstrated that, at least for L. mexicana, PPAR activation, mainly PPARγ, induced macrophage activation through their polarization towards M1 profile with the increase of microbicide activity against intracellular pathogen L. mexicana. PPAR activation induced IL-10 downregulation, whereas the production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 remained high. Moreover, PPAR agonists treatment induced the deactivation of cPLA2-COX-2-prostaglandins pathway together with an increase in TLR4 expression, all of whose criteria meet the M1 macrophage profile. Finally, parasite burden, in treated macrophages, was lower than that in infected nontreated macrophages, most probably associated with the increase of respiratory burst in these treated cells. Based on the above data, we conclude that PPAR agonists used in this work induces M1 macrophages polarization via inhibition of cPLA2 and the increase of aggressive microbicidal activity via reactive oxygen species (ROS) production. PMID:23555077

  10. A snake venom group IIA PLA2 with immunomodulatory activity induces formation of lipid droplets containing 15-d-PGJ2 in macrophages.

    PubMed

    Giannotti, Karina Cristina; Leiguez, Elbio; Carvalho, Ana Eduarda Zulim de; Nascimento, Neide Galvão; Matsubara, Márcio Hideki; Fortes-Dias, Consuelo Latorre; Moreira, Vanessa; Teixeira, Catarina

    2017-06-22

    Crotoxin B (CB) is a catalytically active group IIA sPLA 2 from Crotalus durissus terrificus snake venom. In contrast to most GIIA sPLA 2 s, CB exhibits anti-inflammatory effects, including the ability to inhibit leukocyte functions. Lipid droplets (LDs) are lipid-rich organelles associated with inflammation and recognized as a site for the synthesis of inflammatory lipid mediators. Here, the ability of CB to induce formation of LDs and the mechanisms involved in this effect were investigated in isolated macrophages. The profile of CB-induced 15-d-PGJ 2 (15-Deoxy-Delta-12,14-prostaglandin J 2 ) production and involvement of LDs in 15-d-PGJ 2 biosynthesis were also investigated. Stimulation of murine macrophages with CB induced increased number of LDs and release of 15-d-PGJ 2 . LDs induced by CB were associated to PLIN2 recruitment and expression and required activation of PKC, PI3K, MEK1/2, JNK, iPLA 2 and PLD. Both 15-d-PGJ 2 and COX-1 were found in CB-induced LDs indicating that LDs contribute to the inhibitory effects of CB by acting as platform for synthesis of 15-d-PGJ 2 , a pro-resolving lipid mediator. Together, our data indicate that an immunomodulatory GIIA sPLA 2 can directly induce LD formation and production of a pro-resolving mediator in an inflammatory cell and afford new insights into the roles of LDs in resolution of inflammatory processes.

  11. The role of group IIA secretory phospholipase A2 (sPLA2-IIA) as a biomarker for the diagnosis of sepsis and bacterial infection in adults-A systematic review.

    PubMed

    Tan, Toh Leong; Goh, Yew Yip

    2017-01-01

    This paper investigates the role of Group II Secretory Phospholipase A2 (sPLA2-IIA) as a biomarker for the diagnosis of sepsis and bacterial infection in adults. Sepsis and bacterial infection are common problems encountered by patients in the hospital and often carry adverse outcomes if not managed early. Two independent reviewers conducted a comprehensive search using Ovid MEDLINE published from years 1993 to 2016 and SCOPUS published from year 1985 to 2017 to screen for relevant studies. The main inclusion criteria included adult subjects, patients with suspected or confirmed signs of infection and relevant outcomes which looked into the role of sPLA2-IIA in detecting the presence of sepsis and bacterial infection in the subjects. Four studies met the inclusion criteria. SPLA2-IIA was found to be effective in detecting the presence of sepsis and bacterial infection in adults. The levels of serum sPLA2-IIA also correlated well with the presence of sepsis and bacterial infection. This systematic review highlights the role of sPLA2-IIA as a reliable tool to diagnose sepsis and bacterial infection in adult patients. Nonetheless, further studies should be done in the future to provide more compelling evidence on its application in the clinical setting.

  12. Sulodexide prevents activation of the PLA2/COX-2/VEGF inflammatory pathway in human retinal endothelial cells by blocking the effect of AGE/RAGE.

    PubMed

    Giurdanella, Giovanni; Lazzara, Francesca; Caporarello, Nunzia; Lupo, Gabriella; Anfuso, Carmelina Daniela; Eandi, Chiara M; Leggio, Gian Marco; Drago, Filippo; Bucolo, Claudio; Salomone, Salvatore

    2017-10-15

    Diabetic retinopathy is characterized by the breakdown of endothelial blood-retinal barrier. We tested the hypothesis that sulodexide (SDX), a highly purified glycosaminoglycan composed of 80% iduronylglycosaminoglycan sulfate and 20% dermatan sulfate, protects human retinal endothelial cells (HREC) from high glucose (HG)-induced damage, through the suppression of inflammatory ERK/cPLA2/COX-2/PGE 2 pathway, by blocking the effect of advanced glycation end-products (AGEs). HREC were treated with HG (25mM) or AGEs (glycated-BSA, 2mg/ml) for 48h, with or without SDX (60μg/ml) or aflibercept (AFL, 40μg/ml), a VEGF-trap. SDX protected HREC from HG-induced damage (MTT and LDH release) and preserved their blood-retinal barrier-like properties (Trans Endothelial Electrical Resistance and junction proteins, claudin-5, VE-cadherin and occludin, immunofluorescence and immunoblot) as well as their angiogenic potential (Tube Formation Assay). Both HG and AGEs increased phosphoERK and phospho-cPLA 2 , an effect counteracted by SDX and, less efficiently, by AFL. Both HG and exogenous VEGF (80ng/ml) increased PGE 2 release, an effect partially reverted by SDX for HG and by AFL for VEGF. Analysis of NFκB activity revealed that HG increased the abundance of p65 in the nuclear fraction (nuclear translocation), an effect entirely reverted by SDX, but only partially by AFL. SDX, AFL and SDX+AFL protected HREC even when added 24h after HG. These data show that SDX protects HREC from HG damage and suggest that it counteracts the activation of ERK/cPLA2/COX-2/PGE 2 pathway by reducing AGE-related signaling and downstream NFκB activity. This mechanism, partially distinct from VEGF blockade, may contribute to the therapeutic effect of SDX. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Molecular Cloning and Pharmacological Properties of an Acidic PLA2 from Bothrops pauloensis Snake Venom

    PubMed Central

    Ferreira, Francis Barbosa; Gomes, Mário Sérgio Rocha; Naves de Souza, Dayane Lorena; Gimenes, Sarah Natalie Cirilo; Castanheira, Letícia Eulalio; Borges, Márcia Helena; Rodrigues, Renata Santos; Yoneyama, Kelly Aparecida Geraldo; Homsi Brandeburgo, Maria Inês; Rodrigues, Veridiana M.

    2013-01-01

    In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A2 (PLA2) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA2-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA2-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA2-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA2-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA2s. The phylogenetic analyses showed that BpPLA2-TXI forms a group with other acidic D49 PLA2s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects. PMID:24304676

  14. Computational investigations of physicochemical, pharmacokinetic, toxicological properties and molecular docking of betulinic acid, a constituent of Corypha taliera (Roxb.) with Phospholipase A2 (PLA2).

    PubMed

    Khan, Mohammad Firoz; Nahar, Nusrat; Rashid, Ridwan Bin; Chowdhury, Akhtaruzzaman; Rashid, Mohammad A

    2018-02-02

    noncarcinogenic in mice model. Moreover, molecular docking study of BA with PLA2 revealed that BA interacts with GLY22 & GLY29 through hydrogen bond formation and LEU2, PHE5, HIS6, ALA17, ALA18, HIS47 and TYR51 through different types of hydrophobic interactions. The binding affinity of BA was - 41.00 kJ/mol which is comparable to the binding affinity of potent inhibitor 6-Phenyl-4(R)-(7-Phenyl-heptanoylamino)-hexanoic acid (BR4) (- 33.89 kJ/mol). Our computed properties may assist the development of analytical method to assay BA or to develop BA derivatives with better pharmacokinetic and toxicological profile.

  15. The Leu33/Pro polymorphism (PlA1/PlA2) of the glycoprotein IIIa (GPIIIa) receptor is not related to myocardial infarction in the ECTIM Study. Etude Cas-Temoins de l'Infarctus du Myocarde.

    PubMed

    Herrmann, S M; Poirier, O; Marques-Vidal, P; Evans, A; Arveiler, D; Luc, G; Emmerich, J; Cambien, F

    1997-06-01

    The GPIIb/IIIa receptor complex may contribute to acute coronary syndromes by mediating platelet aggregation. The Leu33/Pro polymorphism (PlA1/PlA2) of the GPIIIa has recently been shown to be associated with CHD in a small case-control study. We have investigated this polymorphism in a large multicenter study of patients with myocardial infarction and controls and found no difference in the distribution of allele and genotype frequencies between cases and controls.

  16. Short-term fenofibrate treatment reduces elevated plasma Lp-PLA2 mass and sVCAM-1 levels in a subcohort of hypertriglyceridemic GOLDN participants

    USDA-ARS?s Scientific Manuscript database

    High levels of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) are associated with inflammation, atherosclerosis, and coronary heart disease events. In addition, Lp-PLA(2) has been linked to classical markers of endothelial activation, including soluble vascular cell adhesion molecule-1 (sVCAM...

  17. Tracking of secretory phospholipase A2 enzyme activity levels from childhood to adulthood: a 21-year cohort.

    PubMed

    Chung, Olivia; Juonala, Markus; Mallat, Ziad; Hutri-Kähönen, Nina; Viikari, Jorma S A; Raitakari, Olli T; Magnussen, Costan G

    2018-02-21

    Secretory phospholipase A2 (sPLA2) enzyme activity is a potential inflammatory biomarker for cardiovascular disease. We examined the tracking, or persistence, of sPLA2 enzyme activity levels from childhood to adulthood, and identify potentially modifiable factors affecting tracking. Prospective cohort of 1735 children (45% females) who had serum sPLA2 enzyme activity levels and other cardiovascular disease risk factors measured in 1980 that were followed-up in 2001. sPLA2 activity tracked from childhood to adulthood for males (r=0.39) and females (r=0.45). Those who decreased body mass index relative to their peers were more likely to resolve elevated childhood sPLA2 levels than have persistent elevated sPLA2 levels in childhood and adulthood. Those who consumed less fruit, and gained more body mass index relative to their peers, began smoking or were a persistent smoker between childhood and adulthood were more likely to develop incident elevated sPLA2 levels than those with persistent not elevated sPLA2 levels. Childhood sPLA2 enzyme activity levels associate with adult sPLA2 levels 21 years later. Healthful changes in modifiable risk factors that occur between childhood and adulthood might prevent children from developing elevated sPLA2 levels in adulthood. Copyright © 2018. Published by Elsevier Editora Ltda.

  18. Associations of maternal PLA2G4C and PLA2G4D polymorphisms with the risk of spontaneous preterm birth in a Chinese population

    PubMed Central

    Liu, Guang-Jian; He, Jian-Rong; Kuang, Ya-Shu; Fan, Xue-Jiao; Li, Wei-Dong; Lu, Jin-Hua; Xia, Xiao-Yan; Liu, Xiao-Dan; Chen, Nian-Nian; Mai, Wei-Bi; Xia, Hui-Min; Qiu, Xiu

    2017-01-01

    Preterm birth is the leading cause of mortality and morbidity in infants. Its etiology is multifactorial with genes and immune homeostasis. The authors investigated whether prostaglandin (PG) synthesis related single nucleotide polymorphisms (SNPs) PLA2G4C rs1366442 and PLA2G4D rs4924618 were associated with the risk of spontaneous preterm birth (SPTB) in a Chinese population of 114 cases of SPTB and 250 controls of term delivery. The risk associations were determined by odds ratios (ORs) and their 95% confidence intervals (CIs) calculated using multivariate logistic regression. Homology modeling was performed to elucidate potential mechanism of the SNP function. The maternal AT/TT genotype of PLA2G4D rs4924618 was associated with a reduced risk of SPTB (OR, 0.61; 95% CI, 0.37–0.99), while no significant association between PLA2G4C rs1366442 and SPTB risk was identified. Structure and sequence analysis revealed that the amino acid substitution introduced by this SNP located at the conserved central core of the catalytic domain of cytosolic phospholipase A2 δ and was close to the active site. These findings suggested that the polymorphism of PLA2G4D rs4924618 may have a protective influence on the SPTB susceptibility in a Chinese population, supporting a role for genetics in the association between PG synthesis and preterm birth. PMID:28440406

  19. Modulation of Phospholipase A2 Activity by Actin and Myosin.

    DTIC Science & Technology

    1987-04-15

    actin. chicken gizzard actin. bovine muscle myosin, chicken gizzard myosin. bovine albumin. phalloidin. and PLA2 derived from bee venom (PLA 2 -B) or...Actin on the Activity of PLA2 Derived from Bee Venom As illustrated in Fig. 4. prior incubation of PLA 2--B with actin (100 /4M) altered the activity...several endothelial cell PLA 2 types might function to initiate the eicosanoid cascade (14). the finding that diverse PLA2 sources ( bee venom and

  20. Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity.

    PubMed

    Rouault, Morgane; Bollinger, James G; Lazdunski, Michel; Gelb, Michael H; Lambeau, Gérard

    2003-10-07

    An increasing number of mammalian secreted phospholipases A(2) (sPLA(2)s) has been identified over the past few years. Here, we report the identification and recombinant expression of a novel sPLA(2)-like protein in mouse and human species that has been called group XIIB (GXIIB). The mature protein has a molecular mass of 19.7 kDa and structural features similar to those of the previously identified GXII sPLA(2), now called GXIIA. Strikingly, the GXIIB sPLA(2) has a mutation in the active site, replacing the canonical histidine by a leucine, suggesting that this sPLA(2) is catalytically inactive. Recombinant expression of human (hGXIIB) and mouse (mGXIIB) sPLA(2)s in Escherichia coli indicates that GXIIB sPLA(2)s display no measurable lipolytic activity on various types of phospholipid substrates. Furthermore, these sPLA(2)-like proteins display relatively weak affinity to phospholipid vesicles. Binding experiments indicate that these proteins are also unable to bind to the well-known M-type sPLA(2) receptor. The RNA tissue distribution of GXIIB sPLA(2)s is distinct from that of other sPLA(2)s including the homologous GXIIA. Strong expression was observed in liver, small intestine, and kidney in both human and mouse species. Interestingly, the expression of the novel sPLA(2) is dramatically decreased in human tumors from the same tissues. The absence of enzymatic activity suggests that the GXIIB sPLA(2)-like proteins probably exert their biological roles by acting as ligands for as yet unidentified receptors.

  1. Association study of bipolar disorder at the phospholipase A2 gene (PLA2A) in the Darier's disease (DAR) region of chromosome 12q23-q24.1.

    PubMed

    Jacobsen, N; Daniels, J; Moorhead, S; Harrison, D; Feldman, E; McGuffin, P; Owen, M J; Craddock, N

    1996-01-01

    We have previously described a pedigree in which affective disorder and Darier's disease cosegregate and, in an independent sample of 45 bipolar pedigrees, reported evidence in support of linkage between a putative susceptibility locus for bipolar disorder and markers in the Darier's disease region of chromosome 12q23-q24.1. The maximum evidence for linkage was given by a polymorphism at the gene encoding secretory phospholipase A2 (PLA2A), a candidate gene for affective disorder. Dawson et al. (Psychiatric Genetics, 5, 177-180) recently reported allelic association with the same polymorphism at PLA2A in a sample of 54 bipolar probands and 56 controls. We have been unable to replicate the association in 127 Caucasian British DSMIII-R bipolar probands and 223 Caucasian British controls. However, on the basis of several lines of evidence discussed in the paper, we believe that this genomic region in general, and the PLA2A gene in particular, are worthy of further study as candidates in the search for susceptibility genes for the functional psychoses.

  2. The standard aqueous stem bark extract of Mangifera indica L. inhibits toxic PLA2 - NN-XIb-PLA2 of Indian cobra venom.

    PubMed

    Dhananjaya, Bhadrapura Lakkappa; Sudarshan, Shivalingaiah; Dongol, Yashad; More, Sunil S

    2016-05-01

    The aqueous extract of Mangifera indica is known to possess diverse medicinal properties, which also includes anti-snake venom activities. However, its inhibitory potency and mechanism of action on multi-toxic snake venom phospholipases A2s are still unknown. Therefore, the objective of this study was to evaluate the modulatory effect of standard aqueous bark extract of M. indica on NN-XIb-PLA2 of Indian cobra venom. The in vitro sPLA2, in situ hemolytic and in vivo edema inhibition effect were carried out as described. Also the effect of substrate and calcium concentration was carried out. M. indica extract dose dependently inhibited the GIA sPLA2 (NN-XIb-PLA2) activity with an IC50 value of 7.6 μg/ml. M. indica extract effectively inhibited the indirect hemolytic activity up to 98% at ∼40 μg/ml concentration. Further, M. indica extract (0-50 μg/ml) inhibited the edema formed in a dose dependent manner. When examined as a function of increased substrate and calcium concentration, there was no relieve of inhibitory effect of M. indica extract on the NN-XIb-PLA2. Further, the inhibition was irreversible as evident from binding studies. The in vitro inhibition is well correlated with in situ and in vivo edema inhibiting activities of M. indica. As the inhibition is independent of substrate and calcium and was irreversible, it can be concluded that M. indica extract mode of inhibition could be due to direct interaction of components present in the extract with the PLA2 enzyme. The aqueous extract of M. indica effectively inhibits svPLA2 enzymatic and its associated toxic activities, which substantiate their anti-snake venom properties. Further in-depth studies on the role and mechanism of the principal constituents present in the extract, responsible for the anti-PLA2 activity will be interesting to develop them into potent antisnake component and also as an anti-inflammatory agent.

  3. Snake Venom PLA2s Inhibitors Isolated from Brazilian Plants: Synthetic and Natural Molecules

    PubMed Central

    Carvalho, B. M. A.; Santos, J. D. L.; Xavier, B. M.; Almeida, J. R.; Resende, L. M.; Martins, W.; Marcussi, S.; Marangoni, S.; Stábeli, R. G.; Calderon, L. A.; Soares, A. M.; Da Silva, S. L.; Marchi-Salvador, D. P.

    2013-01-01

    Ophidian envenomation is an important health problem in Brazil and other South American countries. In folk medicine, especially in developing countries, several vegetal species are employed for the treatment of snakebites in communities that lack prompt access to serum therapy. However, the identification and characterization of the effects of several new plants or their isolated compounds, which are able to inhibit the activities of snake venom, are extremely important and such studies are imperative. Snake venom contains several organic and inorganic compounds; phospholipases A2 (PLA2s) are one of the principal toxic components of venom. PLA2s display a wide variety of pharmacological activities, such as neurotoxicity, myotoxicity, cardiotoxicity, anticoagulant, hemorrhagic, and edema-inducing effects. PLA2 inhibition is of pharmacological and therapeutic interests as these enzymes are involved in several inflammatory diseases. This review describes the results of several studies of plant extracts and their isolated active principles, when used against crude snake venoms or their toxic fractions. Isolated inhibitors, such as steroids, terpenoids, and phenolic compounds, are able to inhibit PLA2s from different snake venoms. The design of specific inhibitors of PLA2s might help in the development of new pharmaceutical drugs, more specific antivenom, or even as alternative approaches for treating snakebites. PMID:24171158

  4. Xenorhabdus nematophilus inhibits p-bromophenacyl bromide (BPB)-sensitive PLA2 of Spodoptera exigua.

    PubMed

    Park, Youngjin; Kim, Yonggyun

    2003-11-01

    Xenorhabdus nematophilus is a Gram-negative symbiotic bacterium of the entomopathogenic nematode, Steinernema carpocapsae. The bacteria delivered into the insect hemocoel by the nematodes cause immunodepression of the target insects to protect host nematodes and themselves from the cellular immune reaction. Previous reports suggest that the immunodepression is caused by inhibition of the eicosanoid pathway that is known to be critically important to mediate cellular immunity. This study focused on the inhibitory effect of X. nematophilus on PLA2 activity of Spodoptera exigua. The PLA2 activity was functionally associated with the activation cascade of prophenoloxidase (pPO). Dexamethasone (DEX), a specific PLA2 inhibitor, inhibited pPO activation completely at the higher doses of approximately 2.4 muM in vitro condition. The inhibitory effect of DEX was reversed by the addition of arachidonic acid, the catalytic product of PLA2. By means of this in vitro PLA2 inhibitor assay system, two different PLA2 inhibitors were used to compare their inhibitory effects on the hemolymph PLA2 of S. exigua. p-Bromophenacyl bromide (BPB), a specific inhibitor of secretory PLA2 (sPLA2), significantly inhibited pPO activation, but methylarachidonyl fluorophosphates (MAFP), a specific inhibitor of cytosolic PLA2 (cPLA2), did not show any inhibitory effect. BPB also inhibited pPO activation of the plasma, though much higher PO activation and its inhibition by BPB was found in the hemocytes. Growth medium of X. nematophilus at the stationary phase had a PLA2 inhibitory effect. Via the in vitro PLA2 inhibitor assay, it was shown that the ethyl ether extract of the medium contained significant PLA2 inhibitor activity. These results indicate that X. nematophilus produces and secretes PLA2 inhibitor, which acts on BPB-susceptible PLA2 of S. exigua. Copyright 2003 Wiley-Liss, Inc.

  5. An association between PLA2G6 and PLA2G4C gene polymorphisms and schizophrenia risk and illness severity in a Croatian population.

    PubMed

    Nadalin, Sergej; Buretić-Tomljanović, Alena

    2017-06-01

    We investigated the allele and genotype frequency of the rs4375 and rs1549637 polymorphisms in phospholipase A2 (PLA2)G6 and PLA2G4C genes in 203 patients with schizophrenia and 191 controls in a Croatian population. We hypothesized that these polymorphic variations might influence the age of schizophrenia onset and Positive and Negative Syndrome Scale psychopathology (PANSS) data. We detected a significant overrepresentation of the PLA2G6-CT and PLA2G4C-AT genotype combination in patients compared with controls (14.7% vs. 7.3%, P < 0.05). The combined PLA2G6/PLA2G4C heterozygosity was associated with about a two-fold higher schizophrenia risk. We found no significant influence of the PLA2G6 and PLA2G4C polymorphisms on mean age at first hospital admission (P > 0.05) and that the investigated polymorphisms significantly influenced the clinical psychopathology only in male patients. The PLA2G4C polymorphism accounted for approximately 12% of negative symptom severity; whereas, the PLA2G6/PLA2G4C interaction contributed to a similar extent to total PANSS symptom variations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Revisiting the use of sPLA2-sensitive liposomes in cancer therapy.

    PubMed

    Pourhassan, Houman; Clergeaud, Gael; Hansen, Anders E; Østrem, Ragnhild G; Fliedner, Frederikke P; Melander, Fredrik; Nielsen, Ole L; O'Sullivan, Ciara K; Kjær, Andreas; Andresen, Thomas L

    2017-09-10

    The first developed secretory phospholipase A 2 (sPLA 2 ) sensitive liposomal cisplatin formulation (LiPlaCis®) is currently undergoing clinical evaluation. In the present study we revisit and evaluate critical preclinical parameters important for the therapeutic potential and safety of platinum drugs, here oxaliplatin (L-OHP), formulated in sPLA 2 sensitive liposomes. We show the mole percentage of negatively charged phospholipid needed to obtain enzyme-sensitivity for saturated systems is ≥25% for 16-carbon chain lipid membranes, and >40% for 18-chain lipid membranes, which was surprising as 25% is used clinically in LiPlaCis®. Efficient sPLA 2 -dependent growth inhibition of colorectal cancer cells was demonstrated in vitro, where cell membrane degradation and cytolysis depends on the sensitivity of the formulation towards the enzyme and is governed by the amount of lysolipids generated and the presence of serum proteins. We found that serum proteins did not affect the lipase activity of the enzyme towards the membranes but instead sequester the lysolipid byproducts consequently inhibiting their detergent-like cytotoxic properties. In vivo therapeutic potential and safety of the liposomes was investigated in nude mice bearing sPLA 2 -deficient FaDu squamous carcinoma and sPLA 2 -expressing Colo205 colorectal adenocarcinoma. After intravenous injections, the tumor growth was suppressed for liposomal L-OHP relative to free drug, but only a weak response was observed for both slow- and fast-releasing sPLA 2 -sensitive formulations compared to non-sensitive liposomes. Also, the mice did not show longer survival. In turn, for the highly sPLA 2 -sensitive liposomes, multiple high doses caused petechial cutaneous hemorrhages, along with multifocal hepatonecrotic lesions, suggestive of premature activation in skin and liver irrespective of sPLA 2 -status of the tumor engraft. These results indicate that although liposomal carriers can improve the antitumor efficacy

  7. sPLA2 and the epidermal barrier

    PubMed Central

    Ilic, Dusko; Bollinger, James M.; Gelb, Michael; Mauro, Theodora M.

    2015-01-01

    The mammalian epidermis provides both an interface and a protective barrier between the organism and its environment. Lipid, processed into water-impermeable bilayers between the outermost layers of the epidermal cells, forms the major barrier that prevents water from exiting the organism, and also prevents toxins and infectious agents from entering. The secretory phospholipase 2 (sPLA2) enzymes control important processes in skin and other organs, including inflammation and differentiation. sPLA2 activity contributes to epidermal barrier formation and homeostasis by generating free fatty acids, which are required both for formation of lamellar membranes and also for acidification of the stratum corneum (SC). sPLA2 is especially important in controlling SC acidification and establishment of an optimum epidermal barrier during the first postnatal week. Several sPLA2 isoforms are present in the epidermis. We find that two of these isoforms, sPLA2 IIA and sPLA2 IIF, localize to the upper stratum granulosum and increase in response to experimental barrier perturbation. sPLA2F−/− mice also demonstrate a more neutral SC pH than do their normal littermates, and their initial recovery from barrier perturbation is delayed. These findings confirm that sPLA2 enzymes perform important roles in epidermal development, and suggest that the sPLA2IIF isoform may be central to SC acidification and barrier function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias. PMID:24269828

  8. Collaborative meta-analysis of individual participant data from observational studies of Lp-PLA2 and cardiovascular diseases.

    PubMed

    Ballantyne, C; Cushman, M; Psaty, B; Furberg, C; Khaw, K T; Sandhu, M; Oldgren, J; Rossi, G P; Maiolino, G; Cesari, M; Lenzini, L; James, S K; Rimm, E; Collins, R; Anderson, J; Koenig, W; Brenner, H; Rothenbacher, D; Berglund, G; Persson, M; Berger, P; Brilakis, E; McConnell, J P; Koenig, W; Sacco, R; Elkind, M; Talmud, P; Rimm, E; Cannon, C P; Packard, C; Barrett-Connor, E; Hofman, A; Kardys, I; Witteman, J C M; Criqui, M; Corsetti, J P; Rainwater, D L; Moss, A J; Robins, S; Bloomfield, H; Collins, D; Packard, C; Wassertheil-Smoller, S; Ridker, P; Ballantyne, C; Cannon, C P; Cushman, M; Danesh, J; Gu, D; Hofman, A; Nelson, J J; Thompson, S; Zalewski, A; Zariffa, N; Di Angelantonio, E; Kaptoge, S; Thompson, A; Thompson, S; Walker, M; Watson, S; Wood, A

    2007-02-01

    A large number of observational epidemiological studies have reported generally positive associations between circulating mass and activity levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) and the risk of cardiovascular diseases. Few studies have been large enough to provide reliable estimates in different circumstances, such as in different subgroups (e.g., by age group, sex, or smoking status) or at different Lp-PLA2 levels. Moreover, most published studies have related disease risk only to baseline values of Lp-PLA2 markers (which can lead to substantial underestimation of any risk relationships because of within-person variability over time) and have used different approaches to adjustment for possible confounding factors. By combination of data from individual participants from all relevant observational studies in a systematic 'meta-analysis', with correction for regression dilution (using available data on serial measurements of Lp-PLA2), the Lp-PLA2 Studies Collaboration will aim to characterize more precisely than has previously been possible the strength and shape of the age and sex-specific associations of plasma Lp-PLA2 with coronary heart disease (and, where data are sufficient, with other vascular diseases, such as ischaemic stroke). It will also help to determine to what extent such associations are independent of possible confounding factors and to explore potential sources of heterogeneity among studies, such as those related to assay methods and study design. It is anticipated that the present collaboration will serve as a framework to investigate related questions on Lp-PLA2 and cardiovascular outcomes. A central database is being established containing data on circulating Lp-PLA2 values, sex and other potential confounding factors, age at baseline Lp-PLA2 measurement, age at event or at last follow-up, major vascular morbidity and cause-specific mortality. Information about any repeat measurements of Lp-PLA2 and potential confounding

  9. Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy

    PubMed Central

    Seitz-Polski, Barbara; Dolla, Guillaume; Payré, Christine; Girard, Christophe A.; Polidori, Joel; Zorzi, Kevin; Birgy-Barelli, Eléonore; Jullien, Perrine; Courivaud, Cécile; Krummel, Thierry; Benzaken, Sylvia; Bernard, Ghislaine; Burtey, Stéphane; Mariat, Christophe; Esnault, Vincent L.M.

    2016-01-01

    The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy. However, the value of anti-PLA2R1 antibody titers in predicting patient outcomes is unknown. Here, we screened serum samples from 50 patients positive for PLA2R1 for immunoreactivity against a series of PLA2R1 deletion mutants covering the extracellular domains. We identified reactive epitopes in the cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) domains and confirmed the reactivity with soluble forms of each domain. We then used ELISAs to stratify 69 patients positive for PLA2R1 by serum reactivity to one or more of these domains: CysR (n=23), CysRC1 (n=14), and CysRC1C7 (n=32). Median ELISA titers measured using the full-length PLA2R1 antigens were not statistically different between subgroups. Patients with anti-CysR–restricted activity were younger (P=0.008), had less nephrotic range proteinuria (P=0.02), and exhibited a higher rate of spontaneous remission (P=0.03) and lower rates of renal failure progression (P=0.002) and ESRD (P=0.01) during follow-up. Overall, 31 of 69 patients had poor renal prognosis (urinary protein/creatinine ratio >4 g/g or eGFR<45 ml/min per 1.73 m2 at end of follow-up). High anti-PLA2R1 activity and epitope spreading beyond the CysR epitope were independent risk factors of poor renal prognosis in multivariable Cox regression analysis. Epitope spreading during follow-up associated with disease worsening (n=3), whereas reverse spreading from a CysRC1C7 profile back to a CysR profile associated with favorable outcome (n=1). We conclude that analysis of the PLA2R1 epitope profile and spreading is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis. PMID:26567246

  10. Regulation of Integrin α6 Recycling by Calcium-independent Phospholipase A2 (iPLA2) to Promote Microglia Chemotaxis on Laminin.

    PubMed

    Lee, Sang-Hyun; Sud, Neetu; Lee, Narae; Subramaniyam, Selvaraj; Chung, Chang Y

    2016-11-04

    Microglia are the immune effector cells that are activated in response to pathological changes in the central nervous system. Microglial activation is accompanied by the alteration of integrin expression on the microglia surface. However, changes of integrin expression upon chemoattractant (ADP) stimulation still remain unknown. In this study, we investigated whether ADP induces the alteration of integrin species on the cell surface, leading to changes in chemotactic ability on different extracellular matrix proteins. Flow cytometry scans and on-cell Western assays showed that ADP stimulation induced a significant increase of α6 integrin-GFP, but not α5, on the surface of microglia cells. Microglia also showed a greater motility increase on laminin than fibronectin after ADP stimulation. Time lapse microscopy and integrin endocytosis assay revealed the essential role of calcium-independent phospholipase A 2 activity for the recycling of α6 integrin-GFP from the endosomal recycling complex to the plasma membrane. Lack of calcium-independent phospholipase A 2 activity caused a reduced rate of focal adhesion formation on laminin at the leading edge. Our results suggest that the alteration of integrin-mediated adhesion may regulate the extent of microglial infiltration into the site of damage by controlling their chemotactic ability. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein.

    PubMed

    Sultan, Md Tipu; Li, Hong-Mei; Lee, Yong Zu; Lim, Soon Sung; Song, Dong-Keun

    2016-03-01

    We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca(2+)]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca(2+) infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 µM) and SKF-96365 (20 µM) signifi cantly inhibited K49-PLA2-induced [Ca(2+)]i increase. These results suggest that K49-PLA2 modulates [Ca(2+)]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

  12. Molecular modeling of the inhibition of enzyme PLA2 from snake venom by dipyrone and 1-phenyl-3-methyl-5-pyrazolone

    NASA Astrophysics Data System (ADS)

    Silva, S. L. Da; Comar, M., Jr.; Oliveira, K. M. T.; Chaar, J. S.; Bezerra, E. R. M.; Calgarotto, A. K.; Baldasso, P. A.; Veber, C. L.; Villar, J. A. F. P.; Oliveira, A. R. M.; Marangoni, S.

    Phospholipases A2 (PLA2) are enzymes that trigger the degradation cascade of the arachidonic acid, leading to the formation of pro-inflammatory eicosanoids. The selective inhibition of PLA2s is crucial in the search for a more efficient anti-inflammatory drug with fewer side effects than the drugs currently used. Hence, we studied the influences caused by two pyrazolonic inhibitors: dipyrone (DIP) and 1-phenyl-3-methyl-5-pyrazolone (PMP) on the kinetic behavior of PLA2 from Crotalus adamanteus venom. Molecular modeling results, by DFT and MM approaches, showed that DIP is strongly associated to the active site of PLA2 through three hydrogen bonds, whereas PMP is associated to the enzyme just through hydrophobic interactions. In addition, only PMP presents an intramolecular hydrogen bond that make difficult the formation of more efficient interactions with PLA2. These results help in the understanding of the experimental observations. Experimentally, the results showed that PLA2 from C. adamanteus present a typical Michaelian behavior. In addition, the calculated kinetic parameters showed that, in the presence of DIP or PMP, the maximum enzymatic velocity (VMAX) value was kept constant, whereas the Michaelis constant (KM) values increased and the inhibition constant (KI) decreased, indicating competitive inhibition. These results show that the phenyl-pyrazolonic structures might help in the development and design of new drugs able to selectively inhibit PLA2.

  13. Alteration of delta-6-desaturase (FADS2), secretory phospholipase-A2 (sPLA2) enzymes by Hot-nature diet with co-supplemented hemp seed, evening primrose oils intervention in multiple sclerosis patients.

    PubMed

    Rezapour-Firouzi, Soheila; Arefhosseini, Seyed Rafie; Ebrahimi-Mamaghani, Mehrangiz; Baradaran, Behzad; Sadeghihokmabad, Elyar; Mostafaei, Somaiyeh; Torbati, Mohammadali; Chehreh, Mahtaj

    2015-10-01

    The effect of nutrition and dietary supplements as environmental factors has been suggested as possible factors affecting both disease risk and progression in on the course of multiple sclerosis with complex genetic-risk profiles. This study was aimed to assess regulation of surface-membrane enzymes such as Delta-6-desaturase (FADS2), secretory Phospholipase A2(sPLA2) by hemp seed and evening primrose oils as well as Hot-natured dietary intervention in relapsing remitting multiple sclerosis (RRMS) patients. In this double blind, randomized trial, 100 RRMS patients with Extended disability status score (EDSS)<6 were allocated into 3 groups: "Group A" who received co-supplemented hemp seed and evening primrose oils along with advised Hot nature diet; "Group B", who received olive oil; "Group C", who received the co-supplemented oils. Clinically EDSS and functional score as well as biochemical parameters [blood cells polyunsaturated fatty acid (PUFA), FADS2, sPLA2] were assessed at baseline and after 6 months. Mean follow-up was 180±2.9SD days (N=65, 23 M and 42 F aged 34.25±8.07 years with disease duration 6.80±4.33 years). There was no significant difference in studies parameters at baseline. After 6 months, significant improvements in EDSS and functional score were found in the groups A and C while EDSS and pyramidal score showed significant increase in group B. Alteration of biochemical parameters showed improvement in groups A and C whereas there was worsening condition for group B after the intervention. The co-supplemented hemp seed and evening primrose oils with Hot nature diet can have beneficial effects in improving clinical symptoms and signs in RRMS patients which were confirmed by regulation of surface-membrane enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum.

    PubMed

    Yun, Bogeon; Lee, HeeJung; Ewing, Heather; Gelb, Michael H; Leslie, Christina C

    2016-10-07

    Cytosolic phospholipase A2α (cPLA2α) mediates agonist-induced release of arachidonic acid from membrane phospholipid for production of eicosanoids. The activation of cPLA2α involves increases in intracellular calcium, which binds to the C2 domain and promotes cPLA2α translocation from the cytosol to membrane to access substrate. The cell permeable pyrrolidine-containing cPLA2α inhibitors including pyrrophenone have been useful to understand cPLA2α function. Although this serine hydrolase inhibitor does not inhibit other PLA2s or downstream enzymes that metabolize arachidonic acid, we reported that it blocks increases in mitochondrial calcium and cell death in lung fibroblasts. In this study we used the calcium indicators G-CEPIA1er and CEPIA2mt to compare the effect of pyrrophenone in regulating calcium levels in the endoplasmic reticulum (ER) and mitochondria in response to A23187 and receptor stimulation. Pyrrophenone blocked calcium release from the ER and concomitant increases in mitochondrial calcium in response to stimulation by ATP, serum and A23187. In contrast, ER calcium release induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin was not blocked by pyrrophenone suggesting specificity for the calcium release pathway. As a consequence of blocking calcium mobilization, pyrrophenone inhibited serum-stimulated translocation of the cPLA2α C2 domain to Golgi. The ability of pyrrophenone to block ER calcium release is an off-target effect since it occurs in fibroblasts lacking cPLA2α. The results implicate a serine hydrolase in regulating ER calcium release and highlight the importance of careful dose-response studies with pyrrophenone to study cPLA2α function. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum

    PubMed Central

    Yun, Bogeon; Lee, HeeJung; Ewing, Heather; Gelb, Michael H.; Leslie, Christina C.

    2016-01-01

    Cytosolic phospholipase A2α (cPLA2α) mediates agonist-induced release of arachidonic acid from membrane phospholipid for production of eicosanoids. The activation of cPLA2α involves increases in intracellular calcium, which binds to the C2 domain and promotes cPLA2α translocation from the cytosol to membrane to access substrate. The cell permeable pyrrolidine-containing cPLA2α inhibitors including pyrrophenone have been useful to understand cPLA2α function. Although this serine hydrolase inhibitor does not inhibit other PLA2s or downstream enzymes that metabolize arachidonic acid, we reported that it blocks increases in mitochondrial calcium and cell death in lung fibroblasts. In this study we used the calcium indicators G-CEPIA1er and CEPIA2mt to compare the effect of pyrrophenone in regulating calcium levels in the endoplasmic reticulum (ER) and mitochondria in response to A23187 and receptor stimulation. Pyrrophenone blocked calcium release from the ER and concomitant increases in mitochondrial calcium in response to stimulation by ATP, serum and A23187. In contrast, ER calcium release induced by the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was not blocked by pyrrophenone suggesting specificity for the calcium release pathway. As a consequence of blocking calcium mobilization, pyrrophenone inhibited serum-stimulated translocation of the cPLA2α C2 domain to Golgi. The ability of pyrrophenone to block ER calcium release is an off-target effect since it occurs in fibroblasts lacking cPLA2α. The results implicate a serine hydrolase in regulating ER calcium release and highlight the importance of careful dose-response studies with pyrrophenone to study cPLA2α function. PMID:27620490

  16. Primary cilium suppression by SREBP1c involves distortion of vesicular trafficking by PLA2G3

    PubMed Central

    Gijs, Hannah Laura; Willemarck, Nicolas; Vanderhoydonc, Frank; Khan, Niamat Ali; Dehairs, Jonas; Derua, Rita; Waelkens, Etienne; Taketomi, Yoshitaka; Murakami, Makoto; Agostinis, Patrizia; Annaert, Wim; Swinnen, Johannes V.

    2015-01-01

    Distortion of primary cilium formation is increasingly recognized as a key event in many human pathologies. One of the underlying mechanisms involves aberrant activation of the lipogenic transcription factor sterol regulatory element–binding protein 1c (SREBP1c), as observed in cancer cells. To gain more insight into the molecular pathways by which SREBP1c suppresses primary ciliogenesis, we searched for overlap between known ciliogenesis regulators and targets of SREBP1. One of the candidate genes that was consistently up-regulated in cellular models of SREBP1c-induced cilium repression was phospholipase A2 group III (PLA2G3), a phospholipase that hydrolyzes the sn-2 position of glycerophospholipids. Use of RNA interference and a chemical inhibitor of PLA2G3 rescued SREBP1c-induced cilium repression. Cilium repression by SREBP1c and PLA2G3 involved alterations in endosomal recycling and vesicular transport toward the cilium, as revealed by aberrant transferrin and Rab11 localization, and was largely mediated by an increase in lysophosphatidylcholine and lysophosphatidylethanolamine levels. Together these findings indicate that aberrant activation of SREBP1c suppresses primary ciliogenesis by PLA2G3-mediated distortion of vesicular trafficking and suggest that PLA2G3 is a novel potential target to normalize ciliogenesis in SREBP1c-overexpressing cells, including cancer cells. PMID:25904332

  17. Liver cirrhosis induces renal and liver phospholipase A2 activity in rats.

    PubMed

    Vishwanath, B S; Frey, F J; Escher, G; Reichen, J; Frey, B M

    1996-07-15

    Maintenance of renal function in liver cirrhosis requires increased synthesis of arachidonic acid derived prostaglandin metabolites. Arachidonate metabolites have been reported to be involved in modulation of liver damage. The purpose of the present study was to establish whether the first enzyme of the prostaglandin cascade synthesis, the phospholipase A2(PLA2) is altered in liver cirrhosis induced by bile duct excision. The mRNA of PLA2(group I and II) and annexin-I a presumptive inhibitor of PLA2 enzyme was measured by PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. The mean mRNA ratio of group II PLA2/GAPDH was increased in liver tissue by 126% (P < 0.001) and in kidney tissue by 263% (P < 0.006) following induction of liver cirrhosis. The increase in group II PLA2 mRNA in cirrhotic animals was reflected by an increase in PLA2 protein and enzyme activity in both liver and kidney tissues. Since the mRNA of group I PLA2 was not detectable and Group IV PLA2 activity measured in liver and kidney tissue samples was very low and not changed following induction of cirrhosis, it is likely that the major PLA2 activity measured in liver and kidney corresponds to group II PLA2 enzyme. The mean mRNA ratio of annexin-I/GAPDH was increased in liver tissue by 115% (P < 0.05) but unchanged in kidney tissue following induction of cirrhosis. The protein content of annexin-I and -V were not affected by bile duct excision in liver and kidney tissue indicating that upregulation of group II PLA2 activity was not due to downregulation of annexin-I or -V. Group II PLA2 activity of glomerular mesangial cells stimulated by interleukin-1 beta was enhanced by bile juice and various bile salts. In conclusion, activity of group II PLA2 is upregulated partly due to enhanced transcription and translation in cirrhosis and is furthermore augmented by elevated levels of bile salts.

  18. 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.

  19. 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

  20. Eight genetic loci associated with variation in lipoprotein-associated phospholipase A2 mass and activity and coronary heart disease: meta-analysis of genome-wide association studies from five community-based studies

    PubMed Central

    Grallert, Harald; Dupuis, Josée; Bis, Joshua C.; Dehghan, Abbas; Barbalic, Maja; Baumert, Jens; Lu, Chen; Smith, Nicholas L.; Uitterlinden, André G.; Roberts, Robert; Khuseyinova, Natalie; Schnabel, Renate B.; Rice, Kenneth M.; Rivadeneira, Fernando; Hoogeveen, Ron C.; Fontes, João Daniel; Meisinger, Christa; Keaney, John F.; Lemaitre, Rozenn; Aulchenko, Yurii S.; Vasan, Ramachandran S.; Ellis, Stephen; Hazen, Stanley L.; van Duijn, Cornelia M.; Nelson, Jeanenne J.; März, Winfried; Schunkert, Heribert; McPherson, Ruth M.; Stirnadel-Farrant, Heide A.; Psaty, Bruce M.; Gieger, Christian; Siscovick, David; Hofman, Albert; Illig, Thomas; Cushman, Mary; Yamamoto, Jennifer F.; Rotter, Jerome I.; Larson, Martin G.; Stewart, Alexandre F.R.; Boerwinkle, Eric; Witteman, Jacqueline C.M.; Tracy, Russell P.; Koenig, Wolfgang; Benjamin, Emelia J.; Ballantyne, Christie M.

    2012-01-01

    Aims Lipoprotein-associated phospholipase A2 (Lp-PLA2) generates proinflammatory and proatherogenic compounds in the arterial vascular wall and is a potential therapeutic target in coronary heart disease (CHD). We searched for genetic loci related to Lp-PLA2 mass or activity by a genome-wide association study as part of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. Methods and results In meta-analyses of findings from five population-based studies, comprising 13 664 subjects, variants at two loci (PLA2G7, CETP) were associated with Lp-PLA2 mass. The strongest signal was at rs1805017 in PLA2G7 [P = 2.4 × 10−23, log Lp-PLA2 difference per allele (beta): 0.043]. Variants at six loci were associated with Lp-PLA2 activity (PLA2G7, APOC1, CELSR2, LDL, ZNF259, SCARB1), among which the strongest signals were at rs4420638, near the APOE–APOC1–APOC4–APOC2 cluster [P = 4.9 × 10−30; log Lp-PLA2 difference per allele (beta): −0.054]. There were no significant gene–environment interactions between these eight polymorphisms associated with Lp-PLA2 mass or activity and age, sex, body mass index, or smoking status. Four of the polymorphisms (in APOC1, CELSR2, SCARB1, ZNF259), but not PLA2G7, were significantly associated with CHD in a second study. Conclusion Levels of Lp-PLA2 mass and activity were associated with PLA2G7, the gene coding for this protein. Lipoprotein-associated phospholipase A2 activity was also strongly associated with genetic variants related to low-density lipoprotein cholesterol levels. PMID:22003152

  1. Expression, purification and refolding of active durum wheat (Triticum durum Desf.) secretory phospholipase A2 from inclusion bodies of Escherichia coli.

    PubMed

    Verlotta, Angelo; Trono, Daniela

    2014-09-01

    Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: inhibition of anticoagulant activity by low molecular weight heparin.

    PubMed

    Dutta, Sumita; Gogoi, Debananda; Mukherjee, Ashis K

    2015-03-01

    In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA2-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 μmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA2-I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA2-I. The NnPLA2-I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA2-I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA2-I did not show cytotoxicity against human glioblastoma U87MG cells

  3. The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2.

    PubMed

    Gora, Sarah; Lambeau, Gerard; Bollinger, James G; Gelb, Michael; Ninio, Ewa; Karabina, Sonia-Athina

    2006-09-01

    Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn-2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A(2), is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A(2) (hGX sPLA(2)) towards PAF. Among different sPLA(2)s, hGX sPLA(2) has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA(2), recombinant hGX sPLA(2) can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA(2) rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA(2) may be a novel player in PAF regulation during inflammatory processes.

  4. Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling

    PubMed Central

    Henderson Jr, William R.; Ye, Xin; Lai, Ying; Ni, Zhanglin; Bollinger, James G.; Tien, Ying-Tzang; Chi, Emil Y.; Gelb, Michael H.

    2013-01-01

    Background Previous work has shown that disruption of the gene for group X secreted phospholipase A2 (sPLA2-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA2s in the mammalian genome, the involvement of other sPLA2s in the asthma model is possible – in particular, the group V sPLA2 (sPLA2-V) that like sPLA2-X is highly active at hydrolyzing membranes of mammalian cells. Methodology and Principal Findings The allergen-driven asthma phenotype was significantly reduced in sPLA2-V-deficient mice but to a lesser extent than observed previously in sPLA2-X-deficient mice. The most striking difference observed between the sPLA2-V and sPLA2-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA2-V−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V−/− mice diminishes Th2 cytokine responses in the airways. Conclusions This paper illustrates the diverse roles of sPLA2s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA2-V as a potential new therapy to treat asthma and other allergic disorders. PMID:23451035

  5. 2-Oxoamides based on dipeptides as selective calcium-independent phospholipase A2inhibitors.

    PubMed

    Smyrniotou, Anneta; Kokotou, Maroula G; Mouchlis, Varnavas D; Barbayianni, Efrosini; Kokotos, George; Dennis, Edward A; Constantinou-Kokotou, Violetta

    2017-02-01

    Calcium-independent phospholipase A 2 (GVIA iPLA 2 ) has recently attracted interest as a medicinal target. The number of known GVIA iPLA 2 inhibitors is limited to a handful of synthetic compounds (bromoenol lactone and polyfluoroketones). To expand the chemical diversity, a variety of 2-oxoamides based on dipeptides and ether dipeptides were synthesized and studied for their in vitro inhibitory activity on human GVIA iPLA 2 and their selectivity over the other major intracellular GIVA cPLA 2 and the secreted GV sPLA 2 . Structure-activity relationship studies revealed the first 2-oxoamide derivative (GK317), which presents potent inhibition of GVIA iPLA 2 (X I (50) value of 0.007) and at the same time significant selectivity over GIVA cPLA 2 and GV sPLA 2 . Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Metalloproteases 2 and 9, Lp-PLA(2) and lipoprotein profile in coronary patients.

    PubMed

    Muzzio, Maria Luz; Miksztowicz, Veronica; Brites, Fernando; Aguilar, Daniel; Repetto, Esteban Martin; Wikinski, Regina; Tavella, Marcelo; Schreier, Laura; Berg, Gabriela Alicia

    2009-01-01

    Many studies suggest that the different steps of the atherosclerotic process may be mediated by metalloproteases (MMPs). MMP-9 and MMP-2, which are highly expressed in the vulnerable regions of the atherosclerotic plaques, have been suggested to be causally involved in plaque rupture. In another manner linked with LDL, lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) hydrolyzes phospholipids generating proinflammatory and proatherogenic products. Our aim was to evaluate plasma activity of MMP-2 and 9, as well as Lp-PLA(2), in subjects with coronary artery stenosis in comparison with controls and to correlate these activities with lipoprotein profile and general biomarkers of inflammation. Forty two subjects who had undergone coronary angiography were divided into two groups: patients with coronary vessels with at least 45% stenosis (CAD [coronary artery disease], n = 24) and patients without angiographically detectable coronary artery disease (controls, n = 18). Plasma activity of MMP-2 and MMP-9 was measured and correlated with markers of systemic inflammation (hs-CRP), subendothelial inflammation (Lp-PLA(2)) and lipoprotein profile. Plasma activity of both MMPs was consistently higher in patients than in controls (p <0.01). Pro-MMP-2 (r = 0.34, p <0.01) and MMP-9 (r = 0.51, p <0.02) activities correlated with apoprotein B. Pro-MMP-2 correlated with hs-CRP (r = 0.47, p <0.01) and inversely with HDL cholesterol (r = -0.35, p <0.02). No differences were observed in Lp-PLA(2) between patients and controls (15.2 +/- 4.0 vs. 15.4 +/- 4.5 micromol/mL/h, p = NS, respectively), and no correlation was observed with MMPs. MMP activity was higher in CAD than in controls. The correlation observed between pro-MMP-2 and high-sensitive C-reactive protein (hs-CRP) may be due to specific systemic inflammatory processes. No correlation was observed between Lp-PLA(2) and MMPs.

  7. Structure-based grafting, mutation, and optimization of peptide inhibitors to fit in the active pocket of human secreted phospholipase A2: find new use of old Peptide agents with anti-inflammatory activity.

    PubMed

    Zhan, Chengye; Li, Shusheng; Zhong, Qiang; Zhou, Daixing

    2015-04-01

    Phospholipase A2 (PLA2) is a key enzyme in the production of diverse mediators of inflammatory conditions, which possesses an open active pocket that is physicochemically compatible with a variety of small-molecule substrates and peptide inhibitors. Although various peptides and peptide analogues have been identified to have inhibitory activity against PLA2 originated from animals and plants, only very few were designed for human secreted PLA2 (hsPLA2), an attractive target of inflammatory arthritis. Considering that the catalytic domains of PLA2 family members across different species are highly conserved in primary sequence, advanced structure, and biological function, in this study, we proposed a synthetic pipeline to implement structure-based grafting, mutation, and optimization of peptide ligands from the snake PLA2-peptide complex crystal structures into the active pocket of apo hsPLA2 structure to computationally generate a large number of potential peptide inhibitors for hsPLA2, and the hsPLA2 inhibitory potency of few highly promising candidates arising from the theoretical analysis was determined. As might be expected, three peptides FLSFK, FLVYK, and FISYR showed relatively high inhibitory capability against hsPLA2, and other three ALSYK, LVFYA, and KGAILGFM were also modestly potent as they can suppress the enzymatic activity with observable doses. Further, the designed peptide FLVYK with highest potency was carried out with structure-guided modification based on its atomic interactions with hsPLA2 using the computationally modeled structure data, consequently resulting in a dual-point mutant ELIYK with significantly increased activity. © 2014 John Wiley & Sons A/S.

  8. Phospholipase A2 activation as a therapeutic approach for cognitive enhancement in early-stage Alzheimer disease.

    PubMed

    Schaeffer, Evelin L; Forlenza, Orestes V; Gattaz, Wagner F

    2009-01-01

    Alzheimer disease (AD) is the leading cause of dementia in the elderly and has no known cure. Evidence suggests that reduced activity of specific subtypes of intracellular phospholipases A2 (cPLA2 and iPLA2) is an early event in AD and may contribute to memory impairment and neuropathology in the disease. The objective of this study was to review the literature focusing on the therapeutic role of PLA2 stimulation by cognitive training and positive modulators, or of supplementation with arachidonic acid (PLA2 product) in facilitating memory function and synaptic transmission and plasticity in either research animals or human subjects. MEDLINE database was searched (no date restrictions) for published articles using the keywords Alzheimer disease (mild, moderate, severe), mild cognitive impairment, healthy elderly, rats, mice, phospholipase A(2), phospholipid metabolism, phosphatidylcholine, arachidonic acid, cognitive training, learning, memory, long-term potentiation, protein kinases, dietary lipid compounds, cell proliferation, neurogenesis, and neuritogenesis. Reference lists of the identified articles were checked to select additional studies of interest. Overall, the data suggest that PLA2 activation is induced in the healthy brain during learning and memory. Furthermore, learning seems to regulate endogenous neurogenesis, which has been observed in AD brains. Finally, PLA2 appears to be implicated in homeostatic processes related to neurite outgrowth and differentiation in both neurodevelopmental processes and response to neuronal injury. The use of positive modulators of PLA2 (especially of cPLA2 and iPLA2) or supplementation with dietary lipid compounds (e.g., arachidonic acid) in combination with cognitive training could be a valuable therapeutic strategy for cognitive enhancement in early-stage AD.

  9. Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Kryukova, Elena V; Spirova, Ekaterina N; Shelukhina, Irina V; Starkov, Vladislav G; Andreeva, Tatyana V; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I; Utkin, Yuri N

    2017-01-01

    Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by

  10. Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors

    PubMed Central

    Vulfius, Catherine A.; Kasheverov, Igor E.; Kryukova, Elena V.; Spirova, Ekaterina N.; Shelukhina, Irina V.; Starkov, Vladislav G.; Andreeva, Tatyana V.; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I.

    2017-01-01

    Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by

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

    PubMed

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

    2001-06-01

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

  12. Calcium-dependent hydrolysis of supported planar lipids was triggered by honey bee venom phospholipase A2 with the right orientation at the interface.

    PubMed

    Kai, Siqi; Li, Xu; Li, Bolin; Han, Xiaofeng; Lu, Xiaolin

    2017-12-20

    Hydrolysis of planar phospholipids catalyzed by honey bee venom phospholipase A 2 (bvPLA 2 ) was studied. Experiments demonstrated that Ca 2+ ions mediated between the lipids and bvPLA 2 , induced reorientation of bvPLA 2 , and activated hydrolysis. One of the hydrolysis products, fatty acids, was desorbed, and the other one, lysophospholipids, self-organized at the interface.

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

    PubMed Central

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

    2011-01-01

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

  14. 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

  15. Expression of a bee venom phospholipase A2 from Apis cerana cerana in the baculovirus-insect cell*

    PubMed Central

    Shen, Li-rong; Ding, Mei-hui; Zhang, Li-wen; Zhang, Wei-guang; Liu, Liang; Li, Duo

    2010-01-01

    Bee venom phospholipase A2 (BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this work, a new BvPLA2 (AccPLA2) gene from the Chinese honeybee (Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector. Tn-5B-4 (Tn) cells were transfected with the recombinant bacmid DNA for expression. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa. Products of hexahistidine AccPLA2 fusion protein accumulated up to 5.32% of the total cellular proteins. The AccPLA2 fusion protein was cross reactive with the anti-AmPLA2 (BvPLA2 of the European honeybee, Apis mellifera) polyclonal serum. The reaction resulted in a double glycosylation band, which agrees with the band generated by the native AmPLA2 in Western blot analysis. The PLA2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg). In summary, the recombinant AccPLA2 protein, a native BvPLA2-like structure with corresponding biological activities, can be glycosylated in Tn cells. These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA2 in the pharmaceutical industry. PMID:20443212

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  18. Primary structures and partial toxicological characterization of two phospholipases A2from Micrurus mipartitus and Micrurus dumerilii coral snake venoms.

    PubMed

    Rey-Suárez, Paola; Núñez, Vitelbina; Saldarriaga-Córdoba, Mónica; Lomonte, Bruno

    2017-06-01

    Snake venom phospholipases A 2 (PLA 2 ) share high sequence identities and a conserved structural scaffold, but show important functional differences. Only a few PLA 2 s 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 PLA 2 s: MmipPLA 2 from Micrurus mipartitus and MdumPLA 2 from Micrurus dumerilii, two species of clinical importance in Colombia. MmipPLA 2 consisted of 119 amino acid residues with a predicted pI of 8.4, whereas MdumPLA 2 consisted of 117 residues with a pI of 5.6. Both PLA 2 s showed the conserved 'group I' cysteine pattern and were enzymatically active, although MdumPLA 2 had higher activity. The two enzymes differed notably in their toxicity, with MmipPLA 2 being highly lethal to mice and mildly myotoxic, whereas MdumPLA 2 was not lethal (up to 3 μg/g body weight) but strongly myotoxic. MdumPLA 2 displayed higher anticoagulant activity than MmipPLA 2 in 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 PLA 2 s and this classification agreed with their divergent biological activities. Overall, these findings highlight the structural and functional diversity of Micrurus venom PLA 2 s. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  19. Antioxidant tempol suppresses heart cytosolic phospholipase A2α stimulated by chronic intermittent hypoxia.

    PubMed

    Míčová, Petra; Klevstig, Martina; Holzerová, Kristýna; Vecka, Marek; Žurmanová, Jitka; Neckář, Jan; Kolář, František; Nováková, Olga; Novotný, Jiří; Hlaváčková, Markéta

    2017-08-01

    Adaptation to chronic intermittent hypoxia (CIH) is associated with reactive oxygen species (ROS) generation implicated in the improved cardiac tolerance against acute ischemia-reperfusion injury. Phospholipases A 2 (PLA 2 s) play an important role in cardiomyocyte phospholipid metabolism influencing membrane homeostasis. Here we aimed to determine the effect of CIH (7000 m, 8 h/day, 5 weeks) on the expression of cytosolic PLA 2 (cPLA 2 α), its phosphorylated form (p-cPLA 2 α), calcium-independent (iPLA 2 ), and secretory (sPLA 2 IIA) at protein and mRNA levels, as well as fatty acids (FA) profile in left ventricular myocardium of adult male Wistar rats. Chronic administration of antioxidant tempol was used to verify the ROS involvement in CIH effect on PLA 2 s expression and phospholipid FA remodeling. While CIH did not affect PLA 2 s mRNA levels, it increased the total cPLA 2 α protein in cytosol and membranes (by 191% and 38%, respectively) and p-cPLA 2 α (by 23%) in membranes. On the contrary, both iPLA 2 and sPLA 2 IIA were downregulated by CIH. CIH further decreased phospholipid n-6 polyunsaturated FA (PUFA) and increased n-3 PUFA proportion. Tempol treatment prevented only CIH-induced cPLA 2 α up-regulation and its phosphorylation on Ser 505 . Our results show that CIH diversely affect myocardial PLA 2 s and suggest that ROS are responsible for the activation of cPLA 2 α under these conditions.

  20. 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

    2018-02-01

    Phospholipases A 2 (PLA 2 s) 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-PLA 2 s 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 PLA 2 s 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 PLA 2 s using acid PLA 2 BthA-I-PLA 2 from B. jararacussu template showed high quality. Both acidic PLA 2 s 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 PLA 2 s 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 Elsevier B.V. All rights reserved.

  1. Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry.

    PubMed

    Cao, Jian; Hsu, Yuan-Hao; Li, Sheng; Woods, Virgil L; Dennis, Edward A

    2013-01-01

    Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), specifically Group VIIA PLA(2), is a member of the phospholipase A(2) superfamily and is found mainly associated with LDL and HDL in human plasma. Lp-PLA(2) is considered as a risk factor, a potential biomarker, a target for therapy in the treatment of cardiovascular disease, and evidence suggests that the level of Lp-PLA(2) in plasma is associated with the risk of future cardiovascular and stroke events. The differential location of the enzyme in LDL/HDL lipoproteins has been suggested to affect Lp-PLA(2) function and/or its physiological role and an abnormal distribution of the enzyme may correlate with diseases. Although a mutagenesis study suggested that a surface helix (residues 362-369) mediates the association between Lp-PLA(2) and HDL, the molecular details and mechanism of association has remained unknown. We have now employed hydrogen deuterium exchange mass spectrometry to characterize the interaction between recombinant human Lp-PLA(2) and human HDL. We have found that specific residues 113-120, 192-204, and 360-368 likely mediate HDL binding. In a previous study, we showed that residues 113-120 are important for Lp-PLA(2)-liposome interactions. We now find that residues 192-204 show a decreased deuteration level when Lp-PLA(2) is exposed to apoA-I, but not apoA-II, the most abundant apoproteins in HDL, and additionally, residues 360-368 are only affected by HDL.The results suggest that apoA-I and phospholipid membranes play crucial roles in Lp-PLA(2) localization to HDL.

  2. Phospholipase A2 in cnidaria.

    PubMed

    Nevalainen, Timo J; Peuravuori, Heikki J; Quinn, Ronald J; Llewellyn, Lyndon E; Benzie, John A H; Fenner, Peter J; Winkel, Ken D

    2004-12-01

    Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.

  3. Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA2 Inhibition

    PubMed Central

    dos Santos, Juliana I.; Cardoso, Fábio F.; Soares, Andreimar M.; dal Pai Silva, Maeli; Gallacci, Márcia; Fontes, Marcos R. M.

    2011-01-01

    Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy. PMID:22205953

  4. Inhibition of secretory phospholipase A2 activity attenuates acute cardiogenic pulmonary edema induced by isoproterenol infusion in mice after myocardial infarction.

    PubMed

    Kawabata, Kenichi; Fujioka, Daisuke; Kobayashi, Tsuyoshi; Saito, Yukio; Obata, Jun-Ei; Nakamura, Takamitsu; Yano, Toshiaki; Watanabe, Kazuhiro; Watanabe, Yosuke; Mishina, Hideto; Kugiyama, Kiyotaka

    2010-10-01

    Several types of secretory phospholipase A2 (sPLA2) are expressed in lung tissue, yielding various eicosanoids that might cause pulmonary edema. This study examined whether inhibition of sPLA2 activity attenuates acute cardiogenic pulmonary edema in mice. Acute cardiogenic pulmonary edema was induced in C57BL/6J male mice by an increase in heart rate with continuous intravenous infusion of isoproterenol (ISP) (10 mg/kg/h) at 2 weeks after the creation of myocardial infarction by left coronary artery ligation. Just before ISP infusion, a single intraperitoneal injection of 100 mg/kg LY374388, a prodrug of LY329722 that inhibits sPLA2 activity, or vehicle was administered. The ISP infusion after myocardial infarction induced interstitial and alveolar edema on lung histology. Furthermore, it increased the lung-to-body weight ratio, pulmonary vascular permeability evaluated by the Evans blue extravasation method, lung activity of sPLA2, and lung content of thromboxane A2 and leukotriene B4. These changes were significantly attenuated by LY374388 treatment. In Kaplan-Meier analysis, the survival rate during the ISP infusion after myocardial infarction was significantly higher in LY374388- than in vehicle-treated mice. Similar results were obtained with another inhibitor of sPLA2 activity, para-bromophenacyl bromide. In conclusion, inhibition of sPLA2 activity suppressed acute cardiogenic pulmonary edema.

  5. Phospholipase A2 activity of peroxiredoxin 6 promotes invasion and metastasis of lung cancer cells.

    PubMed

    Ho, Jin-Nyoung; Lee, Seung Bum; Lee, Seung-Sook; Yoon, Sung Hwan; Kang, Ga Young; Hwang, Sang-Gu; Um, Hong-Duck

    2010-04-01

    Peroxiredoxins (PRDX) are a family of thiol-dependent peroxidases. Among the six mammalian members of this family, PRDX6 is the only protein that additionally exhibits phospholipase A(2) (PLA(2)) activity. The physiologic role of this interesting PRDX6 feature is largely unknown at present. In this study, we show that PRDX6 increases the metastatic potential of lung cancer cells. Functional analyses of the enzymatic activities of PRDX6, using specific pharmacologic inhibitors and mutagenesis studies, reveal that both peroxidase and PLA(2) activities are required for metastasis. Specifically, peroxidase activity facilitates the growth of cancer cells, and PLA(2) activity promotes invasiveness. Further investigation of the latter event discloses that PLA(2) activity promotes accumulation of arachidonic acid, which, in turn, induces the invasive pathway involving p38 kinase, phosphoinositide 3-kinase, Akt, and urokinase-type plasminogen activator. This study is the first to define the functions of the enzymatic activities of PRDX6 in metastasis and to show the involvement of arachidonic acid in PRDX6 action in intact cells. These novel findings provide a significant step toward elucidating the role of PRDX6 in cancer and the mechanism of its action. Mol Cancer Ther; 9(4); 825-32. (c)2010 AACR.

  6. Inhibition of Neurotoxic Secretory Phospholipases A2 Enzymatic, Edematogenic, and Myotoxic Activities by Harpalycin 2, an Isoflavone Isolated from Harpalyce brasiliana Benth

    PubMed Central

    Ximenes, Rafael M.; Rabello, Marcelo M.; Araújo, Renata M.; Silveira, Edilberto R.; Fagundes, Fábio H. R.; Diz-Filho, Eduardo B. S.; Buzzo, Simone C.; Soares, Veronica C. G.; Toyama, Daniela de O.; Gaeta, Henrique H.; Hernandes, Marcelo Z.; Monteiro, Helena S. A.; Toyama, Marcos H.

    2012-01-01

    Secretory phospholipases A2 (sPLA2) exert proinflammatory actions through lipid mediators. These enzymes have been found to be elevated in many inflammatory disorders such as rheumatoid arthritis, sepsis, and atherosclerosis. The aim of this study was to evaluate the effect of harpalycin 2 (Har2), an isoflavone isolated from Harpalyce brasiliana Benth., in the enzymatic, edematogenic, and myotoxic activities of sPLA2 from Bothrops pirajai, Crotalus durissus terrificus, Apis mellifera, and Naja naja venoms. Har2 inhibits all sPLA2 tested. PrTX-III (B. pirajai venom) was inhibited at about 58.7%, Cdt F15 (C. d. terrificus venom) at 78.8%, Apis (from bee venom) at 87.7%, and Naja (N. naja venom) at 88.1%. Edema induced by exogenous sPLA2 administration performed in mice paws showed significant inhibition by Har2 at the initial step. In addition, Har2 also inhibited the myotoxic activity of these sPLA2s. In order to understand how Har2 interacts with these enzymes, docking calculations were made, indicating that the residues His48 and Asp49 in the active site of these enzymes interacted powerfully with Har2 through hydrogen bonds. These data pointed to a possible anti-inflammatory activity of Har2 through sPLA2 inhibition. PMID:22899963

  7. 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.

  8. [Impact of gender on lipoprotein-associated phospholipase A2 activity and association with known cardiovascularrisk factors].

    PubMed

    Jia, Zhang-rong; Zhao, Dong; Qi, Yue; Wang, Wei; Wang, Miao; Sun, Jia-yi; Qin, Lan-ping; Liu, Jing

    2013-11-01

    To explore the impact of gender on lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity and association with known cardiovascular risk factors. Participants in this study were recruited from Beijing sub-cohort from the Chinese Multi-provincial Cohort Study (CMCS) database. A total of 1471 participants with complete laboratory data were included in the study (688 male). Lp-PLA(2) activity was determined by colorimetric assay kit.Lp-PLA(2) activity level and correlation between Lp-PLA(2) activity and known risk factors were compared between men and women. (1) Lp-PLA(2) activity was higher in males than in females [(22.73 ± 8.52) nmol·min(-1)·ml(-1) vs.(20.01 ± 8.06) nmol·min(-1)·ml(-1), P < 0.01].(2) Age, waist circumference, systolic blood pressure, diastolic blood pressure and the prevalence of hypertension were higher in males than in females, while total cholesterol, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were higher in females than in males (P < 0.05 or P < 0.01).(3)Pearson correlation showed that Lp-PLA(2) activity was correlated with lipids ( total cholesterol, LDL-C, HDL-C, and triglyceride), blood pressure (systolic blood pressure and diastolic blood pressure), and adiposity associated parameters (waist circumference and body mass index) in males (all P < 0.01) and was correlated with lipid level (total cholesterol, LDL-C, HDL-C, and triglyceride) and age in females( P < 0.05 or P < 0.01). Correlations with variables associated with obesity or blood pressure in females were much weaker than those in males (in females, r = 0.02-0.08; in males, r = 0.10-0.16).(4)After adjustment for age, waist circumference, systolic blood pressure, glucose, LDL-C, HDL-C, triglyceride and high sensitivity C-reactive protein by multiple logistic regression model, Lp-PLA(2) activity was still significantly higher in males than in females (OR = 1.72, 95% confidence interval = 1.34-2.21, P < 0.01). Lp-PLA(2

  9. Activation of group IVC phospholipase A(2) by polycyclic aromatic hydrocarbons induces apoptosis of human coronary artery endothelial cells.

    PubMed

    Tithof, Patricia K; 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

    2011-06-01

    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 A(2) (PLA(2)) 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 PLA(2) in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA(2) inhibited both (3)H-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 PLA(2). 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 PLA(2).

  10. 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

  11. Enhanced Expression of Lp-PLA2 and Lysophosphatidylcholine in Symptomatic Carotid Atherosclerotic Plaques

    PubMed Central

    Mannheim, Dallit; Herrmann, Joerg; Versari, Daniele; Gössl, Mario; Meyer, Fredric B.; McConnell, Joseph P.; Lerman, Lilach O.; Lerman, Amir

    2015-01-01

    Background and Purpose Circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) has emerged as a novel biomarker for cardiovascular diseases. However, the correlation between the plaque expression of Lp-PLA2 and plaque oxidative stress, inflammation, and stability as well as the clinical presentation remains poorly defined, especially for cerebrovascular disease. Therefore, this study was performed to test the hypothesis that Lp-PLA2 expression is higher in symptomatic than in asymptomatic carotid plaques of patients undergoing carotid endarterectomy. Methods The expression of Lp-PLA2 in 167 carotid artery plaques was determined by immunoblotting and immunostaining. Plaque oxidative stress, inflammation, and stability were quantified by NAD(P)H oxidase p67phox and MMP-2 immunoblotting, oxidized LDL (oxLDL) immunoreactivity, macrophage and Sirius red collagen staining. Lysophosphatidylcholine 16:0 (lysoPC) concentration was measured in 55 plaques using liquid chromatography tandem mass spectrometry. Results Lp-PLA2 expression was significantly higher in plaques of symptomatic patients than asymptomatic patients (1.66±0.19 versus 1.14±0.10, P<0.05) and localized mainly to shoulder and necrotic lipid core areas in colocalization with oxLDL and macrophage content. Similarly, Lp-PLA2 expression was related to collagen content, which was lower in plaques from symptomatic patients than in plaques from asymptomatic patients (9.1±2.2 versus 18.5±1.7% of staining/field, P<0.001). LysoPC plaque concentration was significantly higher in plaques of symptomatic than asymptomatic patients (437.0±57.91 versus 228.84±37.00 mmol/L, P<0.05). Conclusions Symptomatic carotid artery plaques are characterized by increased levels of Lp-PLA2 and its product lysoPC in correlation with markers of tissue oxidative stress, inflammation, and instability. These findings strongly support a role for Lp-PLA2 in the pathophysiology and clinical presentation of cerebrovascular disease

  12. Phospholipase A(2) enzymes in metabolic and cardiovascular diseases.

    PubMed

    Hui, David Y

    2012-06-01

    The phospholipase A2 (PLA2) family of proteins includes lipolytic enzymes that liberate the sn-2 fatty acyl chains from phospholipids to yield nonesterified fatty acids and lysophospholipids. The purpose of this review is to discuss recent findings showing distinct roles of several of these PLA2 enzymes in inflammatory metabolic diseases such as diabetes and atherosclerosis. The group 1B PLA2 digestion of phospholipids in the intestinal lumen facilitates postprandial lysophospholipid absorption, which suppresses hepatic fatty acid oxidation leading to increased VLDL synthesis, decreased glucose tolerance, and promotion of tissue lipid deposition to accentuate diet-induced hyperlipidemia, diabetes, and obesity. Other secretory PLA2s promote inflammatory metabolic diseases by generating bioactive lipid metabolites to induce inflammatory cytokine production, whereas the major intracellular PLA2s, cPLA2α, and iPLA2, generate arachidonic acid and lysophosphatic acid in response to extracellular stimuli to activate leukocyte chemotactic response. Each member of the PLA2 family of enzymes serves a distinct role in generating active lipid metabolites that promote inflammatory metabolic diseases including atherosclerosis, hyperlipidemia, obesity, and diabetes. The development of specific drugs that target one or more of these PLA2 enzymes may be novel strategies for treatment of these chronic inflammatory metabolic disorders.

  13. 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.

  14. Cloning of a novel phospholipase A2 from the cnidarian Adamsia carciniopados.

    PubMed

    Talvinen, Kati A; Nevalainen, Timo J

    2002-07-01

    PLA2 catalytic activity was detected in homogenised tissues, including tentacles and acontia (structures for preying and defence, respectively), of the sea anemone Adamsia carciniopados. Nested reverse transcription polymerase chain reaction (RT PCR) with degenerate primers and rapid amplification of cDNA ends (RACE) were used to clone a novel phospholipase A2 from Adamsia carciniopados (AcPLA2). AcPLA2 contains a putative prepropeptide of 37 residues, ending with a basic doublet followed by a mature protein of 119 amino acids, including 12 cysteines. AcPLA2 displays only 30-42% similarity with other known secretory PLA2s (sPLA2). C-terminal extension, typical of groups II and X PLA2s, is absent. Predicted molecular weight and pI of the mature protein are 13.5 kDa and 9.1, respectively. Structural features and phylogenetic analysis set AcPLA2 apart from the known sPLA2s and define this molecule in the ancient metazoan phylum Cnidaria as a member of a new class of sPLA2s.

  15. Secretory phospholipase A2 hydrolysis of phospholipid analogues is dependent on water accessibility to the active site.

    PubMed

    Peters, Günther H; Møller, Martin S; Jørgensen, Kent; Rönnholm, Petra; Mikkelsen, Mette; Andresen, Thomas L

    2007-05-02

    A new and unnatural type of phospholipids with the head group attached to the 2-position of the glycerol backbone has been synthesized and shown to be a good substrate for secretory phospholipase A2 (sPLA2). To investigate the unexpected sPLA2 activity, we have compared three different phospholipids by using fluorescence techniques and HPLC, namely: (R)-1,2-dipalmitoyl-glycero-3-phosphocholine (hereafter referred to as 1R), (R)-1-O-hexadecyl-2-palmitoyl-glycero-3-phoshocholine (2R), and (S)-1-O-hexadecyl-3-palmitoyl-glycero-2-phosphocholine (3S). Furthermore, to understand the underlying mechanisms for the observed differences, we have performed molecular dynamics simulations to clarify on a structural level the substrate specificity of sPLA2 toward phospholipid analogues with their head groups in the 2-position of the glycerol backbone. We have studied the lipids above 1R, 2R, and 3S as well as their enantiomers 1S, 2S, and 3R. In the simulations of sPLA2-1S and sPLA2-3R, structural distortion in the binding cleft induced by the phospholipids showed that these are not substrates for sPLA2. In the case of the phospholipids 1R, 2R, and 3S, our simulations revealed that the difference observed experimentally in sPLA2 activity might be caused by reduced access of water molecules to the active site. We have monitored the number of water molecules that enter the active site region for the different sPLA2-phospholipid complexes and found that the probability of a water molecule reaching the correct position such that hydrolysis can occur is reduced for the unnatural lipids. The relative water count follows 1R > 2R > 3S. This is in good agreement with experimental data that indicate the same trend for sPLA2 activity: 1R > 2R > 3S.

  16. Phospholipase A(2) activation by poultry particulate matter is mediated through extracellular signal-regulated kinase in lung epithelial cells: regulation of interleukin-8 release.

    PubMed

    Kotha, Sainath R; Piper, Melissa G; Patel, Rishi B; Sliman, Sean; Malireddy, Smitha; Zhao, Lingying; Baran, Christopher P; Nana-Sinkam, Patrick S; Wewers, Mark D; Romberger, Debra; Marsh, Clay B; Parinandi, Narasimham L

    2013-11-01

    The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA2 in A549 cells, which was attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA2 serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA2. The poultry PM-induced cPLA2 serine phosphorylation and IL-8 release were attenuated by AACOCF3, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA2 knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA2 activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.

  17. MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles

    PubMed Central

    Menzel, Nicolas; Fischl, Wolfgang; Hueging, Kathrin; Bankwitz, Dorothea; Frentzen, Anne; Haid, Sibylle; Gentzsch, Juliane; Kaderali, Lars; Bartenschlager, Ralf; Pietschmann, Thomas

    2012-01-01

    Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny. PMID:22911431

  18. Disruption of Golgi morphology and altered protein glycosylation in PLA2G6-associated neurodegeneration.

    PubMed

    Davids, Mariska; Kane, Megan S; He, Miao; Wolfe, Lynne A; Li, Xueli; Raihan, Mohd A; Chao, Katherine R; Bone, William P; Boerkoel, Cornelius F; Gahl, William A; Toro, Camilo

    2016-03-01

    Mutations in PLA2G6, which encodes the calcium-independent phospholipase A2 group VI, cause neurodegeneration and diffuse cortical Lewy body formation by a yet undefined mechanism. We assessed whether altered protein glycosylation due to abnormal Golgi morphology might be a factor in the pathology of this disease. Three patients presented with PLA2G6-associated neurodegeneration (PLAN); two had infantile neuroaxonal dystrophy (INAD) and one had adult-onset dystonia-parkinsonism. We analysed protein N-linked and O-linked glycosylation in cerebrospinal fluid, plasma, urine, and cultured skin fibroblasts using high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization--time of flight/mass spectrometry (MALDI-TOF/MS). We also assessed sialylation and Golgi morphology in cultured fibroblasts by immunofluorescence and performed rescue experiments using a lentiviral vector. The patients with INAD had PLA2G6 mutations NM_003560.2: c.[950G>T];[426-1077dup] and c.[1799G>A];[2221C>T] and the patient with dystonia-parkinsonism had PLA2G6 mutations NM_003560.2: c.[609G>A];[2222G>A]. All three patients had altered Golgi morphology and abnormalities of protein O-linked glycosylation and sialylation in cultured fibroblasts that were rescued by lentiviral overexpression of wild type PLA2G6. Our findings add altered Golgi morphology, O-linked glycosylation and sialylation defects to the phenotypical spectrum of PLAN; these pathways are essential for correct processing and distribution of proteins. Lewy body and Tau pathology, two neuropathological features of PLAN, could emerge from these defects. Therefore, Golgi morphology, O-linked glycosylation and sialylation may play a role in the pathogenesis of PLAN and perhaps other neurodegenerative disorders. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  19. Evidence for inflammation-mediated memory dysfunction in gastropods: putative PLA2 and COX inhibitors abolish long-term memory failure induced by systemic immune challenges

    PubMed Central

    2013-01-01

    Background Previous studies associate lipid peroxidation with long-term memory (LTM) failure in a gastropod model (Lymnaea stagnalis) of associative learning and memory. This process involves activation of Phospholipase A2 (PLA2), an enzyme mediating the release of fatty acids such as arachidonic acid that form the precursor for a variety of pro-inflammatory lipid metabolites. This study investigated the effect of biologically realistic challenges of L. stagnalis host defense response system on LTM function and potential involvement of PLA2, COX and LOX therein. Results Systemic immune challenges by means of β-glucan laminarin injections induced elevated H2O2 release from L. stagnalis circulatory immune cells within 3 hrs of treatment. This effect dissipated within 24 hrs after treatment. Laminarin exposure has no direct effect on neuronal activity. Laminarin injections disrupted LTM formation if training followed within 1 hr after injection but had no behavioural impact if training started 24 hrs after treatment. Intermediate term memory was not affected by laminarin injection. Chemosensory and motor functions underpinning the feeding response involved in this learning model were not affected by laminarin injection. Laminarin’s suppression of LTM induction was reversed by treatment with aristolochic acid, a PLA2 inhibitor, or indomethacin, a putative COX inhibitor, but not by treatment with nordihydro-guaiaretic acid, a putative LOX inhibitor. Conclusions A systemic immune challenge administered shortly before behavioural training impairs associative LTM function in our model that can be countered with putative inhibitors of PLA2 and COX, but not LOX. As such, this study establishes a mechanistic link between the state of activity of this gastropod’s innate immune system and higher order nervous system function. Our findings underwrite the rapidly expanding view of neuroinflammatory processes as a fundamental, evolutionary conserved cause of cognitive and

  20. 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

  1. Group X secreted phospholipase A2 limits the development of atherosclerosis in LDL receptor-null mice.

    PubMed

    Ait-Oufella, Hafid; Herbin, Olivier; Lahoute, Charlotte; Coatrieux, Christelle; Loyer, Xavier; Joffre, Jeremie; Laurans, Ludivine; Ramkhelawon, Bhama; Blanc-Brude, Olivier; Karabina, Sonia; Girard, Christophe A; Payré, Christine; Yamamoto, Kei; Binder, Christoph J; Murakami, Makoto; Tedgui, Alain; Lambeau, Gérard; Mallat, Ziad

    2013-03-01

    Several secreted phospholipases A2 (sPLA2s), including group IIA, III, V, and X, have been linked to the development of atherosclerosis, which led to the clinical testing of A-002 (varespladib), a broad sPLA2 inhibitor for the treatment of coronary artery disease. Group X sPLA2 (PLA2G10) has the most potent hydrolyzing activity toward phosphatidylcholine and is believed to play a proatherogenic role. Here, we show that Ldlr(-/-) mice reconstituted with bone marrow from mouse group X-deficient mice (Pla2g10(-/-)) unexpectedly display a doubling of plaque size compared with Pla2g10(+/+) chimeric mice. Macrophages of Pla2g10(-/-) mice are more susceptible to apoptosis in vitro, which is associated with a 4-fold increase of plaque necrotic core in vivo. In addition, chimeric Pla2g10(-/-) mice show exaggerated T lymphocyte (Th)1 immune response, associated with enhanced T-cell infiltration in atherosclerotic plaques. Interestingly, overexpression of human PLA2G10 in murine bone marrow cells leads to significant reduction of Th1 response and to 50% reduction of lesion size. PLA2G10 expression in bone marrow cells controls a proatherogenic Th1 response and limits the development of atherosclerosis. The results may provide an explanation for the recently reported inefficacy of A-002 (varespladib) to treat patients with coronary artery disease. Indeed, A-002 is a nonselective sPLA2 inhibitor that inhibits both proatherogenic (groups IIA and V) and antiatherogenic (group X) sPLA2s. Our results suggest that selective targeting of individual sPLA2 enzymes may be a better strategy to treat cardiovascular diseases.

  2. Structural and Phylogenetic Studies with MjTX-I Reveal a Multi-Oligomeric Toxin – a Novel Feature in Lys49-PLA2s Protein Class

    PubMed Central

    Cavalcante, Walter L. G.; Fernandez, Roberto M.; Gallacci, Márcia; Soares, Andreimar M.; Oliveira, Cristiano L. P.; Fontes, Marcos R. M.

    2013-01-01

    The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A2, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA2s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA2, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA2s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA2s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities inresponse to environmental changes and adaptation to new preys. PMID:23573271

  3. 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

  4. Purification of a low-molecular-weight phospholipase A(2) associated with soluble high-molecular-weight acidic proteins from rabbit nucleus pulposus and its comparison with a rabbit splenic group IIa phospholipase A(2).

    PubMed

    Tanaka, N; Ishida, T; Hukuda, S; Horiike, K

    2000-06-01

    An intervertebral disc is a large peice of avascular cartilage rich in proteoglycans and water consisting of gelatinous nucleus pulposus and fibrous annulus fibrosus. The soluble fraction of rabbit nucleus pulposus exhibited unusually high Ca(2+)-dependent phospholipase A(2) (PLA(2)) activity (about 70% of the total PLA(2) activity). The soluble PLA(2) activity was 6-7-fold higher than those of rabbit annulus fibrosus and spleen. The PLA(2) was bound to an anion-exchange column at pH 7.4, and eluted near the void volume as a broad peak on gel-filtration on a TSKgel SuperSW3000 column developed with a buffer containing 0.1-0.2 M salt. When the gel-filtration column was developed in the presence of 1 M salt, almost all the PLA(2) activity was eluted near the total available volume. The soluble PLA(2) was purified to near homogeneity. A Ca(2+)-dependent PLA(2) was also purified from the fractions extracted with 1 M KBr from nucleus pulposus. For comparison, we purified a Ca(2+)-dependent PLA(2) from the KBr fraction of spleen. The splenic PLA(2) was identical to a group IIa PLA(2), as judged from its N-terminal amino acid sequences and mass spectra. On SDS-polyacrylamide gel electrophoresis the enzymes purified from the soluble and KBr fractions of nucleus pulposus both gave a major 15. 7-kDa band at the same position as splenic group IIa PLA(2). These results suggest that group IIa PLA(2) is associated with soluble high-molecular-weight proteins, most likely proteoglycans, in the extracellular matrix of rabbit nucleus pulposus.

  5. Secreted Phospholipases A2 from Animal Venoms in Pain and Analgesia

    PubMed Central

    Zambelli, Vanessa O.; Picolo, Gisele; Fernandes, Carlos A. H.

    2017-01-01

    Animal venoms comprise a complex mixture of components that affect several biological systems. Based on the high selectivity for their molecular targets, these components are also a rich source of potential therapeutic agents. Among the main components of animal venoms are the secreted phospholipases A2 (sPLA2s). These PLA2 belong to distinct PLA2s groups. For example, snake venom sPLA2s from Elapidae and Viperidae families, the most important families when considering envenomation, belong, respectively, to the IA and IIA/IIB groups, whereas bee venom PLA2 belongs to group III of sPLA2s. It is well known that PLA2, due to its hydrolytic activity on phospholipids, takes part in many pathophysiological processes, including inflammation and pain. Therefore, secreted PLA2s obtained from animal venoms have been widely used as tools to (a) modulate inflammation and pain, uncovering molecular targets that are implicated in the control of inflammatory (including painful) and neurodegenerative diseases; (b) shed light on the pathophysiology of inflammation and pain observed in human envenomation by poisonous animals; and, (c) characterize molecular mechanisms involved in inflammatory diseases. The present review summarizes the knowledge on the nociceptive and antinociceptive actions of sPLA2s from animal venoms, particularly snake venoms. PMID:29311537

  6. Isolation and Functional Characterization of an Acidic Myotoxic Phospholipase A2 from Colombian Bothrops asper Venom

    PubMed Central

    Posada Arias, Silvia; Rey-Suárez, Paola; Pereáñez J, Andrés; Acosta, Cristian; Rojas, Mauricio; Ferreira Jr, Rui Seabra; Núñez, Vitelbina

    2017-01-01

    Myotoxic phospholipases A2 (PLA2) are responsible for many clinical manifestations in envenomation by Bothrops snakes. A new myotoxic acidic Asp49 PLA2 (BaCol PLA2) was isolated from Colombian Bothrops asper venom using reverse-phase high performance liquid chromatography (RP-HPLC). BaCol PLA2 had a molecular mass of 14,180.69 Da (by mass spectrometry) and an isoelectric point of 4.4. The complete amino acid sequence was obtained by cDNA cloning (GenBank accession No. MF319968) and revealed a mature product of 124 amino acids with Asp at position 49. BaCol PLA2 showed structural homology with other acidic PLA2 isolated from Bothrops venoms, including a non-myotoxic PLA2 from Costa Rican B. asper. In vitro studies showed cell membrane damage without exposure of phosphatidylserine, an early apoptosis hallmark. BaCol PLA2 had high indirect hemolytic activity and moderate anticoagulant action. In mice, BaCol PLA2 caused marked edema and myotoxicity, the latter seen as an increase in plasma creatine kinase and histological damage to gastrocnemius muscle fibers that included vacuolization and hyalinization necrosis of the sarcoplasm. PMID:29072602

  7. Evidence for distinct mechanisms of uptake and antitumor activity of secretory phospholipase A2 responsive liposome in prostate cancer.

    PubMed

    Mock, J N; Costyn, L J; Wilding, S L; Arnold, R D; Cummings, B S

    2013-01-01

    Secretory phospholipase A(2) (sPLA(2)) cleave phospholipids at sn-2 ester bonds, releasing lysophospholipids and fatty acids, and are over expressed in several pathologies, including inflammation, arthritis, sepsis and breast and prostate cancers. Herein we evaluated the therapeutic activity of liposomes engineered to be responsive to different sPLA(2) isoforms compared to clinically used long-circulating (pegylated) sterically stabilized liposomes (SSL) in vitro and in vivo, and assessed differences in roles of sPLA(2) in the mechanism of uptake and delivery of these nanoparticles. Exposing sPLA(2) responsive liposomes (SPRL) to sPLA(2) increased the release of intraluminal entrapped contents in a time-dependent manner that was inhibited by the sPLA(2) inhibitor LY3117273. Treatment of prostate cancer cells with doxorubicin encapsulated in SSL and SPRL resulted in cytotoxicity in LNCaP, DU-145 and PC-3 cells lines comparable to free drug. Interestingly, cytotoxicity was not altered by sPLA(2) inhibition. Tracking of drug and liposome delivery using fluorescence microscopy and flow cytometry, we demonstrated that drug uptake was liposome-dependent, as encapsulation of doxorubicin in SPRL resulted in 1.5 to 2-fold greater intracellular drug levels compared to SSL. Liposome uptake was cell-dependent and did not correlate to doxorubicin uptake; however, doxorubicin uptake was generally greatest in PC-3 cells, followed by DU-145 cells and then LNCaP cells. In almost all cases, uptake of one of our formulations, SPRL-E, was greater than SSL. The therapeutic activity of SPRL in vivo was demonstrated using a mouse xenograft model of human prostate cancer, which showed that doxorubicin entrapped within SPRL decreased tumor growth compared to SSL, suggesting that SPRL are more effective at slowing tumor growth than a SSL formulation similar to the FDA approved DOXIL™. Collectively, these data show the therapeutic activity of SPRL compared to SSL, yield insights into the

  8. Evidence for distinct mechanisms of uptake and antitumor activity of secretory phospholipase A2 responsive liposome in prostate cancer

    PubMed Central

    Mock, JN; Costyn, LJ; Wilding, SL; Arnold, RD; Cummings, BS

    2014-01-01

    Secretory phospholipase A2 (sPLA2) cleave phospholipids at sn-2 ester bonds, releasing lysophospholipids and fatty acids, and are over expressed in several pathologies, including inflammation, arthritis, sepsis and breast and prostate cancers. Herein we evaluated the therapeutic activity of liposomes engineered to be responsive to different sPLA2 isoforms compared to clinically used long-circulating (pegylated) sterically stabilized liposomes (SSL) in vitro and in vivo, and assess differences in role of sPLA2 in the mechanism of uptake and delivery of these nanoparticles. Exposing sPLA2 responsive liposomes (SPRL) to sPLA2 increased the release of intraluminal entrapped contents in a time-dependent manner that was inhibited by the sPLA2 inhibitor LY3117272. Treatment of prostate cancer cells with doxorubicin encapsulated in SSL and SPRL resulted in cytotoxicity in LNCaP, DU-145 and PC-3 cells lines comparable to free drug. Interestingly, cytotoxicity was not altered by sPLA2 inhibition. Tracking of drug and liposome delivery using fluorescence microscopy and flow cytometry, we demonstrated that drug uptake was liposome-dependent, as encapsulation of doxorubicin in SPRL resulted in 1.5 to 2-fold greater intracellular drug levels compared to SSL. Liposome uptake was cell-dependent and did not correlate to doxorubicin uptake; however, doxorubicin uptake was generally greatest in PC-3 cells, followed by DU-145 cells and then LNCaP cells. In almost all cases, uptake of one of our formulations, SPRL-E, was greater than SSL. The therapeutic activity of SPRL in vivo was demonstrated using a mouse xenograft model of human prostate cancer, which showed that doxorubicin entrapped within SPRL decreased tumor growth compared to SSL, suggesting that SPRL are more effective at slowing tumor growth than a SSL formulation similar to the FDA approved DOXIL™. Collectively, these data show the therapeutic activity of SPRL compared to SSL, yield insights into the mechanisms of action

  9. α-Synuclein-Induced Synapse Damage in Cultured Neurons Is Mediated by Cholesterol-Sensitive Activation of Cytoplasmic Phospholipase A2

    PubMed Central

    Bate, Clive; Williams, Alun

    2015-01-01

    The accumulation of aggregated forms of the α-synuclein (αSN) is associated with the pathogenesis of Parkinson’s disease (PD) and Dementia with Lewy Bodies. The loss of synapses is an important event in the pathogenesis of these diseases. Here we show that aggregated recombinant human αSN, but not βSN, triggered synapse damage in cultured neurons as measured by the loss of synaptic proteins. Pre-treatment with the selective cytoplasmic phospholipase A2 (cPLA2) inhibitors AACOCF3 and MAFP protected neurons against αSN-induced synapse damage. Synapse damage was associated with the αSN-induced activation of synaptic cPLA2 and the production of prostaglandin E2. The activation of cPLA2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B or Hexa-PAF) also protect neurons against αSN-induced synapse damage. αSN-induced synapse damage was also reduced in neurons pre-treated with the cholesterol synthesis inhibitor (squalestatin). These results are consistent with the hypothesis that αSN triggered synapse damage via hyperactivation of cPLA2. They also indicate that αSN-induced activation of cPLA2 is influenced by the cholesterol content of membranes. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse damage seen during PD. PMID:25761116

  10. α-Synuclein-induced synapse damage in cultured neurons is mediated by cholesterol-sensitive activation of cytoplasmic phospholipase A2.

    PubMed

    Bate, Clive; Williams, Alun

    2015-03-09

    The accumulation of aggregated forms of the α-synuclein (αSN) is associated with the pathogenesis of Parkinson's disease (PD) and Dementia with Lewy Bodies. The loss of synapses is an important event in the pathogenesis of these diseases. Here we show that aggregated recombinant human αSN, but not βSN, triggered synapse damage in cultured neurons as measured by the loss of synaptic proteins. Pre-treatment with the selective cytoplasmic phospholipase A2 (cPLA2) inhibitors AACOCF3 and MAFP protected neurons against αSN-induced synapse damage. Synapse damage was associated with the αSN-induced activation of synaptic cPLA2 and the production of prostaglandin E2. The activation of cPLA2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B or Hexa-PAF) also protect neurons against αSN-induced synapse damage. αSN-induced synapse damage was also reduced in neurons pre-treated with the cholesterol synthesis inhibitor (squalestatin). These results are consistent with the hypothesis that αSN triggered synapse damage via hyperactivation of cPLA2. They also indicate that αSN-induced activation of cPLA2 is influenced by the cholesterol content of membranes. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse damage seen during PD.

  11. Adenoviruses-mediated RNA interference targeting cytosolic phospholipase A2α attenuates focal ischemic brain damage in mice.

    PubMed

    Wu, Huijun; Liu, Hui; Zuo, Fengtong; Zhang, Lihua

    2018-04-01

    Cerebral ischemia injury is a clinical, frequently occurring disease, which causes a heavy burden on society and families. It has been demonstrated that cytosolic phospholipase A2α (cPLA2α) is significant in neurological injury caused by ischemic brain injury, and inhibition of cPLA2α may reduce stroke injury. In the present study, the role of cPLA2α was investigated in a mouse model of middle cerebral artery occlusion and/or reperfusion (MCAO/R) using an effective cPLA2α inhibitor and adenoviruses‑mediated RNA interference. The most effective recombinant adenovirus encoding cPLA2α small interfering RNA (pAd‑siRNA‑cPLA2α) was constructed and selected. MCAO/R surgery is used to construct the model of focal ischemic brain damage in mice. Adenoviruses‑mediated RNA interference targeting cPLA2α was administered by stereotactic surgery 2 h before the MCAO/R. The expression/activity of cPLA2α and cPLA2α‑derived injurious lipid mediators was assessed. pAd‑siRNA‑cPLA2α‑treated animals (RNA interference; RNAi group) were compared with pAd-siRNA-control-treated animals (negative group) with regard to neurological deficit, motor function, pathological changes, apoptosis, and infarct volume. The RNAi group animals reduced the expression level of cPLA2α, as determined by western blotting and reverse transcription‑quantitative polymerase chain reaction, the improvement of locomotor function was evaluated by rotarod test, and the decrease of apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end‑labeling staining. The decreased infarct areas were evaluated by 2,3,5‑triphenyltetrazolium chloride staining. The expression levels of prostaglandin E2, leukotrienes B4, lysophosphatidylcholine and free fatty acids were reduced in the RNAi group when compared with the negative control group. Thus, the data indicates that the expression level of cPLA2α was effectively controlled by pAd‑siRNA‑cPLA2α treatment. pAd‑siRNA‑cPLA

  12. Neutrophil-Derived Cytosolic PLA2α Contributes to Bacterial-Induced Neutrophil Transepithelial Migration.

    PubMed

    Yonker, Lael M; Pazos, Michael A; Lanter, Bernard B; Mou, Hongmei; Chu, Kengyeh K; Eaton, Alexander D; Bonventre, Joseph V; Tearney, Guillermo J; Rajagopal, Jayaraj; Hurley, Bryan P

    2017-10-15

    Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa- induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil - epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell - derived epithelial cultures and micro-optical coherence tomography, a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection. Copyright © 2017 by The American

  13. Secreted phospholipases A(2), a new class of HIV inhibitors that block virus entry into host cells.

    PubMed

    Fenard, D; Lambeau, G; Valentin, E; Lefebvre, J C; Lazdunski, M; Doglio, A

    1999-09-01

    Mammalian and venom secreted phospholipases A(2) (sPLA(2)s) have been associated with a variety of biological effects. Here we show that several sPLA(2)s protect human primary blood leukocytes from the replication of various macrophage and T cell-tropic HIV-1 strains. Inhibition by sPLA(2)s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA(2)s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA(2)s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA(2) have no effect on HIV-1 infection, suggesting that sPLA(2) catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA(2)s to host cells. Indeed, of 11 sPLA(2)s from venom and mammalian tissues assayed, 4 venom sPLA(2)s were found to be very potent HIV-1 inhibitors (ID(50) < 1 nM) and also to bind specifically to host cells with high affinities (K(0.5) < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA(2)s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA(2)s are being characterized in humans.

  14. Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells

    PubMed Central

    Fenard, David; Lambeau, Gérard; Valentin, Emmanuel; Lefebvre, Jean-Claude; Lazdunski, Michel; Doglio, Alain

    1999-01-01

    Mammalian and venom secreted phospholipases A2 (sPLA2s) have been associated with a variety of biological effects. Here we show that several sPLA2s protect human primary blood leukocytes from the replication of various macrophage and T cell–tropic HIV-1 strains. Inhibition by sPLA2s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA2s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA2s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA2 have no effect on HIV-1 infection, suggesting that sPLA2 catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA2s to host cells. Indeed, of 11 sPLA2s from venom and mammalian tissues assayed, 4 venom sPLA2s were found to be very potent HIV-1 inhibitors (ID50 < 1 nM) and also to bind specifically to host cells with high affinities (K0.5 < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA2s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA2s are being characterized in humans. PMID:10487775

  15. Neuroprotective effects of bee venom phospholipase A2 in the 3xTg AD mouse model of Alzheimer's disease.

    PubMed

    Ye, Minsook; Chung, Hwan-Suck; Lee, Chanju; Yoon, Moon Sik; Yu, A Ram; Kim, Jin Su; Hwang, Deok-Sang; Shim, Insop; Bae, Hyunsu

    2016-01-16

    Alzheimer's disease (AD) is a severe neuroinflammatory disease. CD4(+)Foxp3(+) regulatory T cells (Tregs) modulate various inflammatory diseases via suppressing Th cell activation. There are increasing evidences that Tregs have beneficial roles in neurodegenerative diseases. Previously, we found the population of Treg cells was significantly increased by bee venom phospholipase A2 (bvPLA2) treatment in vivo and in vitro. To examine the effects of bvPLA2 on AD, bvPLA2 was administered to 3xTg-AD mice, mouse model of Alzheimer's disease. The levels of amyloid beta (Aβ) deposits in the hippocampus, glucose metabolism in the brain, microglia activation, and CD4(+) T cell infiltration were analyzed to evaluate the neuroprotective effect of bvPLA2. bvPLA2 treatment significantly enhanced the cognitive function of the 3xTg-AD mice and increased glucose metabolism, as assessed with 18F-2 fluoro-2-deoxy-D-glucose ([F-18] FDG) positron emission tomography (PET). The levels of Aβ deposits in the hippocampus were dramatically decreased by bvPLA2 treatment. This neuroprotective effect of bvPLA2 was associated with microglial deactivation and reduction in CD4(+) T cell infiltration. Interestingly, the neuroprotective effects of bvPLA2 were abolished in Treg-depleted mice. The present studies strongly suggest that the increase of Treg population by bvPLA2 treatment might inhibit progression of AD in the 3xTg AD mice.

  16. Using Hydrogen/Deuterium Exchange Mass Spectrometry to Define the Specific Interactions of the Phospholipase A2 Superfamily with Lipid Substrates, Inhibitors, and Membranes*

    PubMed Central

    Cao, Jian; Burke, John E.; Dennis, Edward A.

    2013-01-01

    The phospholipase A2 (PLA2) superfamily consists of 16 groups and many subgroups and constitutes a diverse set of enzymes that have a common catalytic activity due to convergent evolution. However, different PLA2 types have unique three-dimensional structures and catalytic residues as well as specific tissue localization and distinct biological functions. Understanding how the different PLA2 enzymes associate with phospholipid membranes, specific phospholipid substrate molecules, and inhibitors on a molecular basis has advanced in recent years due to the introduction of hydrogen/deuterium exchange mass spectrometry. Its theory, practical considerations, and application to understanding PLA2/membrane interactions are addressed. PMID:23209293

  17. Using hydrogen/deuterium exchange mass spectrometry to define the specific interactions of the phospholipase A2 superfamily with lipid substrates, inhibitors, and membranes.

    PubMed

    Cao, Jian; Burke, John E; Dennis, Edward A

    2013-01-18

    The phospholipase A(2) (PLA(2)) superfamily consists of 16 groups and many subgroups and constitutes a diverse set of enzymes that have a common catalytic activity due to convergent evolution. However, different PLA(2) types have unique three-dimensional structures and catalytic residues as well as specific tissue localization and distinct biological functions. Understanding how the different PLA(2) enzymes associate with phospholipid membranes, specific phospholipid substrate molecules, and inhibitors on a molecular basis has advanced in recent years due to the introduction of hydrogen/deuterium exchange mass spectrometry. Its theory, practical considerations, and application to understanding PLA(2)/membrane interactions are addressed.

  18. Cooperation of TLR2 with MyD88, PI3K, and Rac1 in Lipoteichoic Acid–Induced cPLA2/COX-2–Dependent Airway Inflammatory Responses

    PubMed Central

    Lee, I-Ta; Lee, Chiang-Wen; Tung, Wei-Hsuan; Wang, Shyi-Wu; Lin, Chih-Chung; Shu, Jwu-Ching; Yang, Chuen-Mao

    2010-01-01

    Lipoteichoic acid (LTA) plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. Cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and interleukin (IL)-6 have been demonstrated to engage in airway inflammation. In this study, LTA-induced cPLA2 and COX-2 expression and PGE2 or IL-6 synthesis were attenuated by transfection with siRNAs of TLR2, MyD88, Akt, p42, p38, JNK2, and p65 or pretreatment with the inhibitors of PI3K (LY294002), p38 (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-κB (helenalin) in human tracheal smooth muscle cells (HTSMCs). LTA also induced cPLA2 and COX-2 expression and leukocyte count in bronchoalveolar lavage fluid in mice. LTA-regulated PGE2 or IL-6 production was inhibited by pretreatment with the inhibitors of cPLA2 (AACOCF3) and COX-2 (NS-398) or transfection with cPLA2 siRNA or COX-2 siRNA, respectively. LTA-stimulated NF-κB translocation or cPLA2 phosphorylation was attenuated by pretreatment with LY294002, SB202190, U0126, or SP600125. Furthermore, LTA could stimulate TLR2, MyD88, PI3K, and Rac1 complex formation. We also demonstrated that Staphylococcus aureus could trigger these responses through a similar signaling cascade in HTSMCs. It was found that PGE2 could directly stimulate IL-6 production in HTSMCs or leukocyte count in bronchoalveolar lavage fluid in mice. These results demonstrate that LTA-induced MAPKs activation is mediated through the TLR2/MyD88/PI3K/Rac1/Akt pathway, which in turn initiates the activation of NF-κB, and ultimately induces cPLA2/COX-2–dependent PGE2 and IL-6 generation. PMID:20167866

  19. Elevated PLA2G7 gene promoter methylation as a gender-specific marker of aging increases the risk of coronary heart disease in females.

    PubMed

    Jiang, Danjie; Zheng, Dawei; Wang, Lingyan; Huang, Yi; Liu, Haibo; Xu, Leiting; Liao, Qi; Liu, Panpan; Shi, Xinbao; Wang, Zhaoyang; Sun, Lebo; Zhou, Qingyun; Li, Ni; Xu, Limin; Le, Yanping; Ye, Meng; Shao, Guofeng; Duan, Shiwei

    2013-01-01

    PLA2G7 gene product is a secreted enzyme whose activity is associated with coronary heart disease (CHD). The goal of our study is to investigate the contribution of PLA2G7 promoter DNA methylation to the risk of CHD. Using the bisulphite pyrosequencing technology, PLA2G7 methylation was measured among 36 CHD cases and 36 well-matched controls. Our results indicated that there was a significant association between PLA2G7 methylation and CHD (adjusted P = 0.025). Significant gender-specific correlation was observed between age and PLA2G7 methylation (males: adjusted r = -0.365, adjusted P = 0.037; females: adjusted r = 0.373, adjusted P = 0.035). A breakdown analysis by gender showed that PLA2G7 methylation was significantly associated with CHD in females (adjusted P = 0.003) but not in males. A further two-way ANOVA analysis showed there was a significant interaction between gender and status of CHD for PLA2G7 methylation (gender*CHD: P = 6.04E-7). Moreover, PLA2G7 methylation is associated with the levels of total cholesterols (TC, r = 0.462, P = 0.009), triglyceride (TG, r = 0.414, P = 0.02) and Apolipoprotein B (ApoB, r = 0.396, P = 0.028) in females but not in males (adjusted P>0.4). Receiver operating characteristic (ROC) curves showed that PLA2G7 methylation could predict the risk of CHD in females (area under curve (AUC) = 0.912, P = 2.40E-5). Our results suggest that PLA2G7 methylation changes with aging in a gender-specific pattern. The correlation between PLA2G7 methylation and CHD risk in females is independent of other parameters including age, smoking, diabetes and hypertension. PLA2G7 methylation might exert its effects on the risk of CHD by regulating the levels of TC, TG, and ApoB in females. The gender disparities in the PLA2G7 methylation may play a role in the molecular mechanisms underlying the pathophysiology of CHD.

  20. Phospholipases A(2) in the genome of the sea anemone Nematostella vectensis.

    PubMed

    Nevalainen, Timo J

    2008-09-01

    The genome of the sea anemone Nematostella vectensis (Nv) (Cnidaria, Anthozoa) was sequenced recently (Putnam et al., Science 317: 86, 2007). In the current study, 22 proteins of Nv were identified as putative phospholipases A(2) (PLA(2)) that showed up to 40-50% sequence identity with secreted or intracellular PLA(2)s including those of humans. Nv1-Nv6 PLA(2)s have identity with secreted human group (G)IB and GIIA PLA(2)s and PLA(2)s of the sea anemones Adamsia carciniopados and Urticina crassicornis. Nv7 and Nv8 PLA(2)s have identity with human and bee venom GIII PLA(2)s and Nv9 PLA(2) with GXIIA PLA(2). Nv10-Nv13 PLA(2)s show identity with GIX PLA(2) of Conus magus and bacterial PLA(2)s but no significant identity with any human PLA(2). Nv14 has identity with intracellular GIV PLA(2), Nv15 with GVII PLA(2), Nv16 and Nv17 with GVIII PLA(2), Nv18-Nv20 with GVI PLA(2), and Nv21 and Nv22 with patatin, respectively. The observations indicate that the cnidarian phospholipasome contains a rich array of orthologs of most types of animal PLA(2)s, and that many of the PLA(2)-driven vital functions prevail in these ancient metazoans. Cnidarian PLA(2)s may be considered as evolutionary precursors of PLA(2)s of higher animals.

  1. Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding.

    PubMed

    Martín, Rubén; Cordova, Claudia; Nieto, Maria L

    2012-07-02

    Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer's disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells. Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events. The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM inhibitor (TAPI-1), and a HB

  2. Role of phospholipases A2 in diabetic retinopathy: in vitro and in vivo studies.

    PubMed

    Lupo, Gabriella; Motta, Carla; Giurdanella, Giovanni; Anfuso, Carmelina Daniela; Alberghina, Mario; Drago, Filippo; Salomone, Salvatore; Bucolo, Claudio

    2013-12-01

    Diabetic retinopathy is one of the leading causes of blindness and the most common complication of diabetes with no cure available. We investigated the role of phospholipases A2 (PLA2) in diabetic retinopathy using an in vitro blood-retinal barrier model (BRB) and an in vivo streptozotocin (STZ)-induced diabetic model. Mono- and co-cultures of endothelial cells (EC) and pericytes (PC), treated with high or fluctuating concentrations of glucose, to mimic the diabetic condition, were used. PLA2 activity, VEGF and PGE2 levels and cell proliferation were measured, with or without PLA2 inhibition. Diabetes was induced in rats by STZ injection and PLA2 activity along with VEGF, TNFα and ICAM-1 levels were measured in retina. High or fluctuating glucose induced BRB breakdown, and increased PLA2 activity, PGE2 and VEGF in EC/PC co-cultures; inhibition of PLA2 in mono- or co-cultures treated with high or fluctuating glucose dampened PGE2 and VEGF production down to the levels of controls. High or fluctuating glucose increased EC number and reduced PC number in co-cultures; these effects were reversed after transfecting EC with small interfering RNA targeted to PLA2. PLA2 and COX-2 protein expressions were significantly increased in microvessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNFα that were reduced by treatment with a cPLA2 inhibitor. In conclusion, the present findings indicate that PLA2 upregulation represents an early step in glucose-induced alteration of BRB, possibly upstream of VEGF; thus, PLA2 may be an interesting target in managing diabetic retinopathy. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. The importance of age and statin therapy in the interpretation of Lp-PLA(2) in ACS patients, and relation to CRP.

    PubMed

    Franeková, J; Kettner, J; Kubíček, Z; Jabor, A

    2015-01-01

    C-reactive protein (CRP) is a marker of arterial inflammation while lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is related to plaque instability. The aim of this study was to evaluate the correlation between the risk of unstable plaque presenting as acute coronary syndrome (ACS) and Lp-PLA(2), and to assess the influence of statins on interpretation of Lp-PLA(2). A total of 362 consecutive patients presenting to the emergency department (ED) with acute chest pain suggestive of ACS were evaluated by cardiologists as STEMI, NSTEMI, or unstable angina, and non-ACS. Serum biomarkers measured on admission: troponin I, C-reactive protein (Abbott), and Lp-PLA(2) (DiaDexus). Four groups were defined according to the final diagnosis and history of statin medication: ACS/statin-; ACS/statin+; non-ACS/statin-; non-ACS/statin+. Lp-PLA(2) was highest in ACS/statin- group; statins decreased Lp-PLA(2) both in ACS and non-ACS of about 20 %. Lp-PLA(2) was higher in ACS patients in comparison with non-ACS patients group without respect to statin therapy (p<0.001). Lp-PLA(2) predicted worse outcome (in terms of acute coronary syndrome) effectively in patients up to 62 years; limited prediction was found in older patients. C-reactive protein (CRP) failed to discriminate four groups of patients. Statin therapy and age should be taken into consideration while interpreting Lp-PLA(2) concentrations and lower cut-off values should be used for statin-treated persons.

  4. 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.

  5. 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

    2017-07-01

    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. Copyright © 2017. Published by Elsevier B.V.

  6. Association between PLA2G12A Polymorphisms and Schizophrenia in a Han Chinese Population from Northeast China.

    PubMed

    Yang, Guang; Xu, Hongqin; Zhang, Huiping; Yu, Qiong; Wu, Yanhua; Shi, Jieping; Rao, Wenwang; You, Yueyue; Kou, Changgui; Yu, Yaqin

    2016-01-01

    The purpose of this study was to explore the association between single nucleotide polymorphisms (SNPs) in the phospholipase A2 (PLA2), group XIIA gene (PLA2G12A) and schizophrenia. This study included 1,063 schizophrenia patients and 1,103 healthy controls from a Han Chinese Population in Northeast China. Four tagSNPs (rs11728699 in intron 1, synonymous rs2285714 in exon 3, rs3087494 in the 3' UTR, and rs7694620 in the downstream region) in PLA2G12A were selected, and they were genotyped by the MALDI-TOF-MS technology. The Chi-square (χ2) test and haplotype analysis were performed to analyze the association of PLA2G12A SNPs and schizophrenia using the software packages SPSS 16.0 and Haploview 4.2. Among the four tagSNPs, only SNP rs3087494 in the 3' UTR of PLA2G12A showed significant differences in both allele frequencies (χ2 = 20.136, P<0.001) compared to healthy controls. The minor allele G of SNP rs3087494 is potentially a predictive factor for schizophrenia (OR = 0.753, 95% CI: 0.665-0.882). The frequency distribution of haplotypes consisting of specific alleles of two SNPs (rs7694620-rs3087494 or rs3087494-rs2285714), three SNPs (rs7694620-rs3087494-rs2285714 or rs3087494-rs2285714-rs11728699), or all four SNPs (rs7694620-rs3087494-rs2285714-rs11728699) was significantly different between schizophrenia patients and control subjects (P<0.001). Our study demonstrated that PLA2G12A SNPs or haplotypes might influence the susceptibility to schizophrenia in the Han Chinese population from Northeast China.

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

    PubMed

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

    2016-07-08

    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.

  8. 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

  9. 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

  10. Antitumoral Potential of Tunisian Snake Venoms Secreted Phospholipases A2

    PubMed Central

    Zouari-Kessentini, Raoudha; Srairi-Abid, Najet; Bazaa, Amine; El Ayeb, Mohamed; Luis, Jose; Marrakchi, Naziha

    2013-01-01

    Phospholipases type A2 (PLA2s) are the most abundant proteins found in Viperidae snake venom. They are quite fascinating from both a biological and structural point of view. Despite similarity in their structures and common catalytic properties, they exhibit a wide spectrum of pharmacological activities. Besides being hydrolases, secreted phospholipases A2 (sPLA2) are an important group of toxins, whose action at the molecular level is still a matter of debate. These proteins can display toxic effects by different mechanisms. In addition to neurotoxicity, myotoxicity, hemolytic activity, antibacterial, anticoagulant, and antiplatelet effects, some venom PLA2s show antitumor and antiangiogenic activities by mechanisms independent of their enzymatic activity. This paper aims to discuss original finding against anti-tumor and anti-angiogenic activities of sPLA2 isolated from Tunisian vipers: Cerastes cerastes and Macrovipera lebetina, representing new tools to target specific integrins, mainly, α5β1 and αv integrins. PMID:23509718

  11. Phospholipase A2 activity during the replication cycle of the flavivirus West Nile virus.

    PubMed

    Liebscher, Susann; Ambrose, Rebecca L; Aktepe, Turgut E; Mikulasova, Andrea; Prier, Julia E; Gillespie, Leah K; Lopez-Denman, Adam J; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Mackenzie, Jason M

    2018-04-30

    Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.

  12. ALTERATION IN THE ACTIVATION STATE OF NEW INFLAMMATION-ASSOCIATED TARGETS BY PHOSPHOLIPASE A2-ACTIVATING PROTEIN (PLAA)

    PubMed Central

    Zhang, Fan; Sha, Jian; Wood, Thomas. G.; Galindo, Cristi. L.; Garner, Harold R.; Burkart, Mark. F.; Suarez, Giovanni; Sierra, Johanna. C.; Agar, Stacy. L.; Peterson, Johnny W.; Chopra, Ashok K.

    2009-01-01

    Phospholipase A2 (PLA2)-activating protein (PLAA) is a novel signaling molecule that regulates the production of prostaglandins (PGE2) and tumor necrosis factor (TNF)-α. To characterize the function of native PLAA in situ, we generated HeLa (Tet-off) cells overexpressing plaa (plaahigh) and control (plaalow) cells, with the plaa gene in opposite orientation in the latter construct. The plaahigh cells produced significantly more PGE2 and interleukin (IL)-6 compared to plaalow cells in response to TNF-α. There was increased activation and/or expression of cytosolic PLA2, cyclooxgenase-2, and NF-κB after induction of plaahigh cells with TNF-α compared to the respective plaalow cells. Microarray analysis of plaahigh cells followed by functional assays revealed increased production of proinflammatory cytokine IL-32 and a decrease in the production of annexin A4 and clusterin compared to plaalow cells. We demonstrated the role of annexin A4 as an inhibitor of PLA2 and showed that addition of exogeneous clusterin limited the production of PGE2 from plaahigh cells. To understand regulation of plaa gene expression, we used a luciferase reporter system in HeLa cells and identified one stimulatory element, with Sp1 binding sites, and one inhibitory element, in exon 1 of the plaa gene. By using decoy DNA oligonucleotides to Sp1 and competitive binding assays, we showed that Sp1 maintains basal expression of the plaa gene and binds to the above-mentioned stimulatory element. We demonstrated for the first time that the induction of native PLAA by TNF-α can perpetuate inflammation by enhancing activation of PLA2 and NF-κB. PMID:18291623

  13. Inhibition of Human Group IIA-Secreted Phospholipase A2 and THP-1 Monocyte Recruitment by Maslinic Acid.

    PubMed

    Yap, Wei Hsum; Ahmed, Nafees; Lim, Yang Mooi

    2016-10-01

    Maslinic acid is a natural pentacyclic triterpenoid which has anti-inflammatory properties. A recent study showed that secretory phospholipase A2 (sPLA2) may be a potential binding target of maslinic acid. The human group IIA (hGIIA)-sPLA2 is found in human sera and their levels are correlated with severity of inflammation. This study aims to determine whether maslinic acid interacts with hGIIA-sPLA2 and inhibits inflammatory response induced by this enzyme. It is shown that maslinic acid enhanced intrinsic fluorescence of hGIIA-sPLA2 and inhibited its enzyme activity in a concentration-dependent manner. Molecular docking revealed that maslinic acid binds to calcium binding and interfacial phospholipid binding site, suggesting that it inhibit access of catalytic calcium ion for enzymatic reaction and block binding of the enzyme to membrane phospholipid. The hGIIA-sPLA2 enzyme is also responsible in mediating monocyte recruitment and differentiation. Results showed that maslinic acid inhibit hGIIA-sPLA2-induced THP-1 cell differentiation and migration, and the effect observed is specific to hGIIA-sPLA2 as cells treated with maslinic acid alone did not significantly affect the number of adherent and migrated cells. Considering that hGIIA-sPLA2 enzyme is known to hydrolyze glyceroacylphospholipids present in lipoproteins and cell membranes, maslinic acid may bind and inhibit hGIIA-sPLA2 enzymatic activity, thereby reduces the release of fatty acids and lysophospholipids which stimulates monocyte migration and differentiation. This study is the first to report on the molecular interaction between maslinic acid and inflammatory target hGIIA-sPLA2 as well as its effect towards hGIIA-sPLA2-induced THP-1 monocyte adhesive and migratory capabilities, an important immune-inflammation process in atherosclerosis.

  14. Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

    PubMed

    Bhowmick, Rudra; Clark, Stacie; Bonventre, Joseph V; Leong, John M; McCormick, Beth A

    2017-11-01

    Pulmonary infection by Streptococcus pneumoniae is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A 3 (HXA 3 ) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with S. pneumoniae As phospholipase A 2 (PLA 2 ) promotes the release of AA, we investigated the role of PLA 2 in local and systemic disease during S. pneumoniae infection. The group IVA cytosolic isoform of PLA 2 (cPLA 2 α) was activated upon S. pneumoniae infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked S. pneumoniae -induced PMN transepithelial migration in vitro Genetic ablation of the cPLA 2 isoform cPLA 2 α dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with S. pneumoniae The cPLA 2 α-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild-type mice. Our data suggest that cPLA 2 α plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following S. pneumoniae lung challenge. Copyright © 2017 American Society for Microbiology.

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

    PubMed

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

    2012-12-14

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

  16. In Vitro Antiplasmodial Activity of Phospholipases A2 and a Phospholipase Homologue Isolated from the Venom of the Snake Bothrops asper

    PubMed Central

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

    2012-01-01

    The antimicrobial and antiparasite activity of phospholipase A2 (PLA2) from snakes and bees has been extensively explored. We studied the antiplasmodial effect of the whole venom of the snake Bothrops asper and of two fractions purified by ion-exchange chromatography: one containing catalytically-active phospholipases A2 (PLA2) (fraction V) and another containing a PLA2 homologue devoid of enzymatic activity (fraction VI). The antiplasmodial effect was assessed on in vitro cultures of Plasmodium falciparum. The whole venom of B. asper, as well as its fractions V and VI, were active against the parasite at 0.13 ± 0.01 µg/mL, 1.42 ± 0.56 µg/mL and 22.89 ± 1.22 µg/mL, respectively. Differences in the cytotoxic activity on peripheral blood mononuclear cells between the whole venom and fractions V and VI were observed, fraction V showing higher toxicity than total venom and fraction VI. Regarding toxicity in mice, the whole venom showed the highest lethal effect in comparison to fractions V and VI. These results suggest that B. asper PLA2 and its homologue have antiplasmodial potential. PMID:23242318

  17. Differential effects of phorbol 12-myristate 13-acetate and diacylglycerols on thromboxane A2-independent phospholipase A2 activation in collage-stimulated human platelets.

    PubMed

    Reddy, S; Rao, G H; Murthy, M

    1994-04-01

    We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC8 and OAG, and 1,3-DiC8 (a poor activator of PKC) on thromboxane A2 (TxA2)-independent phospholipase A2 (PLA2) activation in human platelets using collagen and A23187 as agonists. We measured PLA2 activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA2 synthesis, rise in cytosolic Ca2+, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13.85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC8, OAG, and 1,3-DiC8 increased TxA2-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC8, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC8) in priming TxA2-independent PLA2 activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA2-dependent IP3-mediated rise in cytosolic Ca2+ may not be obligatory for priming PLA2 activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC8, OAG, and 1,3-DiC8 likely enhanced PLA2 activation via intracellular Ca2+ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA1, or nonspecific PLA2. Since both 1,2-DiC8 and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA1 may not

  18. Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom.

    PubMed

    Castro, K L; Duarte, C G; Ramos, H R; Machado de Avila, R A; Schneider, F S; Oliveira, D; Freitas, C F; Kalapothakis, E; Ho, P L; Chávez-Olortegui, C

    2015-01-01

    The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

    PubMed Central

    Son, Dong Ju; Akiba, Satoshi; Hong, Jin Tae; Yun, Yeo Pyo; Hwang, Seock Yeon; Park, Young Hyun; Lee, Sung Eun

    2014-01-01

    PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum) and long pepper (Piper longum), was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX)-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA) metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2), COX-1, COX-2, and thromboxane A2 (TXA2) synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PG)E2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms. PMID:25153972

  20. Revealing the functional structure of a new PLA2 K49 from Bothriopsis taeniata snake venom employing automatic "de novo" sequencing using CID/HCD/ETD MS/MS analyses.

    PubMed

    Carregari, Victor Corasolla; Dai, Jie; Verano-Braga, Thiago; Rocha, Thalita; Ponce-Soto, Luis Alberto; Marangoni, Sergio; Roepstorff, Peter

    2016-01-10

    Snake venoms are composed of approximately 90% of proteins with several pharmacological activities having high potential in research as biological tools. One of the most abundant compounds is phospholipases A2 (PLA2), which are the most studied venom protein due to their wide pharmacological activity. Using a combination of chromatographic steps, a new PLA2 K49 was isolated and purified from the whole venom of the Bothriopsis taeniata and submitted to analyses mass spectrometry. An automatic “de novo” sequencing of this new PLA2 K49 denominated Btt-TX was performed using Peaks Studio 6 for analysis of the spectra. Additionally, a triplex approach CID/HCD/ETD has been performed, to generate higher coverage of the sequence of the protein. Structural studies correlating biological activities were made associating specific Btt-TX regions and myotoxic activity. Lysine acetylation was performed to better understand the mechanism of membrane interaction, identifying the extreme importance of the highly hydrophobic amino acids L, P and F for disruption of the membrane. Our myotoxical studies show a possible membrane disruption mechanism by Creatine Kinase release without a noticeable muscle damage, that probably occurred without phospholipid hydrolyses, but with a probable penetration of the hydrophobic amino acids present in the C-terminal region of the protein.

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

  2. [Carboxyl-11 C]Labelling of Four High-Affinity cPLA2α Inhibitors and Their Evaluation as Radioligands in Mice by Positron Emission Tomography.

    PubMed

    Fisher, Martin J; McMurray, Lindsay; Lu, Shuiyu; Morse, Cheryl L; Liow, Jeih-San; Zoghbi, Sami S; Kowalski, Aneta; Tye, George L; Innis, Robert B; Aigbirhio, Franklin I; Pike, Victor W

    2018-01-22

    Cytosolic phospholipase A2α (cPLA2α) may play a critical role in neuropsychiatric and neurodegenerative disorders associated with oxidative stress and neuroinflammation. An effective PET radioligand for imaging cPLA2α in living brain might prove useful for biomedical research, especially on neuroinflammation. We selected four high-affinity (IC 50 2.1-12 nm) indole-5-carboxylic acid-based inhibitors of cPLA2α, namely 3-isobutyryl-1-(2-oxo-3-(4-phenoxyphenoxy)propyl)-1H-indole-5-carboxylic acid (1); 3-acetyl-1-(2-oxo-3-(4-(4-(trifluoromethyl)phenoxy)phenoxy)propyl)-1H-indole-5-carboxylic acid (2); 3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-(2-oxo-3-(4-phenoxyphenoxy)propyl)-1H-indole-5-carboxylic acid (3); and 3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-(3-(4-octylphenoxy)-2-oxopropyl)-1H-indole-5-carboxylic acid (4), for labelling in carboxyl position with carbon-11 (t 1/2 =20.4 min) to provide candidate PET radioligands for imaging brain cPLA2α. Compounds [ 11 C]1-4 were obtained for intravenous injection in adequate overall yields (1.1-5.5 %) from cyclotron-produced [ 11 C]carbon dioxide and with moderate molar activities (70-141 GBq μmol -1 ) through the use of Pd 0 -mediated [ 11 C]carbon monoxide insertion on iodo precursors. Measured logD 7.4 values were within a narrow moderate range (1.9-2.4). After intravenous injection of [ 11 C]1-4 in mice, radioactivity uptakes in brain peaked at low values (≤0.8 SUV) and decreased by about 90 % over 15 min. Pretreatments of the mice with high doses of the corresponding non-radioactive ligands did not alter brain time-activity curves. Brain uptakes of radioactivity after administration of [ 11 C]1 to wild-type and P-gp/BCRP dual knock-out mice were similar (peak 0.4 vs. 0.5 SUV), indicating that [ 11 C]1 and others in this structural class, are not substrates for efflux transporters. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The bactericidal effect of human secreted group IID phospholipase A2 results from both hydrolytic and non-hydrolytic activities.

    PubMed

    Fonseca-Maldonado, Raquel; Ferreira, Tatiana Lopes; Ward, Richard J

    2012-06-01

    The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca(2+)-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10μg/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca(2+)-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca(2+)-independent damaging activity may play a role in the bactericidal function of the protein. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  4. Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages* ♦

    PubMed Central

    Sokolowska, Milena; Chen, Li-Yuan; Eberlein, Michael; Martinez-Anton, Asuncion; Liu, Yueqin; Alsaaty, Sara; Qi, Hai-Yan; Logun, Carolea; Horton, Maureen; Shelhamer, James H.

    2014-01-01

    Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4−/− and Myd88−/− mice, but not in Cd44−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2high and COX1/ALOX15/ALOX5/LTA4Hlow gene and PGE2/PGD2/15-HETEhigh and LXA4low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism. PMID:24366870

  5. 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.

  6. BK-induced cytosolic phospholipase A2 expression via sequential PKC-delta, p42/p44 MAPK, and NF-kappaB activation in rat brain astrocytes.

    PubMed

    Hsieh, Hsi-Lung; Wu, Cheng-Ying; Hwang, Tsong-Long; Yen, Mao-Hsiung; Parker, Peter; Yang, Chuen-Mao

    2006-01-01

    Bradykinin (BK), an inflammatory mediator, has been shown to induce cytosolic phospholipase A2 (cPLA2) expression implicating in inflammatory responses in various cell types. However, the detailed mechanisms underlying BK-induced cPLA2 expression in astrocytes remain unclear. RT-PCR and Western blotting analysis showed that BK induced the expression of cPLA2 mRNA and protein, which was inhibited by Hoe140, suggesting the involvement of B2 BK receptors, confirmed by immunofluorescence staining using anti-B2 BK receptor antibody. BK-induced cPLA2 expression and phosphorylation of p42/p44 MAPK was attenuated by PD98059, indicating the involvement of MEK1/2-p42/p44 MAPK in these responses. BK-induced cPLA2 expression might be due to the translocation of NF-kappaB into nucleus which was inhibited by Hoe140, helenalin, and PD98059, implying the involvement of NF-kappaB. Moreover, BK-induced cPLA2 expression was attenuated by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was supported by the transfection with a dominant negative plasmid of PKC-delta significantly attenuated BK-induced response. In addition, BK-stimulated translocation of PKC-delta from cytosol to membrane fraction was inhibited by rottlerin but not by PD98059, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Accordingly, BK-induced phosphorylation of p42/p44 MAPK was attenuated by rottlerin but not by helenalin. These results suggest that in RBA-1 cells, BK-induced cPLA2 expression was sequentially mediated through activation of PKC-delta, p42/p44 MAPK, and NF-kappaB. Understanding the regulation of cPLA2 expression induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases. Copyright 2005 Wiley-Liss, Inc.

  7. 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

  8. An in vitro model for synaptic loss in neurodegenerative diseases suggests a neuroprotective role for valproic acid via inhibition of cPLA2 dependent signalling.

    PubMed

    Williams, Robin S B; Bate, Clive

    2016-02-01

    Many neurodegenerative diseases present the loss of synapses as a common pathological feature. Here we have employed an in vitro model for synaptic loss to investigate the molecular mechanism of a therapeutic treatment, valproic acid (VPA). We show that amyloid-β (Aβ), isolated from patient tissue and thought to be the causative agent of Alzheimer's disease, caused the loss of synaptic proteins including synaptophysin, synapsin-1 and cysteine-string protein from cultured mouse neurons. Aβ-induced synapse damage was reduced by pre-treatment with physiologically relevant concentrations of VPA (10 μM) and a structural variant propylisopropylacetic acid (PIA). These drugs also reduced synaptic damage induced by other neurodegenerative-associated proteins α-synuclein, linked to Lewy body dementia and Parkinson's disease, and the prion-derived peptide PrP82-146. Consistent with these effects, synaptic vesicle recycling was also inhibited by these proteins and protected by VPA and PIA. We show a mechanism for this damage through aberrant activation of cytoplasmic phospholipase A2 (cPLA2) that is reduced by both drugs. Furthermore, Aβ-dependent cPLA2 activation correlates with its accumulation in lipid rafts, and is likely to be caused by elevated cholesterol (stabilising rafts) and decreased cholesterol ester levels, and this mechanism is reduced by VPA and PIA. Such observations suggest that VPA and PIA may provide protection against synaptic damage that occurs during Alzheimer's and Parkinson's and prion diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. 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

  10. Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes.

    PubMed

    Perumal Samy, R; Gopalakrishnakone, P; Thwin, M M; Chow, T K V; Bow, H; Yap, E H; Thong, T W J

    2007-03-01

    Venoms of snakes, scorpions, bees and purified venom phospholipase A(2) (PLA(2)) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms. Thirty-four crude venoms, nine purified PLA(2)s and two L-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 microg ml(-1)). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was -S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160-1.25 microg ml(-1)). A stronger effect was noted with the viperidae venoms (20 microg ml(-11)) as compared with elapidae venoms (40 microg ml(-1)). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin). The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (-) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA(2) enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms. The activity of LAAO and PLA(2) enzymes may be associated with the antibacterial activity of snake venoms.

  11. The Absence of Myocardial Calcium-Independent Phospholipase A2γ Results in Impaired Prostaglandin E2 Production and Decreased Survival in Mice with Acute Trypanosoma cruzi Infection

    PubMed Central

    Eickhoff, Christopher S.; Hoft, Daniel F.; Ford, David A.; Gross, Richard W.; McHowat, Jane

    2013-01-01

    Cardiomyopathy is a serious complication of Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi. The parasite often infects cardiac myocytes, causing the release of inflammatory mediators, including eicosanoids. A recent study from our laboratory demonstrated that calcium-independent phospholipase A2γ (iPLA2γ) accounts for the majority of PLA2 activity in rabbit ventricular myocytes and is responsible for arachidonic acid (AA) and prostaglandin E2 (PGE2) release. Thus, we hypothesized that cardiac iPLA2γ contributes to eicosanoid production in T. cruzi infection. Inhibition of the isoform iPLA2γ or iPLA2β, with the R or S enantiomer of bromoenol lactone (BEL), respectively, demonstrated that iPLA2γ is the predominant isoform in immortalized mouse cardiac myocytes (HL-1 cells). Stimulation of HL-1 cells with thrombin, a serine protease associated with microthrombus formation in Chagas' disease and a known activator of iPLA2, increased AA and PGE2 release, accompanied by platelet-activating factor (PAF) production. Similarly, T. cruzi infection resulted in increased AA and PGE2 release over time that was inhibited by pretreatment with (R)-BEL. Further, T. cruzi-infected iPLA2γ-knockout (KO) mice had lower survival rates and increased tissue parasitism compared to wild-type (WT) mice, suggesting that iPLA2γ-KO mice were more susceptible to infection than WT mice. A significant increase in iPLA2 activity was observed in WT mice following infection, whereas iPLA2γ-KO mice showed no alteration in cardiac iPLA2 activity and produced less PGE2. In summary, these studies demonstrate that T. cruzi infection activates cardiac myocyte iPLA2γ, resulting in increased AA and PGE2 release, mediators that may be essential for host survival during acute infection. Thus, these studies suggest that iPLA2γ plays a cardioprotective role during the acute stage of Chagas' disease. PMID:23429536

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

    SciTech Connect

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy

    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 structuremore » 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.« less

  13. Biological and biochemical characterization of two new PLA2 isoforms Cdc-9 and Cdc-10 from Crotalus durissus cumanensis snake venom.

    PubMed

    Romero-Vargas, Frey Francisco; Ponce-Soto, Luis Alberto; Martins-de-Souza, Daniel; Marangoni, Sergio

    2010-01-01

    This work reports the purification, biological characterization and amino acid sequence of two new basic PLA(2) isoforms, Cdc-9 and Cdc-10, purified from the Crotalus durissus cumanensis venom by one step analytical chromatography reverse phase HPLC. The molecular masses of the PLA(2) were 14,175+/-2.7 Da for Cdc-9 and 14,228+/-3.5 Da for Cdc-10 both deduced by primary structure and confirmed by MALDI-TOF. The isoforms presented an amino acid sequence of 122 amino acid residues, being Cdc-9: SLVQFNKMIK FETRKSGLPF YAAYGCYCGW GGQRPKDATD RCCFVHDCCY GKVAKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLS TYKNEYMFYP DSRCREPPEY TC with pI value of 8.25 and Cdc-10: SLLQFNKMIK FETRKSGVPF YAAYGCYCGW GGRRPKDPTD RCCFVHDCCY GKLTKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLN TYKNEYMFYP DSRCRGPPEY TC with a pI value of 8.46, showing highly conserved Ca(2+)-binding and catalytic sites. The PLA(2) activity decreased when the isoforms Cdc-9 and Cdc-10 were incubated with 4-bromophenacyl bromide (p-BPB), anhydrous acetic acid and p-nitrobenzene sulfonyl fluoride (NBSF) when compared with the activity of both native isoforms. In mice, the PLA(2) isoforms Cdc-9 and Cdc-10 induced myonecrosis and edema. Myotoxic and edema activities were reduced after treatment of the isoforms with p-BPB; acetylation of the lysine residues and the treatment of PLA(2) with NBSF have also induced edema reduction. However, p-BPB strongly diminishes the local and systemic myotoxic effects.

  14. 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.

  15. Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: biochemical and toxicological characterization.

    PubMed

    Fernández, Julián; Gutiérrez, José María; Angulo, Yamileth; Sanz, Libia; Juárez, Paula; Calvete, Juan J; Lomonte, Bruno

    2010-03-01

    Phospholipases A(2) (PLA(2)) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA(2)s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA(2)-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA(2)-II is monomeric, with a mass of 14,212 +/- 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA(2)-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA(2)-II also differed from other acidic PLA(2)s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 microg (5.9 microg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA(2)-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA(2)-II revealed that acidic and basic PLA(2)s form two different antigenic groups in B. asper venom. Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.

  16. Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells: role of ERK1/2, p38 MAPK, and NF-kappaB.

    PubMed

    Lin, Chih-Chung; Lin, Wei-Ning; Wang, Wei-Jung; Sun, Chi-Chin; Tung, Wei-Hsuan; Wang, Hui-Hsin; Yang, Chuen-Mao

    2009-06-01

    Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in inflammatory diseases such as atherosclerosis and angiogenesis. As extracellular nucleotides such as ATP have been shown to act via activation of P(2) purinoceptors implicated in various inflammatory diseases, we hypothesized that extracellular nucleotides contribute to vascular diseases via upregulated expression of inflammatory proteins, such as cyclooxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) in VSMCs. Western blotting, promoter assay, RT-PCR, and PGE2 immunoassay revealed that ATPgammaS induced expression of COX-2 and prostaglandin (PGE2) synthesis through the activation of p42/p44 MAPK (mitogen-activated protein kinase), p38 MAPK, and nuclear factor-kappaB (NF-kappaB). These responses were attenuated by inhibitors of MAPK/ERK kinase (MEK1/2; U0126), p38 MAPK (SB202190), and NF-kappaB (helenalin), or by tranfection with dominant negative mutants of p42, p38, IkappaB kinase (IKK)alpha, and IKKbeta. Furthermore, the ATPgammaS-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaBalpha was blocked by U0126 and helenalin. In addition, the ATPgammaS-stimulated cPLA2 expression was inhibited by U0126, SB202190, helenalin, celecoxib (a selective COX-2 inhibitor), and PGE2 receptor antagonists (AH6809, GW627368X, and SC-19220). However, the inhibitory effect of celecoxib on cPLA2 expression was reversed by addition of exogenous PGE2. Our results suggest that in VSMCs, activation of p42/p44 MAPK, p38 MAPK, and NF-kappaB is essential for ATPgammaS-induced COX-2 expression and PGE2 synthesis. Newly synthesized PGE2 was observed to act as an autocrine signal contributing to cPLA2 expression, which may be implicated in inflammatory responses. Collectively, our findings provide insights into the correlation between COX-2 and cPLA2 expression in ATPgammaS-stimulated VSMCs with similar molecular mechanisms and functional coupling to amplify the

  17. Group VI phospholipases A2: homeostatic phospholipases with significant potential as targets for novel therapeutics.

    PubMed

    Wilkins, W P; Barbour, S E

    2008-08-01

    Group VI phospholipase A2 (PLA2) is a family of acyl hydrolases that targets the sn-2 fatty acid on the glycerophospholipid (GPL) backbone. These enzymes are grouped together based on structural homologies and catalytic activities that are independent of calcium and hence are also called the iPLA(2)s. Although the best characterized of these enzymes, iPLA2beta and iPLA2gamma, have long been proposed as homeostatic enzymes involved in basal GPL metabolism, recent studies indicate roles for these enzymes in biomedically relevant processes as well. For example, iPLA2 modulates calcium homeostasis by promoting replenishment of intracellular calcium stores. This function is likely of importance in the pathogenesis of Duchenne muscular dystrophy and potentially allergy as well. iPLA2 has a variety of roles in bacterial pathogenesis and the host response against bacterial and fungal infections. These characteristics suggest that the enzyme as a potential target to control infectious diseases. iPLA2 is linked to both proliferation and chemotherapy-induced apoptosis of tumor cells. As such, the enzyme is a potential target for cancer chemotherapy. Recent studies indicate essential roles for iPLA2 in glucose homeostasis, maintenance of energy balance, adipocyte development, and hepatic lipogenesis. Thus, the enzyme is an attractive target for drugs to control type II diabetes, fatty liver disease, and other manifestations of the metabolic syndrome. Several recent studies have associated iPLA2 inactivation with neurodegenerative diseases, suggesting the possibility that products of the iPLA2 reaction as potential treatments for these disorders. Together, these observations suggest iPLA2 as a novel and important target for drug development. However given the ubiquitous expression of the enzyme and its roles in basal GPL metabolism, drug strategies targeting iPLA2 must exhibit exquisite selectivity to avoid undesired side effects. Furthermore, the cell-specific nature of many iPLA

  18. Snake venom phospholipases A(2): a novel tool against bacterial diseases.

    PubMed

    Samy, R P; Gopalakrishnakone, P; Stiles, B G; Girish, K S; Swamy, S N; Hemshekhar, M; Tan, K S; Rowan, E G; Sethi, G; Chow, V T K

    2012-01-01

    The majority of snake venom phospholipases A(2) (svPLA(2)s) are toxic and induce a wide spectrum of biological effects. They are cysteine-rich proteins that contain 119-134 amino acids and share similar structures and functions. About 50% of the residues are incorporated into α-helices, whereas only 10% are in β-sheets. Fourteen conserved cysteines form a network of seven disulfide bridges that stabilize the tertiary structure. They show a high degree of sequence and structural similarity, and are believed to have a common calcium- dependent catalytic mechanism. Additionally, svPLA(2)s display an array of biological actions that are either dependent or independent of catalysis. The PLA(2)s of mammalian origin also exert potent bactericidal activity by binding to anionic surfaces and enzymatic degradation of phospholipids in the target membranes, preferentially of Gram-positive species. The bactericidal activity against Gram-negatives by svPLA(2) requires a synergistic action with bactericidal/permeability-increasing protein (BPI), but is equally dependent on enzymatic- based membrane degradation. Several hypotheses account for the bactericidal properties of svPLA(2)s, which include "fatal depolarization" of the bacterial membrane, creation of physical holes in the membrane, scrambling of normal distribution of lipids between the bilayer leaflets, and damage of critical intracellular targets after internalization of the peptide. The present review discusses several svPLA(2)s and derived peptides that exhibit strong bactericidal activity. The reports demonstrate that svPLA(2)-derived peptides have the potential to counteract microbial infections. In fact, the C-terminal cationic/hydrophobic segment (residues 115-129) of svPLA(2)s is bactericidal. Thus identification of the bactericidal sites in svPLA(2)s has potential for developing novel antimicrobials.

  19. Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants.

    PubMed

    Chacur, M; Longo, I; Picolo, G; Gutiérrez, J M; Lomonte, B; Guerra, J L; Teixeira, C F P; Cury, Y

    2003-05-01

    The ability of Lys49 and Asp49 phospholipases A(2) (PLA(2)), from Bothrops asper snake venom, to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of both toxins (5-20 micro g/paw) caused hyperalgesia, which peaked 1h after injections. Incubation of both proteins with heparin, prior to their injection, partially reduced this response. Chemical modification of Asp49 PLA(2) with p-bromophenacyl bromide (p-BPB), which abrogates its PLA(2) activity, also abolished hyperalgesia. Intraplantar injection of a synthetic peptide corresponding to the C-terminal sequence 115-129 of Lys49 PLA(2), caused hyperalgesia of similar time course, but varying magnitude, than that induced by the native protein. In contrast, a homologous peptide derived from the Asp49 PLA(2) did not show any nociceptive effect. Hyperalgesia induced by both PLA(2)s was blocked by the histamine and serotonin receptor antagonists promethazine and methysergide, respectively, by the bradykinin B(2) receptor antagonist HOE 140 and by antibodies to tumor necrosis factor alfa (TNFalpha) and interleukin 1 (IL-1). Pretreatment with guanethidine, atenolol, prazosin and yohimbine, inhibitors of sympathomimetic amines, or with indomethacin, inhibitor of the cyclo-oxygenase pathway, reduced Lys49 PLA(2)-induced hyperalgesia without interfering with the nociceptive activity of Asp49 PLA(2). The hyperalgesic response to both myotoxins was not modified by pretreatment with celecoxib, an inhibitor of the cyclo-oxygenase type II, by zileuton, an inhibitor of the lipoxygenase pathway or by N(g)-methyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthase. These results suggest that Asp49 and Lys49 PLA(2)s are important hyperalgesic components of B. asper venom, and that Lys49 and Asp49 PLA(2)s exert their algogenic actions through different molecular mechanisms.

  20. L-carnitine is essential to beta-oxidation of quarried fatty acid from mitochondrial membrane by PLA(2).

    PubMed

    Yano, Hiromi; Oyanagi, Eri; Kato, Yasuko; Samejima, Yoshiyuki; Sasaki, Junzo; Utsumi, Kozo

    2010-09-01

    Mitochondrial beta-oxidation is an important system involved in the energy production of various cells. In this system, the function of L-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O(2) consumption without substrates, is caused by L-carnitine treatment. In this study, we investigated whether L-carnitine is essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A(2) (PLA(2)) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and L-carnitine. The effect of L-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with L-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of L-carnitine. Moreover, the L-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA(2) inhibitors were treated before ADP treatment. The L-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that L-carnitine might be essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by PLA(2).

  1. 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

  2. Anti-PLA2 action test of Casearia sylvestris Sw.

    PubMed

    Raslan, D S; Jamal, C M; Duarte, D S; Borges, M H; De Lima, M E

    2002-01-01

    Casearia sylvestris (Flacourtiaceae) is a plant which grows in the wild. The crude extract and pure substances from this plant induced partial inhibition of the PLA: (phospholipase A2) activity of snake venoms and some purified toxins. C. sylvestris extract efficiently neutralized the hemorrhagic and myotoxic activities caused by crude venoms and toxins.

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

    PubMed Central

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

    2014-01-01

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

  4. The interaction of phospholipase A2with oxidized phospholipids at the lipid-water surface with different structural organization.

    PubMed

    Litvinko, N M; Skorostetskaya, L A; Gerlovsky, D O

    2018-03-01

    Phospholipase A 2 (PLA 2 IB) activity towards UV-irradiated (λ=180-400nm) phospholipids in comparison to non-irradiated ones was investigated using phosphatidylcholine (PC) liposomes and mixed micelles of phosphatidylcholine and sodium deoxycholate as a membrane model. PLA 2 activity, determined by spectral changes of hemoglobin (Hb) under the interaction with fatty acids (product of the phospholipolysis), correlated well with the phospholipid peroxidation degree. The present work is the first study that determines the degree of oxidation of non-fragmented OxPCs, on the base of PLA 2 activity. Fragmented OxPLs have been determined as usually by analysis of MDA using spectroscopy at 532nm. Antioxidant Trolox and human blood serum reduced observed exceeding of PLA 2 activity toward OxPLs, what makes this model perspective for determining the total antioxidant activity of biological liquids. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. 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

  6. 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.

  7. Quantitative trait locus mapping in mice identifies phospholipase Pla2g12a as novel atherosclerosis modifier.

    PubMed

    Nicolaou, Alexandros; Northoff, Bernd H; Sass, Kristina; Ernst, Jana; Kohlmaier, Alexander; Krohn, Knut; Wolfrum, Christian; Teupser, Daniel; Holdt, Lesca M

    2017-10-01

    In a previous work, a female-specific atherosclerosis risk locus on chromosome (Chr) 3 was identified in an intercross of atherosclerosis-resistant FVB and atherosclerosis-susceptible C57BL/6 (B6) mice on the LDL-receptor deficient (Ldlr -/- ) background. It was the aim of the current study to identify causative genes at this locus. We established a congenic mouse model, where FVB.Chr3 B6/B6 mice carried an 80 Mb interval of distal Chr3 on an otherwise FVB.Ldlr -/- background, to validate the Chr3 locus. Candidate genes were identified using genome-wide expression analyses. Differentially expressed genes were validated using quantitative PCRs in F0 and F2 mice and their functions were investigated in pathophysiologically relevant cells. Fine-mapping of the Chr3 locus revealed two overlapping, yet independent subloci for female atherosclerosis susceptibility: when transmitted by grandfathers to granddaughters, the B6 risk allele increased atherosclerosis and downregulated the expression of the secreted phospholipase Pla2g12a (2.6 and 2.2 fold, respectively); when inherited by grandmothers, the B6 risk allele induced vascular cell adhesion molecule 1 (Vcam1). Down-regulation of Pla2g12a and up-regulation of Vcam1 were validated in female FVB.Chr3 B6/B6 congenic mice, which developed 2.5 greater atherosclerotic lesions compared to littermate controls (p=0.039). Pla2g12a was highly expressed in aortic endothelial cells in vivo, and knocking-down Pla2g12a expression by RNAi in cultured vascular endothelial cells or macrophages increased their adhesion to ECs in vitro. Our data establish Pla2g12a as an atheroprotective candidate gene in mice, where high expression levels in ECs and macrophages may limit the recruitment and accumulation of these cells in nascent atherosclerotic lesions. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-09

    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.

  10. Lipoprotein-associated phospholipase A2 and risks of coronary heart disease and ischemic stroke in the general population: A systematic review and meta-analysis.

    PubMed

    Li, Dongze; Wei, Wei; Ran, Xun; Yu, Jing; Li, Hui; Zhao, Lizhi; Zeng, Honglian; Cao, Yu; Zeng, Zhi; Wan, Zhi

    2017-08-01

    This study investigated the associations between lipoprotein-associated phospholipase A2 (Lp-PLA2) and the risks of coronary heart disease (CHD) and ischemic stroke (IS) in the general population. PubMed, Embase, and the Cochrane Library databases were searched for prospective cohort studies published prior to June 2016. Multivariate-adjusted hazard ratios (HRs) with 95% confidence intervals (CIs) for CHD and IS risks according to Lp-PLA2 activity or mass were extracted, pooled, and weighted using random-effects modeling. Twelve studies examining Lp-PLA2 activity or mass and long-term risks of CHD and IS were included. Combined HRs for CHD and IS risks for the highest category referring to lowest category of Lp-PLA2 were 1.46 (95% CI: 1.20-1.78, P<0.001) and 1.58 (95% CI: 1.21-2.07, P=0.001), respectively. The same patterns were observed for both mass and activity, with the exception of those for CHD. For every 1-standard deviation (SD) increase in Lp-PLA2 activity, CHD risk increased by 12% (HR: 1.12, 95% CI: 1.05-1.22, P=0.002); no association between 1-SD increases in Lp-PLA2 activity and IS was observed. Lp-PLA2 mass was associated with CHD risk (HR: 1.02-1.24, 95% CI: 1.02-1.24, P=0.021). Lp-PLA2 mass per 1-SD increase was not associated with IS risk. Greater Lp-PLA2 activity or mass was associated with an increased risk of CHD and IS; however, additional well-designed trials are warranted to confirm this association. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Secretory phospholipases A(2) isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E2 and D2 and expression of cyclooxygenases.

    PubMed

    Moreira, Vanessa; Gutiérrez, José Maria; Soares, Andreimar Martins; Zamunér, Stella Regina; Purgatto, Eduardo; Teixeira, Catarina de Fátima Pereira

    2008-09-01

    The effects of myotoxin III (MT-III), a phospholipase A(2) (sPLA2) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA(2)s promoted the synthesis of PGD2 and PGE2, with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA(2)s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. Inhibition of cPLA2 by AACOCF3, but not of iPLA2 by PACOCF3 or BEL, significantly reduced PGD2, PGE2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA2 inhibition did not modify the effects of CB, whereas iPLA2 inhibition reduced PGD2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs' synthesis are triggered by these snake venom sPLA(2)s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA(2)s in vivo.

  12. 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

  13. Activation and induction of cytosolic phospholipase A2 by TNF-α mediated through Nox2, MAPKs, NF-κB, and p300 in human tracheal smooth muscle cells.

    PubMed

    Lee, Chiang-Wen; Lin, Chih-Chung; Lee, I-Ta; Lee, Hui-Chun; Yang, Chuen-Mao

    2011-08-01

    Cytosolic phospholipase A(2) (cPLA(2)) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA(2) expression and PGE(2) synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we report that TNF-α-induced cPLA(2) protein and mRNA expression, PGE(2) production, and phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which were attenuated by pretreatment with a ROS scavenger [N-acetyl-L-cysteine, (NAC)] and the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNA of Nox2, p47(phox) , MEK1, p42, p38, or JNK2. TNF-α-induced cPLA(2) expression was also inhibited by pretreatment with a selective NF-κB inhibitor [helenalin (HLN)] or transfection with dominant negative mutants of NF-κB inducing kinase (NIK) or IκB kinase (IKK)α/β. TNF-α-induced NF-κB translocation was blocked by pretreatment with NAC, DPI, APO, or HLN, but not by U0126, SB202190, or SP600125. In addition, pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA blocked cPLA(2) expression and PGE(2) synthesis induced by TNF-α. We further confirmed that p300 was associated with the cPLA(2) promoter which was dynamically linked to histone H4 acetylation stimulated by TNF-α, determined by chromatin immunoprecipitation assay. Association of p300 and histone H4 to cPLA(2) promoter was inhibited by U0126, SB202190, and SP600125. These results suggested that in HTSMCs, activation of p47(phox) , MAPKs, NF-κB, and p300 is essential for TNF-α-induced cPLA(2) expression and PGE(2) release. Copyright © 2010 Wiley-Liss, Inc.

  14. Cigarette smoke extract induces cytosolic phospholipase A2 expression via NADPH oxidase, MAPKs, AP-1, and NF-kappaB in human tracheal smooth muscle cells.

    PubMed

    Cheng, Shin-Ei; Luo, Shue-Fen; Jou, Mei-Jie; Lin, Chih-Chung; Kou, Yu Ru; Lee, I-Ta; Hsieh, Hsi-Lung; Yang, Chuen-Mao

    2009-04-01

    Up-regulation of cytosolic phospholipase A2 (cPLA2) by cigarette smoke extract (CSE) may play a critical role in airway inflammatory diseases. However, the mechanisms underlying CSE-induced cPLA2 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. CSE induced cPLA2 protein and mRNA expression, and ROS generation was attenuated by pretreatment with a reactive oxygen species (ROS) scavenger (N-acetylcysteine), or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, apocynin) and transfection with p47phox siRNA, suggesting that CSE-induced cPLA2 expression was mediated through NADPH oxidase activation and ROS production in HTSMCs. Furthermore, CSE-induced cPLA2 expression was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), which were further confirmed by transfection with siRNAs of JNK1, p42, and p38 to down-regulate the expression of respective proteins and reduce cPLA2 expression. Induction of cPLA2 by CSE was attenuated by selective inhibitors of NF-kappaB (helenalin) and AP-1 (curcumin). Moreover, promoter assays revealed that increases of cPLA2, NF-kappaB, and AP-1 luciferase activities stimulated by CSE were attenuated by these inhibitors. These results suggest that in HTSMCs, CSE induced NADPH oxidase activation leading to phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK. These reactions induced nuclear transcription NF-kappaB and AP-1 activities which were essential for CSE-induced cPLA2 gene expression.

  15. Recombinant production and properties of binding of the full set of mouse secreted phospholipases A2 to the mouse M-type receptor.

    PubMed

    Rouault, Morgane; Le Calvez, Catherine; Boilard, Eric; Surrel, Fanny; Singer, Alan; Ghomashchi, Farideh; Bezzine, Sofiane; Scarzello, Sabine; Bollinger, James; Gelb, Michael H; Lambeau, Gérard

    2007-02-13

    To date, 12 secreted phospholipases A2 (sPLA2s) have been identified in the mouse species and divided into three structural collections (I/II/V/X, III, and XII). On the basis of their different molecular properties and tissue distributions, each sPLA2 is likely to exert distinct functions by acting as an enzyme or ligand for specific soluble proteins or receptors, among which the M-type receptor is the best-characterized target. Here, we present the properties of binding of the full set of mouse sPLA2s to the mouse M-type receptor. All enzymes have been produced in Escherichia coli or insect cells, and their properties of binding to the cloned and native M-type receptor have been determined. sPLA2s IB, IIA, IIE, IIF, and X are high-affinity ligands (K0.5 = 0.3-3 nM); sPLA2s IIC and V are low-affinity ligands (K0.5 = 30-75 nM), and sPLA2s IID, III, XIIA, and XIIB bind only very weakly or do not bind to the M-type receptor (K0.5 > 100 nM). Three exogenous parvoviral group XIII PLA2s and two fungal group XIV sPLA2s do not bind to the receptor. Together, these results indicate that the mouse M-type receptor is selective for only a subset of mouse sPLA2s from the group I/II/V/X structural collection. Binding of mouse sPLA2s to a recombinant soluble mouse M-type receptor leads in all cases to inhibition of enzymatic activity, and the extent of deglycosylation of the receptor decreases yet does not abolish sPLA2 binding. The physiological meaning of binding of sPLA2 to the M-type receptor is discussed on the basis of our current knowledge of sPLA2 functions.

  16. Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2alpha in regulating interfacial kinetics and binding and cellular function.

    PubMed

    Tucker, Dawn E; Ghosh, Moumita; Ghomashchi, Farideh; Loper, Robyn; Suram, Saritha; John, Bonnie St; Girotti, Milena; Bollinger, James G; Gelb, Michael H; Leslie, Christina C

    2009-04-03

    Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA(2)alpha (IMLF(-/-)) were reconstituted with wild type and cPLA(2)alpha mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA(2)alpha translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA(2)alpha to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA(2)alpha to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF(-/-) expressing phosphorylation site (S505A) and PIP(2) binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA(2)alpha, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA(2)alpha reduced activation by PIP(2) but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA(2)alpha hydrolytic activity.

  17. A Lys49-PLA2 myotoxin of Bothrops asper triggers a rapid death of macrophages that involves autocrine purinergic receptor signaling

    PubMed Central

    Tonello, F; Simonato, M; Aita, A; Pizzo, P; Fernández, J; Lomonte, B; Gutiérrez, J M; Montecucco, C

    2012-01-01

    Lys49-PLA2 myotoxins, an important component of various viperid snake venoms, are a class of PLA2-homolog proteins deprived of catalytic activity. Similar to enzymatically active PLA2 (Asp49) and to other classes of myotoxins, they cause severe myonecrosis. Moreover, these toxins are used as tools to study skeletal muscle repair and regeneration, a process that can be very limited after snakebites. In this work, the cytotoxic effect of different myotoxins, Bothrops asper Lys49 and Asp49-PLA2, Notechis scutatus notexin and Naja mossambica cardiotoxin, was evaluated on macrophages, cells that have a key role in muscle regeneration. Only the Lys49-myotoxin was found to trigger a rapid asynchronous death of mouse peritoneal macrophages and macrophagic cell lines through a process that involves ATP release, ATP-induced ATP release and that is inhibited by various purinergic receptor antagonists. ATP leakage is induced also at sublytical doses of the Lys49-myotoxin, it involves Ca2+ release from intracellular stores, and is reduced by inhibitors of VSOR and the maxi-anion channel. The toxin-induced cell death is different from that caused by high concentration of ATP and appears to be linked to localized purinergic signaling. Based on present findings, a mechanism of cell death is proposed that can be extended to other cytolytic proteins and peptides. PMID:22764102

  18. A Lys49-PLA2 myotoxin of Bothrops asper triggers a rapid death of macrophages that involves autocrine purinergic receptor signaling.

    PubMed

    Tonello, F; Simonato, M; Aita, A; Pizzo, P; Fernández, J; Lomonte, B; Gutiérrez, J M; Montecucco, C

    2012-07-05

    Lys49-PLA(2) myotoxins, an important component of various viperid snake venoms, are a class of PLA(2)-homolog proteins deprived of catalytic activity. Similar to enzymatically active PLA(2) (Asp49) and to other classes of myotoxins, they cause severe myonecrosis. Moreover, these toxins are used as tools to study skeletal muscle repair and regeneration, a process that can be very limited after snakebites. In this work, the cytotoxic effect of different myotoxins, Bothrops asper Lys49 and Asp49-PLA(2), Notechis scutatus notexin and Naja mossambica cardiotoxin, was evaluated on macrophages, cells that have a key role in muscle regeneration. Only the Lys49-myotoxin was found to trigger a rapid asynchronous death of mouse peritoneal macrophages and macrophagic cell lines through a process that involves ATP release, ATP-induced ATP release and that is inhibited by various purinergic receptor antagonists. ATP leakage is induced also at sublytical doses of the Lys49-myotoxin, it involves Ca(2+) release from intracellular stores, and is reduced by inhibitors of VSOR and the maxi-anion channel. The toxin-induced cell death is different from that caused by high concentration of ATP and appears to be linked to localized purinergic signaling. Based on present findings, a mechanism of cell death is proposed that can be extended to other cytolytic proteins and peptides.

  19. 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

  20. Perfluorodecanoic acid (PFDA) promotes gastric cell proliferation via sPLA2-IIA

    PubMed Central

    Zhong, Ning; Liu, Fengyan; Zhang, Hanyu; Xu, Mengchen; Liu, Rutao; Han, Mingyong; Tian, Xingsong; Jia, Jihui; Chang, Lap Kam; Guo, Liang-Hong; Liu, Shili

    2017-01-01

    The association of perfluorodecanoicacid (PFDA) with tumor promotion and associated effects is not clear. Given that PDFA is mostly consumed with food and drinking water, we evaluated the effects of PFDA on a gastric cell line. When added to cell cultures, PFDA significantly increased growth rate and colony forming ability compared with control treatment. We found that suppression of cell senescence, but not apoptosis or autophagy was associated with PFDA-induced promotion of cell amount. To determine the molecular mechanism that was involved, DNA microarray assays was used to analyze changes in gene expression in response to PFDA treatment. Data analysis demonstrated that the vascular endothelial growth factor signaling pathway had the lowest p-value, with sPLA2-IIA (pla2g2a) exhibits the most altered expression pattern within the pathway. Moreover, sPLA2-IIA and its transcription factor TCF4, known as a direct target and a binding partner of Wnt/β-catenin signaling in gastric cells respectively, were the third and second most varied genes globally. Cells transfected with expression plasmids pENTER-tcf4 and pENTER-pla2g2a show reduced cell proliferation by more than 60% and 30% respectively. Knockdown with sPLA2-IIA siRNA provided additional evidence that sPLA2-IIA was a mediator of PFDA-induced cell senescence suppression. The results suggest for the first time that PFDA induced suppression of cell senescence through inhibition of sPLA2-IIA protein expression and might increased the proliferative capacity of an existing tumor. PMID:28881615

  1. Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro.

    PubMed

    Miyabara, E H; Baptista, I L; Lomonte, B; Selistre-de-Araújo, H S; Gutiérrez, J M; Moriscot, A S

    2006-07-01

    Previous studies have shown that calcineurin activity plays a critical role in the myotoxic activity induced by crotoxin (CTX), a group II phospholipase A(2) (PLA(2)) with neurotoxic and myotoxic actions. In order to address whether calcineurin is also important for the activity of non-neurotoxic group II PLA(2) myotoxins we have compared the effects of calcineurin inhibition on the myotoxic capacity of CTX and the non-neurotoxic PLA(2)s, myotoxin II (Mt II) and myotoxin III (Mt III) from Bothrops asper venom. Rats were treated with cyclosporin A (CsA) or FK506, calcineurin inhibitors, and received an intramuscular injection of either CTX, Mt II or Mt III into the tibialis anterior. Animals were killed 24 h after injection of toxins. Tibialis anterior was removed and stored in liquid nitrogen. Myofibers in culture were also treated with CsA or FK506 and exposed to CTX, Mt II and Mt III. It was observed that, in contrast to CTX, CsA and FK506 do not attenuate myotoxic effects induced by both Mt II and Mt III in vivo and in vitro. The results of the present study suggest that calcineurin is not essential for the myotoxic activity of Mt II and Mt III, indicating that distinct intracellular pathways might be involved in myonecrosis induced by neurotoxic CTX and non-neurotoxic Bothrops sp. PLA(2) myotoxins. Alternatively, calcineurin dependent fast fiber type shift might render the muscle resistant to the action of CTX, without affecting its susceptibility to Bothrops sp. myotoxins.

  2. A Small Phospholipase A2-α from Castor Catalyzes the Removal of Hydroxy Fatty Acids from Phosphatidylcholine in Transgenic Arabidopsis Seeds1[OPEN

    PubMed Central

    Bayon, Shen; Chen, Guanqun; Weselake, Randall J.; Browse, John

    2015-01-01

    Ricinoleic acid, an industrially useful hydroxy fatty acid (HFA), only accumulates to high levels in the triacylglycerol fraction of castor (Ricinus communis) endosperm, even though it is synthesized on the membrane lipid phosphatidylcholine (PC) from an oleoyl ester. The acyl chains of PC undergo intense remodeling through the process of acyl editing. The identities of the proteins involved in this process, however, are unknown. A phospholipase A2 (PLA2) is thought to be involved in the acyl-editing process. We show here a role for RcsPLA2α in the acyl editing of HFA esterified to PC. RcsPLA2α was identified by its high relative expression in the castor endosperm transcriptome. Coexpression in Arabidopsis (Arabidopsis thaliana) seeds of RcsPLA2α with the castor fatty acid hydroxylase RcFAH12 led to a dramatic decrease in seed HFA content when compared with RcFAH12 expression alone in both PC and the neutral lipid fraction. The low-HFA trait was heritable and gene dosage dependent, with hemizygous lines showing intermediate HFA levels. The low seed HFA levels suggested that RcsPLA2α functions in vivo as a PLA2 with HFA specificity. Activity assays with yeast (Saccharomyces cerevisiae) microsomes showed a high specificity of RcsPLA2α for ricinoleic acid, superior to that of the endogenous Arabidopsis PLA2α. These results point to RcsPLA2α as a phospholipase involved in acyl editing, adapted to specifically removing HFA from membrane lipids in seeds. PMID:25667315

  3. Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper.

    PubMed Central

    Lizano, S; Lomonte, B; Fox, J W; Gutiérrez, J M

    1997-01-01

    A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23-25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23-25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I-IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood. PMID:9307037

  4. 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

  5. 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.

  6. Potentiation of cytokine induction of group IIA phospholipase A(2) in rat mesangial cells by ATP and adenosine via the A2A adenosine receptor.

    PubMed

    Scholz-Pedretti, K; Pfeilschifter, J; Kaszkin, M

    2001-01-01

    1. In rat mesangial cells extracellular nucleotides were found to increase arachidonic acid release by a cytosolic phospholipase A(2) through the P2Y(2) purinergic receptor. 2. In this study we investigated the effects of ATP and UTP on interleukin-1ss (IL-1ss)-induced mRNA expression and activity of group IIA phospholipase A(2) (sPLA(2)-IIA) in rat mesangial cells. 3. Treatment of cells for 24 h with extracellular ATP potentiated IL-1ss-stimulated sPLA(2)-IIA induction, whereas UTP had no effect. 4. We obtained the following evidence that the P2Y(2) receptor is not involved in the potentiation of sPLA(2)-IIA induction: (i) ATP-gamma-S had no enhancing effect; (ii) suramin, a P(2) receptor antagonist, did not inhibit ATP-mediated potentiation; (iii) inhibition of degradation of extracellular nucleotides by the 5'-ectonucleotidase inhibitor AOPCP did not enhance sPLA(2)-IIA induction and (iv) adenosine deaminase treatment completely abolished the ATP-mediated potentiation of sPLA(2)-IIA induction. 5. In contrast, treatment of mesangial cells with adenosine or the A2A receptor agonist CGS 21680 mimicked the effects of ATP in enhancing IL-1ss-stimulated sPLA(2)-IIA induction, whereas the specific A2A receptor antagonist ZM 241385 completely abolished the potentiating effect of ATP or adenosine. 6. The protein kinase A inhibitor Rp-8-Br-cyclic AMPS dose-dependently inhibited the enhancing effect of ATP or adenosine indicating the participation of an adenosine receptor-mediated cyclic AMP-dependent signalling pathway. 7. These data indicate that ATP mediates proinflammatory long-term effects in rat mesangial cells via its degradation product adenosine through the A2A receptor resulting in potentiation of sPLA(2)-IIA induction.

  7. In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2

    PubMed Central

    Guillaume, Carole; Payré, Christine; Jemel, Ikram; Jeammet, Louise; Bezzine, Sofiane; Naika, Gajendra S.; Bollinger, James; Grellier, Philippe; Gelb, Michael H.; Schrével, Joseph

    2015-01-01

    We have previously shown that secreted phospholipases A2 (sPLA2s) from animal venoms inhibit the in vitro development of Plasmodium falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA2s in host defense against P. falciparum has not been investigated. We show here that 4 out of 10 human sPLA2s, namely, hGX, hGIIF, hGIII, and hGV, exhibit potent in vitro anti-Plasmodium properties with half-maximal inhibitory concentrations (IC50s) of 2.9 ± 2.4, 10.7 ± 2.1, 16.5 ± 9.7, and 94.2 ± 41.9 nM, respectively. Other human sPLA2s, including hGIIA, are inactive. The inhibition is dependent on sPLA2 catalytic activity and primarily due to hydrolysis of plasma lipoproteins from the parasite culture. Accordingly, purified lipoproteins that have been prehydrolyzed by hGX, hGIIF, hGIII, and hGV are more toxic to P. falciparum than native lipoproteins. However, the total enzymatic activities of human sPLA2s on purified lipoproteins or plasma did not reflect their inhibitory activities on P. falciparum. For instance, hGIIF is 9-fold more toxic than hGV but releases a lower quantity of nonesterified fatty acids (NEFAs). Lipidomic analyses of released NEFAs from lipoproteins demonstrate that sPLA2s with anti-Plasmodium properties are those that release polyunsaturated fatty acids (PUFAs), with hGIIF being the most selective enzyme. NEFAs purified from lipoproteins hydrolyzed by hGIIF were more potent at inhibiting P. falciparum than those from hGV, and PUFA-enriched liposomes hydrolyzed by sPLA2s were highly toxic, demonstrating the critical role of PUFAs. The selectivity of sPLA2s toward low- and high-density (LDL and HDL, respectively) lipoproteins and their ability to directly attack parasitized erythrocytes further explain their anti-Plasmodium activity. Together, our findings indicate that 4 human sPLA2s are active against P

  8. In vitro anti-Plasmodium falciparum properties of the full set of human secreted phospholipases A2.

    PubMed

    Guillaume, Carole; Payré, Christine; Jemel, Ikram; Jeammet, Louise; Bezzine, Sofiane; Naika, Gajendra S; Bollinger, James; Grellier, Philippe; Gelb, Michael H; Schrével, Joseph; Lambeau, Gérard; Deregnaucourt, Christiane

    2015-06-01

    We have previously shown that secreted phospholipases A2 (sPLA2s) from animal venoms inhibit the in vitro development of Plasmodium falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA2s in host defense against P. falciparum has not been investigated. We show here that 4 out of 10 human sPLA2s, namely, hGX, hGIIF, hGIII, and hGV, exhibit potent in vitro anti-Plasmodium properties with half-maximal inhibitory concentrations (IC50s) of 2.9 ± 2.4, 10.7 ± 2.1, 16.5 ± 9.7, and 94.2 ± 41.9 nM, respectively. Other human sPLA2s, including hGIIA, are inactive. The inhibition is dependent on sPLA2 catalytic activity and primarily due to hydrolysis of plasma lipoproteins from the parasite culture. Accordingly, purified lipoproteins that have been prehydrolyzed by hGX, hGIIF, hGIII, and hGV are more toxic to P. falciparum than native lipoproteins. However, the total enzymatic activities of human sPLA2s on purified lipoproteins or plasma did not reflect their inhibitory activities on P. falciparum. For instance, hGIIF is 9-fold more toxic than hGV but releases a lower quantity of nonesterified fatty acids (NEFAs). Lipidomic analyses of released NEFAs from lipoproteins demonstrate that sPLA2s with anti-Plasmodium properties are those that release polyunsaturated fatty acids (PUFAs), with hGIIF being the most selective enzyme. NEFAs purified from lipoproteins hydrolyzed by hGIIF were more potent at inhibiting P. falciparum than those from hGV, and PUFA-enriched liposomes hydrolyzed by sPLA2s were highly toxic, demonstrating the critical role of PUFAs. The selectivity of sPLA2s toward low- and high-density (LDL and HDL, respectively) lipoproteins and their ability to directly attack parasitized erythrocytes further explain their anti-Plasmodium activity. Together, our findings indicate that 4 human sPLA2s are active against P

  9. Suppressive effect of secretory phospholipase A2 inhibitory peptide on interleukin-1β-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts, and its antiarthritic activity in hTNFtg mice

    PubMed Central

    2009-01-01

    Introduction Secretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis. Methods Gene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1β stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)α, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively. Results PIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1β-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1β-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice. Conclusions Our results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential

  10. RNase 7 but not psoriasin nor sPLA2-IIA associates with Mycobacterium tuberculosis during airway epithelial cell infection.

    PubMed

    Torres-Juarez, Flor; Touqui, Lousseine; Leon-Contreras, Juan; Rivas-Santiago, Cesar; Enciso-Moreno, Jose A; Hernández-Pando, Rogelio; Rivas-Santiago, Bruno

    2018-03-01

    Tuberculosis is a disease caused by Mycobacterium tuberculosis (Mtb). Innate immunity is the first line of defense against Mtb and malfunctions in any of its components are associated with the susceptibility to the disease. Epithelial products such as host defense peptides (HDPs) are the first molecules produced to counteract the infection. Although a wide variety of HDPs are produced by epithelial cells only a few of them have been studied during Mtb infection. Here, we assessed the expression and production of the HDPs psoriasin, secreted phospholipases A2 (sPLA2-IIA) and Ribonuclease (RNase) 7 in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells and from the human cell line THP1 after Mtb in vitro infection. Results show that psoriasin and sPLA2-IIA were not induced by Mtb in any of the evaluated cells, while RNase 7 was overexpressed in infected airway epithelial cells. Intracellular analysis by flow cytometry demonstrated that the highest levels of RNase 7 were observed 6 h post-infection and the induction was dependent on direct interaction between airway epithelial cells and Mtb. In addition, analysis by electron microscopy showed that RNase 7 was capable of attaching to the cell wall of intracellular mycobacteria. Our studies suggest that the induction of RNase 7 in response to Mtb could have a role in anti-mycobacterial immunity, which needs to be studied as an innate immune mechanism.

  11. Effects of cholesterol on physical properties of human erythrocyte membranes: impact on susceptibility to hydrolysis by secretory phospholipase A2.

    PubMed

    Heiner, Anne L; Gibbons, Elizabeth; Fairbourn, Jeremy L; Gonzalez, Laurie J; McLemore, Chisako O; Brueseke, Taylor J; Judd, Allan M; Bell, John D

    2008-04-15

    The ability of secretory phospholipase A(2) (sPLA(2)) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA(2) activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA(2). To test this hypothesis, defined amounts of cholesterol were removed from erythrocyte membranes using methyl-beta-cyclodextrin. Treatment of cells with methyl-beta-cyclodextrin increased the hydrolysis rate and total substrate hydrolyzed by sPLA(2). In general, this effect of cholesterol removal was more pronounced at higher temperatures. Comparison of the level of membrane order (assessed with the fluorescent probe laurdan) with hydrolysis rate revealed that sPLA(2) activity was greatly enhanced upon significant reductions in lipid order. Additional treatment of the cells with calcium ionophore further enhanced the hydrolysis rate and altered the relationship with membrane order. These data demonstrated that interactions with sPLA(2) observed in artificial bilayers apply to biological membranes. It is also proposed that the high level of cholesterol in erythrocyte membranes is a protective mechanism to guard against hydrolytic enzymes.

  12. Interfacial recognition by bee venom phospholipase A2: insights into nonelectrostatic molecular determinants by charge reversal mutagenesis.

    PubMed

    Ghomashchi, F; Lin, Y; Hixon, M S; Yu, B Z; Annand, R; Jain, M K; Gelb, M H

    1998-05-12

    The basis for tight binding of bee venom phospholipase A2 (bvPLA2) to anionic versus zwitterionic phospholipid interfaces is explored by charge reversal mutagenesis of basic residues (lysines/arginines to glutamates) on the putative membrane binding surface. Single-site mutants and, surprisingly, multisite mutants (2-5 of the 6 basic residues mutated) are fully functional on anionic vesicles. Mutants bind tightly to anionic vesicles, and active-site substrate and Ca2+ binding are not impaired. Multisite mutants undergo intervesicle exchange slightly faster than wild type, especially in the presence of salt. It is estimated that electrostatic contribution to interfacial binding is modest, perhaps 2-3 kcal/mol of the estimated 15 kcal/mol. Elution properties of bvPLA2 from HPLC columns containing solid phases of tightly packed monolayers of phosphocholine amphiphiles suggest that ionic effects provide a modest portion of the interfacial binding energy and that this contribution decreases as the number of cationic residues mutated is increased. These results are consistent with the observation that Gila monster venom PLA2 (Pa2), which is homologous to bvPLA2, has high activity on anionic vesicles despite the fact that it has only a single basic residue on its putative interfacial recognition face. Results with bvPLA2 mutants show that manoalogue and 12-epi-scalaradial inactivate bvPLA2 by modification of K94. Also, deletion of the large beta-loop (residues 99-118) is without consequence for interfacial binding and catalysis of bvPLA2. All together, the preferential binding of bvPLA2 to anionic vesicles versus phosphatidylcholine vesicles is mainly due to factors other than electrostatics. Therefore hydrogen-bonding and hydrophobic interactions must provide a major portion of the interfacial binding energy, and this is consistent with recent spectroscopic studies.

  13. 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

  14. Role of Phospholipase A2 in Retrograde Transport of Ricin

    PubMed Central

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

    2011-01-01

    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 A2 (PLA2) as an important factor in ricin retrograde transport. Inhibition of PLA2 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 PLA2 inhibition. Confocal microscopy analysis shows that ricin is still colocalized with the trans-Golgi marker TGN46 in the presence of PLA2 inhibitor, but less is colocalized with the cis-Golgi marker GM130. We propose that PLA2 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. PMID:22069763

  15. Contributions of residues of pancreatic phospholipase A2 to interfacial binding, catalysis, and activation.

    PubMed

    Yu, B Z; Rogers, J; Tsai, M D; Pidgeon, C; Jain, M K

    1999-04-13

    Primary rate and equilibrium parameters for 60 site-directed mutants of bovine pancreatic phospholipase A2 (PLA2) are analyzed so incremental contributions of the substitution of specific residues can be evaluated. The magnitude of the change is evaluated so a functional role in the context of the N- and C-domains of PLA2 can be assigned, and their relationship to the catalytic residues and to the i-face that makes contact with the interface. The effect of substitutions and interfacial charge is characterized by the equilibrium dissociation constant for dissociation of the bound enzyme from the interface (Kd), the dissociation constant for dissociation of a substrate mimic from the active site of the bound enzyme (KL), and the interfacial Michaelis constants, KM and kcat. Activity is lost (>99.9%) on the substitution of H48 and D49, the catalytic residues. A more than 95% decrease in kcat is seen with the substitution of F5, I9, D99, A102, or F106, which form the substrate binding pocket. Certain residues, which are not part of the catalytic site or the substrate binding pocket, also modulate kcat. Interfacial anionic charge lowers Kd, and induces kcat activation through K56, K53, K119, or K120. Significant changes in KL are seen by the substitution of N6, I9, F22, Y52, K53, N71, Y73, A102, or A103. Changes in KM [=(k2+k-1)/k1] are attributed to kcat (=k2) and KL (=k-1/k1). Some substitutions change more than one parameter, implying an allosteric effect of the binding to the interface on KS, and the effect of the interfacial anionic charge on kcat. Interpreted in the context of the overall structure, results provide insights into the role of segments and domains in the microscopic events of catalytic turnover and processivity, and their allosteric regulation. We suggest that the interfacial recognition region (i-face) of PLA2, due to the plasticity of certain segments and domains, exercises an allosteric control on the substrate binding and chemical step.

  16. Molecular Characterization of Lys49 and Asp49 Phospholipases A2 from Snake Venom and Their Antiviral Activities against Dengue virus

    PubMed Central

    Cecilio, Alzira B.; Caldas, Sergio; De Oliveira, Raiana A.; Santos, Arthur S. B.; Richardson, Michael; Naumann, Gustavo B.; Schneider, Francisco S.; Alvarenga, Valeria G.; Estevão-Costa, Maria I.; Fuly, Andre L.; Eble, Johannes A.; Sanchez, Eladio F.

    2013-01-01

    We report the detailed molecular characterization of two PLA2s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA2s, named BlK-PLA2 and BlD-PLA2, are composed of 121 and 122 amino acids determined by automated sequencing of the native proteins and peptides produced by digestion with trypsin. They contain fourteen cysteines with pIs of 9.05 and 8.18 for BlK- and BlD-PLA2s, and show a high degree of sequence similarity to homologous snake venom PLA2s, but may display different biological effects. Molecular masses of 13,689.220 (Lys49) and 13,978.386 (Asp49) were determined by mass spectrometry. DENV causes a prevalent arboviral disease in humans, and no clinically approved antiviral therapy is currently available to treat DENV infections. The maximum non-toxic concentration of the proteins to LLC-MK2 cells determined by MTT assay was 40 µg/mL for Bl-PLA2s (pool) and 20 µg/mL for each isoform. Antiviral effects of Bl-PLA2s were assessed by quantitative Real-Time PCR. Bl-PLA2s were able to reduce DENV-1, DENV-2, and DENV-3 serotypes in LLC-MK2 cells infection. Our data provide further insight into the structural properties and their antiviral activity against DENV, opening up possibilities for biotechnological applications of these Bl-PLA2s as tools of research. PMID:24131891

  17. Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins.

    PubMed

    Melo, P A; Ownby, C L

    1999-01-01

    We examined the ability of wedelolactone, heparin and para-bromophenacyl bromide to antagonize the myotoxic activity in mice of venoms from Crotalus viridis viridis and Agkistrodon contortrix laticinctus and two phospholipase A2 myotoxins, CVV myotoxin and ACL myotoxin, isolated from them. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours and histological changes in extensor digitorum longus muscle (EDL) at three hours after injection of the test solution. Both heparin and wedelolactone independently reduced the myotoxic effect of both crude venoms and both myotoxins, but wedelolactone was more effective. Wedelolactone plus heparin reduced the myotoxic effect of CVV myotoxin more than either antagonist alone. The PLA2 inhibitor, para-bromophenacyl bromide (pBPB), reduced the myotoxic effect of both myotoxins more than either wedelolactone or heparin. On the other hand, the myotoxic effect of polylysine was not reduced by either wedelolactone or para-bromophenacyl bromide, but it was reduced by heparin. These results indicate that wedelolactone, para-bromophenacyl bromide and heparin are antagonists of these two phospholipase A2 myotoxins, and that antagonism by the first two compounds may be due to a more specific interaction with these proteins than that by the latter.

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

    PubMed

    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

    2015-11-01

    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. © FASEB.

  19. Biochemical Characterization and Pharmacological Properties of New Basic PLA2 BrTX-I Isolated from Bothrops roedingeri (Roedinger's Lancehead) Mertens, 1942, Snake Venom

    PubMed Central

    Gomes Heleno, Mauricio Aurelio; Baldasso, Paulo Aparecido; Ponce-Soto, Luis Alberto; Marangoni, Sérgio

    2013-01-01

    BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on μ-Bondapak C-18 column. A molecular mass of 14358.69 Da 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 total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35–45°C, and required Ca2+. In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNFα were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively. PMID:23509747

  20. Varespladib (LY315920) Appears to Be a Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-Referral Treatment for Envenomation.

    PubMed

    Lewin, Matthew; Samuel, Stephen; Merkel, Janie; Bickler, Philip

    2016-08-25

    Snakebite remains a neglected medical problem of the developing world with up to 125,000 deaths each year despite more than a century of calls to improve snakebite prevention and care. An estimated 75% of fatalities from snakebite occur outside the hospital setting. Because phospholipase A2 (PLA2) activity is an important component of venom toxicity, we sought candidate PLA2 inhibitors by directly testing drugs. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib showed high-level secretory PLA2 (sPLA2) inhibition at nanomolar and picomolar concentrations against 28 medically important snake venoms from six continents. In vivo proof-of-concept studies with varespladib had striking survival benefit against lethal doses of Micrurus fulvius and Vipera berus venom, and suppressed venom-induced sPLA2 activity in rats challenged with 100% lethal doses of M. fulvius venom. Rapid development and deployment of a broad-spectrum PLA2 inhibitor alone or in combination with other small molecule inhibitors of snake toxins (e.g., metalloproteases) could fill the critical therapeutic gap spanning pre-referral and hospital setting. Lower barriers for clinical testing of safety tested, repurposed small molecule therapeutics are a potentially economical and effective path forward to fill the pre-referral gap in the setting of snakebite.

  1. Varespladib (LY315920) Appears to Be a Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-Referral Treatment for Envenomation

    PubMed Central

    Lewin, Matthew; Samuel, Stephen; Merkel, Janie; Bickler, Philip

    2016-01-01

    Snakebite remains a neglected medical problem of the developing world with up to 125,000 deaths each year despite more than a century of calls to improve snakebite prevention and care. An estimated 75% of fatalities from snakebite occur outside the hospital setting. Because phospholipase A2 (PLA2) activity is an important component of venom toxicity, we sought candidate PLA2 inhibitors by directly testing drugs. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib showed high-level secretory PLA2 (sPLA2) inhibition at nanomolar and picomolar concentrations against 28 medically important snake venoms from six continents. In vivo proof-of-concept studies with varespladib had striking survival benefit against lethal doses of Micrurus fulvius and Vipera berus venom, and suppressed venom-induced sPLA2 activity in rats challenged with 100% lethal doses of M. fulvius venom. Rapid development and deployment of a broad-spectrum PLA2 inhibitor alone or in combination with other small molecule inhibitors of snake toxins (e.g., metalloproteases) could fill the critical therapeutic gap spanning pre-referral and hospital setting. Lower barriers for clinical testing of safety tested, repurposed small molecule therapeutics are a potentially economical and effective path forward to fill the pre-referral gap in the setting of snakebite. PMID:27571102

  2. 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.

  3. Elevated inflammatory Lp-PLA2 and IL-6 link e-waste Pb toxicity to cardiovascular risk factors in preschool children.

    PubMed

    Lu, Xueling; Xu, Xijin; Zhang, Yu; Zhang, Yuling; Wang, Chenyang; Huo, Xia

    2018-03-01

    Cardiovascular toxicity of lead (Pb) manifests primarily as an effect on blood pressure and eventual increased risk of atherosclerosis and cardiovascular events. Therefore, we investigated vascular inflammatory biomarkers and cardiovascular effects of Pb-exposed children. A total of 590 children (3-7 years old) were recruited from Guiyu (n = 337), an electronic waste (e-waste)-exposed group, and Haojiang (n = 253), a reference group, from November to December 2016. We measured child blood Pb levels (BPbs), and systolic and diastolic blood pressure. Pulse pressure was calculated for the latter two. Serum biomarkers including lipid profiles and inflammatory cytokines, and plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) were detected. Unadjusted regression analysis illustrated that higher ln-transformed BPb associated with lower systolic blood pressure and pulse pressure. After adjustment for various confounders, the relational degree of lnBPb and blood pressure measures became slightly attenuated or not significant. Elevated BPb was associated with higher Lp-PLA2, interleukin (IL)-6, triglycerides (TG) and lower high-density lipoprotein (HDL). Lp-PLA2 remained inversely associated with pulse pressure and HDL, but positively with ratios of total cholesterol to HDL (Tc/HDL) and low-density lipoprotein to HDL (LDL/HDL). IL-6 was associated negatively with systolic blood pressure, pulse pressure and HDL, and positively associated with TG, Tc/HDL and LDL/HDL. The mediation effect of biomarkers on the association of BPb with pulse pressure was insignificant except for Lp-PLA2. Available data supports the conclusion that e-waste-exposed children with higher BPbs and concomitant abnormal measures of cardiovascular physiology have an augmented prevalence of vascular inflammation, as well as lipid disorder. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. The expression of genes encoding for COX-2, MPO, iNOS, and sPLA2-IIA in patients with recurrent depressive disorder.

    PubMed

    Gałecki, Piotr; Gałecka, Elżbieta; Maes, Michael; Chamielec, Marcelina; Orzechowska, Agata; Bobińska, Kinga; Lewiński, Andrzej; Szemraj, Janusz

    2012-05-01

    There is evidence that inflammation, oxidative and nitrosative stress (IO&NS) play a role in the pathophysiology of depression. There are also data indicating altered inflammatory gene expression in depressive disorder and that genetic variants of IO&NS genes are associated with increased risk of the disease in question. The aim of this study was to explore mRNA expression of four IO&NS genes PTGS2, MPO, NOS2A, and PLA2G2A coding respectively: cyclooxygenase-2 (COX-2), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and secretory phospholipase A2 type IIA (sPLA2-IIA). Expression of the mRNA was determined using quantitative real-time PCR, in peripheral blood cells of patients with recurrent depressive disorder (rDD) and normal controls. The mRNA expressions of the genes encoding for COX-2, MPO, iNOS and sPLA2-IIA were significantly increased in the peripheral blood cells of depressed patients versus controls. Patients were treated with antidepressants. Our results indicate and may confirm the role of peripheral IO&NS pathways in the pathophysiology of depression. The results represent a promising way to investigate biological markers of depression. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Venomic Analysis of the Poorly Studied Desert Coral Snake, Micrurus tschudii tschudii, Supports the 3FTx/PLA2 Dichotomy across Micrurus Venoms

    PubMed Central

    Sanz, Libia; Pla, Davinia; Pérez, Alicia; Rodríguez, Yania; Zavaleta, Alfonso; Salas, Maria; Lomonte, Bruno; Calvete, Juan J.

    2016-01-01

    The venom proteome of the poorly studied desert coral snake Micrurus tschudii tschudii was unveiled using a venomic approach, which identified ≥38 proteins belonging to only four snake venom protein families. The three-finger toxins (3FTxs) constitute, both in number of isoforms (~30) and total abundance (93.6% of the venom proteome), the major protein family of the desert coral snake venom. Phospholipases A2 (PLA2s; seven isoforms, 4.1% of the venom proteome), 1–3 Kunitz-type proteins (1.6%), and 1–2 l-amino acid oxidases (LAO, 0.7%) complete the toxin arsenal of M. t. tschudii. Our results add to the growing evidence that the occurrence of two divergent venom phenotypes, i.e., 3FTx- and PLA2-predominant venom proteomes, may constitute a general trend across the cladogenesis of Micrurus. The occurrence of a similar pattern of venom phenotypic variability among true sea snake (Hydrophiinae) venoms suggests that the 3FTx/PLA2 dichotomy may be widely distributed among Elapidae venoms. PMID:27338473

  6. Elevated expression of lipoprotein-associated phospholipase A2 in calcific aortic valve disease: implications for valve mineralization.

    PubMed

    Mahmut, Ablajan; Boulanger, Marie-Chloé; El Husseini, Diala; Fournier, Dominique; Bouchareb, Rihab; Després, Jean-Pierre; Pibarot, Philippe; Bossé, Yohan; Mathieu, Patrick

    2014-02-11

    This study sought to document the presence and role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in calcific aortic valve disease (CAVD). CAVD is a chronic disorder characterized by pathological mineralization and remodeling. Studies have indicated that human CAVD tissues are infiltrated by lipids and that inflammation may play a role in the pathobiology. We hypothesized that Lp-PLA2 (encoded by the PLA2G7 gene) is expressed in CAVD and may play a role in the mineralization of valve interstitial cells. We have documented the expression of the phospholipase A2 family of genes in aortic valves by using a transcriptomic assay. Messenger ribonucleic acid and protein expression were confirmed in aortic valves explanted from 60 patients by quantitative polymerase chain reaction and immunohistochemistry, respectively. The effect of lysophosphatidylcholine, the product of Lp-PLA2 activity, was documented on the mineralization of valve interstitial cell cultures. Transcriptomic analyses of CAVD and control nonmineralized aortic valves revealed that Lp-PLA2 was increased by 4.2-fold in mineralized aortic valves. Higher expression of Lp-PLA2 in stenotic aortic valves was confirmed by quantitative polymerase chain reaction, immunohistochemistry, and enzymatic Lp-PLA2 activity. The number of Lp-PLA2 transcripts correlated with several indexes of tissue remodeling. In vitro, lysophosphatidylcholine increased the expression of alkaline phosphatase, the ectonucleotide pyrophosphatase/phosphodiesterase 1 enzyme, sodium-dependent phosphate cotransporter 1 (encoded by the SLC20A1 gene), and osteopontin. We then showed that lysophosphatidylcholine-induced mineralization involved ectonucleotidase enzyme as well as apoptosis through a protein-kinase-A-dependent pathway. Together, these results demonstrated that Lp-PLA2 is highly expressed in CAVD, and it plays a role in the mineralization of valve interstitial cells. Further work is necessary to document whether Lp-PLA2 could

  7. The role of calcium-independent phospholipase A2γ in modulation of aqueous humor drainage and Ca2+ sensitization of trabecular meshwork contraction

    PubMed Central

    Pattabiraman, Padmanabhan P.; Lih, Fred B.; Tomer, Kenneth B.

    2012-01-01

    The contractile and relaxation characteristics of trabecular meshwork (TM) are presumed to influence aqueous humor (AH) drainage and intraocular pressure. The mechanisms underlying regulation of TM cell contractile properties, however, are not well understood. This study investigates the role of calcium-independent phospholipase A2 (iPLA2), which controls eicosanoid synthesis, in regulation of TM cell contraction and AH outflow using mechanism-based isoform specific inhibitors (R)-bromoenol lactone (R-BEL, iPLA2γ specific) and (S)-bromoenol lactone (S-BEL, iPLA2β specific). Immunohistochemical analysis revealed intense staining for both iPLA2β and γ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA2γ by R-BEL or small interfering RNA-mediated silencing of iPLA2γ expression induced dramatic changes in TM cell morphology, and decreased actin stress fibers, focal adhesions, and myosin light-chain (MLC) phosphorylation. AH outflow facility increased progressively and significantly in enucleated porcine eyes perfused with R-BEL. This response was associated with a significant decrease in TM tissue MLC phosphorylation and alterations in the morphology of aqueous plexi in R-BEL-perfused eyes. In contrast, S-BEL did not affect either of these parameters. Additionally, R-BEL-induced cellular relaxation of the TM was associated with a significant decrease in the levels of active Rho GTPase, phospho-MLC phosphatase, phospho-CPI-17, and arachidonic acid. Taken together, these observations demonstrate that iPLA2γ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA2γ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways. PMID:22237407

  8. Novel Intergenically Spliced Chimera,NFATC3-PLA2G15, Is Associated with Aggressive T-ALL Biology and Outcome.

    PubMed

    Bond, Jonathan; Tran Quang, Christine; Hypolite, Guillaume; Belhocine, Mohamed; Bergon, Aurélie; Cordonnier, Gaëlle; Ghysdael, Jacques; Macintyre, Elizabeth; Boissel, Nicolas; Spicuglia, Salvatore; Asnafi, Vahid

    2018-03-01

    Leukemias are frequently characterized by the expression of oncogenic fusion chimeras that normally arise due to chromosomal rearrangements. Intergenically spliced chimeric RNAs (ISC) are transcribed in the absence of structural genomic changes, and aberrant ISC expression is now recognized as a potential driver of cancer. To better understand these potential oncogenic drivers, high-throughput RNA sequencing was performed on T-acute lymphoblastic leukemia (T-ALL) patient specimens ( n = 24), and candidate T-ALL-related ISCs were identified ( n = 55; a median of 4/patient). In-depth characterization of the NFATC3-PLA2G15 chimera, which was variably expressed in primary T-ALL, was performed. Functional assessment revealed that the fusion had lower activity than wild-type NFATC3 in vitro , and T-ALLs with elevated NFATC3-PLA2G15 levels had reduced transcription of canonical NFAT pathway genes in vivo Strikingly, high expression of the NFATC3-PLA2G15 chimera correlated with aggressive disease biology in murine patient-derived T-ALL xenografts, and poor prognosis in human T-ALL patients. Mol Cancer Res; 16(3); 470-5. ©2018 AACR . ©2018 American Association for Cancer Research.

  9. Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

    PubMed

    Pabbidi, M R; Ji, X; Maxwell, J T; Mignery, G A; Samarel, A M; Lipsius, S L

    2016-01-01

    We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of

  10. Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes

    PubMed Central

    Ji, X.; Maxwell, J. T.; Mignery, G. A.; Samarel, A. M.; Lipsius, S. L.

    2016-01-01

    We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in–PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in–PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In–PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in–PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the

  11. TNF-α and intPLA2 genes' polymorphism in anorexia nervosa.

    PubMed

    Slopien, Agnieszka; Rybakowski, Filip; Dmitrzak-Weglarz, Monika; Czerski, Piotr; Hauser, Joanna; Komorowska-Pietrzykowska, Renata; Rajewski, Andrzej

    2004-12-01

    The aim of this study was the assessment of -308G/A tumor necrosis factor (TNF)-α gene polymorphism and intPLA2 gene polymorphism in patients with anorexia nervosa (AN) and healthy controls. We studied 91 non-related patients with AN and 144 healthy women (blood donors and students). The mean age of women from study group was 18.22 years (SD ± 3.13 years) and from control group was 31.71 years (SD ± 8.22). Gene polymorphisms were studied with the use of polymerase chain reaction-restriction fragment length polymorphism method. TNF-α gene polymorphism consists of G/A substitution in -308 promoter region. IntPLA2 gene polymorphism is related to intron 1, in which restrictive region is found and recognized by BanI enzyme. We did not obtain statistically significant differences in the frequency of genotypes and alleles of -308G/A TNF-α polymorphism between the study and control groups (genotypes: P = 0.106, alleles: P = 0.076). We did analogous analysis in the restrictive and bulimic subgroups. We did not observe statistically relevant differences in the frequency of genotypes (P = 0.700) and alleles (P = 0.305). We did not obtain statistically relevant difference in the frequency of genotypes and alleles of intPLA2 gene between the study group and controls (genotypes: P = 0.300, alleles: P = 0.331). We did analogous analysis in both subgroups of AN. We did not observe statistically relevant differences in the frequency of genotypes (P = 0.344) and alleles (P = 0.230). There was no statistically relevant trend for the association between TNF-α polymorphism and AN. We did not find association between studied polymorphism of intPLA2 gene and risk of AN.

  12. 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

  13. The C-terminal flavin domain of gp91phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A2 regulation.

    PubMed

    Pessach, Itai; Shmelzer, Zeev; Leto, Thomas L; Dinauer, Mary C; Levy, Rachel

    2006-09-01

    We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient PLB-985 cells and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and NADPH-dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91(phox) (residues 283-570), which contains the NADPH binding and flavin adenine dinucleotide-reducing center, to determine if this portion is regulated by AA. The gp91(phox) C-terminal reductase domain was expressed in X-CGD PLB-985 cells lacking normal gp91(phox) (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabilized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA(2) protein and activity, as well as AA release after stimulation, supported NADPH-dependent diaphorase activity after stimulation, similar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47(phox) and p67(phox) to the membrane fractions of both stimulated cell types indicated that the gp91(phox) C-terminal region is sufficient to anchor the cytosolic oxidase components to the membranes. cPLA(2) translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane-bound oxidase complex encompassing the flavin domain of gp91(phox) provides a docking site for cPLA(2) but is not the site of AA-based regulation of oxidase activity.

  14. 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.

  15. Effects of lifestyle counseling and combination lipid-modifying therapy on lipoprotein-associated phospholipase A2 mass concentration.

    PubMed

    Reddy, Kota J; Singh, Manmeet; Batsell, Richard R; Bangit, Joey R; Miraskar, Rekha A; Zaheer, Misbah S; Cockerham, Carol; Wegner, Michael

    2009-08-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA(2)) is a novel inflammatory biomarker that is associated with increased cardiovascular disease risk independent of and additive to traditional risk factors. Lp-PLA(2) activity is correlated with the degree of inflammation in the atherosclerotic plaque. In human blood, approximately 80% of Lp-PLA(2) is associated with low-density lipoproteins (LDL). Thus, it is hypothesized that changes in Lp-PLA(2) should imitate the changes in the LDL cholesterol. In this present study, we examined the efficacy of lifestyle intervention and combination lipid-lowering therapy on reducing the Lp-PLA(2) levels and determined the relationship between changes in LDL-C and Lp-PLA(2). This retrospective chart review study includes two hundred forty eight patients (58% men and 42% women) who completed the life style intervention in combination with pharmacologic therapy for an average period of 10.5 months. Life style modification included diet and exercise counseling. Combination therapy included omega 3 fish oil (2000mg/d), extended-release niacin (500-1000mg/d), ezetimibe (10mg/d), fenofibrate 160mg/d and colesevelam HCI (1850mg/d), as well as statins. The statins used were either simvastatin (20-40mg/d) or rosuvastatin (5-20mg/d). Sixty five percent (n=161) received low to medium doses of simvastatin, whereas 35% (n=87) received low to medium doses of rosuvastatin. The study revealed a 32.5% reduction in mean Lp-PLA(2) values (baseline 181.1±41.5 vs 122.1±28.1 ng/mL after treatment; P<.001). The change observed in LDL-C was 41%, (baseline 126.2±43 vs 73.9±37.7mg/dL after treatment), which also was significant (P < .001). However, a Pearson correlation test analysis revealed only a weak positive association between changes in Lp-PLA(2) and LDL-C (r(2)=0.052, P < .001). Lp-PLA(2) is reduced with the use of life style counseling and combination lipid lowering therapy. Results also revealed that changes in Lp-PLA(2) may be partially

  16. Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis.

    PubMed

    Le, Zhen; Li, XingZhang; Yuan, Peng; Liu, Pi; Huang, Chunhong

    2015-12-15

    Phospholipase A2 (PLA2) is a calcium-dependent enzyme that is involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Snake venom are known containing PLA2s (svPLA2s) which exhibit a wide variety of pharmacological effects including neurotoxicity, cardiotoxicity, myotoxicity and hemorrhage. Therefore, inhibition of svPLA2 would be advantageous to successful envenomation treatment. A gamma type PLI (PLA2 inhibitor) has been extracted from the serum of Sinonatrix annularis, a non-venomous snake indigenous to China. This showed strong inhibition of Deinagkistrrodon acutus PLA2, however, the PLIγ level in the serum and snake resource are not sufficiently sustainable for further research. To overcome these limitations, we constructed a His6-PLIγ pET28 fusion expression vector and transformed Escherichia coli BL21. To improve the expression of PLIγ, an orthogonal experiment [L16(4)(5)] was performed to optimize induction parameters. The optimized condition was determined to be: induction by 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) for 6 h to the recombinant BL21 after its OD600 was 0.8, with continuous shaking cultivation at 190 rpm and 35 °C. Under these conditions, the amount of expressed protein could reach 57 mg/L. The His6-PLIγ was purified by nickel affinity chromatography and renatured by On-column refolding. The resulting PLIγ showed a good inhibitory effect of enzymatic activities to venom PLA2 isolated from D. acutus. Moreover, the PLIγ had a wide anti-hemorrhage activities to D. acutus, Naja atra and Agkistrodon halys venom. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

    PubMed

    Donnini, Sandra; Finetti, Federica; Francese, Simona; Boscaro, Francesca; Dani, Francesca R; Maset, Fabio; Frasson, Roberta; Palmieri, Michele; Pazzagli, Mario; De Filippis, Vincenzo; Garaci, Enrico; Ziche, Marina

    2011-12-01

    Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

  18. Lipoprotein associated phospholipase A(2): role in atherosclerosis and utility as a biomarker for cardiovascular risk.

    PubMed

    Colley, Kenneth J; Wolfert, Robert L; Cobble, Michael E

    2011-03-01

    Atherosclerosis and its clinical manifestations are widely prevalent throughout the world. Atherogenesis is highly complex and modulated by numerous genetic and environmental risk factors. A large body of basic scientific and clinical research supports the conclusion that inflammation plays a significant role in atherogenesis along the entire continuum of its progression. Inflammation adversely impacts intravascular lipid handling and metabolism, resulting in the development of macrophage foam cell, fatty streak, and atheromatous plaque formation. Given the enormous human and economic cost of myocardial infarction, ischemic stroke, peripheral arterial disease and amputation, and premature death and disability, considerable effort is being committed to refining our ability to correctly identify patients at heightened risk for atherosclerotic vascular disease and acute cardiovascular events so that they can be treated earlier and more aggressively. Serum markers of inflammation have emerged as an important component of risk factor burden. Lipoprotein-associated phospholipase A2 (Lp-PLA(2)) potentiates intravascular inflammation and atherosclerosis. A variety of epidemiologic studies support the utility of Lp-PLA(2) measurements for estimating and further refining cardiovascular disease risk. Drug therapies to inhibit Lp-PLA(2) are in development and show considerable promise, including darapladib, a specific molecular inhibitor of the enzyme. In addition to substantially inhibiting Lp-PLA(2) activity, darapladib reduces progression of the necrotic core volume of human coronary artery atheromatous plaque. The growing body of evidence points to an important role and utility for Lp-PLA(2) testing in preventive and personalized clinical medicine.

  19. 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.

  20. PLA2G3, a gene involved in oxidative stress induced death, is associated with Alzheimer's disease.

    PubMed

    Martínez-García, Ana; Sastre, Isabel; Recuero, María; Aldudo, Jesús; Vilella, Elisabet; Mateo, Ignacio; Sánchez-Juan, Pascual; Vargas, Teo; Carro, Eva; Bermejo-Pareja, Félix; Rodríguez-Rodríguez, Eloy; Combarros, Onofre; Rosich-Estrago, Marcel; Frank, Ana; Valdivieso, Fernando; Bullido, María J

    2010-01-01

    Oxidative stress, which plays a critical role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), is intimately linked to aging, the best established risk factor for AD. Studies in neuronal cells subjected to oxidative stress, mimicking such stress in AD brains, are therefore of great interest. PLA2G3 is the most overexpressed gene in a human neuronal model of oxidative stress induced by the free radical-generating xanthine/xanthine oxidase (X-XOD) system, which provokes apoptotic cell death. In this work, we describe that PLA2G3 gene silencing produced a marked inhibition of X-XOD induced cell death, and that PLA2G3 polymorphisms are associated with AD in a Spanish case-control sample. The capacity to respond to oxidative stress may therefore modulate the risk of AD, and PLA2G3 is a potential target to regulate neuronal damage induced by free radicals.

  1. Kinetic characterization, optimum conditions for catalysis and substrate preference of secretory phospholipase A2 from Glycine max in model membrane systems.

    PubMed

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

    2015-01-01

    Two secretory phospholipase A2 (sPLA2s) from Glycine max, GmsPLA2-IXA-1 and GmsPLA2-XIB-2, have been purified as recombinant proteins and the activity was evaluated in order to obtain the optimum conditions for catalysis using mixed micelles and lipid monolayers as substrate. Both sPLA2s showed a maximum enzyme activity at pH 7 and a requirement of Ca(2+) in the micromolar range. These parameters were similar to those found for animal sPLA2s but a surprising optimum temperature for catalysis at 60 °C was observed. The effect of negative interfacial charges on the hydrolysis of organized substrates was evaluated through initial rate measurements using short chain phospholipids with different head groups. The enzymes showed subtle differences in the specificity for phospholipids with different head groups (DLPC, DLPG, DLPE, DLPA) in presence or absence of NaCl. Both recombinant enzymes showed lower activity toward anionic phospholipids and a preference for the zwitterionic ones. The values of the apparent kinetic parameters (Vmax and KM) demonstrated that these enzymes have more affinity for phosphatidylcholine compared with phosphatidylglycerol, in contrast with the results observed for pancreatic sPLA2. A hopping mode of catalysis was proposed for the action of these sPLA2 on mixed phospholipid/triton micelles. On the other hand, Langmuir-monolayers assays indicated an optimum lateral surface pressure for activity in between 13 and 16 mN/m for both recombinant enzymes. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  2. 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. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  3. The assembly of very low density lipoproteins in rat hepatoma McA-RH7777 cells is inhibited by phospholipase A2 antagonists.

    PubMed

    Tran, K; Wang, Y; DeLong, C J; Cui, Z; Yao, Z

    2000-08-11

    In McA-RH7777 cells, the oleate-stimulated assembly and secretion of very low density lipoproteins (VLDL) was associated with enhanced deacylation of phospholipids, which was markedly decreased by inactivation of the cellular phospholipase A(2). Treatment of the cells with antagonists or antisense oligonucleotide of the Ca(2+)-independent phospholipase A(2) (iPLA(2)) significantly inhibited secretion of apoB100-VLDL and triglyceride. Similar inhibitory effect of the iPLA(2) antagonists was observed on apoB48-VLDL secretion, but secretion of high density lipoprotein particles (such as apoAI- and apoB48-high density lipoprotein) or proteins in general was unaffected. The iPLA(2) antagonist did not affect the synthesis of apoB100 or triglyceride, nor did it affect the activities of phospholipase D, phosphatidate phosphohydrolase, or microsomal triglyceride transfer protein. Inactivation of iPLA(2) resulted in impaired apoB100-VLDL assembly as shown by decreased apoB100-VLDL and triglyceride within the microsomal lumen, with concomitant increase in apoB100 association with the microsomal membranes. The inhibitory effect of iPLA(2) antagonists on apoB100-VLDL assembly/secretion could be abated by pretreatment of cells with oleate. Analysis of molecular species of microsomal phosphatidylcholine and phosphatidylethanolamine by electron spray tandem mass spectrometry revealed that the enrichment of oleoyl moieties was altered by the treatment of iPLA(2) antagonist. These results suggest that the oleate-induced VLDL assembly/secretion may depend upon the establishment of membrane glycerolipids enriched in oleoyl chain, a process mediated by the iPLA(2) activity.

  4. Identification of Epithelial Phospholipase A2 Receptor 1 as a Potential Target in Asthma.

    PubMed

    Nolin, James D; Ogden, H Luke; Lai, Ying; Altemeier, William A; Frevert, Charles W; Bollinger, James G; Naika, Gajendra S; Kicic, Anthony; Stick, Stephen M; Lambeau, Gerard; Henderson, William R; Gelb, Michael H; Hallstrand, Teal S

    2016-12-01

    Secreted phospholipase A 2 s (sPLA 2 s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA 2 s function as enzymes, some of the sPLA 2 s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA 2 s. We sought to examine the expression of PLA2R1 in the airway epithelium of human subjects with asthma and the function of the murine Pla2r1 gene in a model of asthma. Expression of PLA2R1 in epithelial brushings was assessed in two distinct cohorts of children with asthma by microarray and quantitative PCR, and immunostaining for PLA2R1 was conducted on endobronchial tissue and epithelial brushings from adults with asthma. C57BL/129 mice deficient in Pla2r1 (Pla2r1 -/- ) were characterized in an ovalbumin (OVA) model of allergic asthma. PLA2R1 was differentially overexpressed in epithelial brushings of children with atopic asthma in both cohorts. Immunostaining for PLA2R1 in endobronchial tissue localized to submucosal glandular epithelium and columnar epithelial cells. After OVA sensitization and challenge, Pla2r1 -/- mice had increased airway hyperresponsiveness, as well as an increase in cellular trafficking of eosinophils to the peribronchial space and bronchoalveolar lavage fluid, and an increase in airway permeability. In addition, Pla2r1 -/- mice had more dendritic cells in the lung, higher levels of OVA-specific IgG, and increased production of both type-1 and type-2 cytokines by lung leukocytes. PLA2R1 is increased in the airway epithelium in asthma, and serves as a regulator of airway hyperresponsiveness, airway permeability, antigen sensitization, and airway inflammation.

  5. Identification of Epithelial Phospholipase A2 Receptor 1 as a Potential Target in Asthma

    PubMed Central

    Nolin, James D.; Ogden, H. Luke; Lai, Ying; Altemeier, William A.; Frevert, Charles W.; Bollinger, James G.; Naika, Gajendra S.; Kicic, Anthony; Stick, Stephen M.; Lambeau, Gerard; Henderson, William R.; Gelb, Michael H.

    2016-01-01

    Secreted phospholipase A2s (sPLA2s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA2s function as enzymes, some of the sPLA2s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA2s. We sought to examine the expression of PLA2R1 in the airway epithelium of human subjects with asthma and the function of the murine Pla2r1 gene in a model of asthma. Expression of PLA2R1 in epithelial brushings was assessed in two distinct cohorts of children with asthma by microarray and quantitative PCR, and immunostaining for PLA2R1 was conducted on endobronchial tissue and epithelial brushings from adults with asthma. C57BL/129 mice deficient in Pla2r1 (Pla2r1−/−) were characterized in an ovalbumin (OVA) model of allergic asthma. PLA2R1 was differentially overexpressed in epithelial brushings of children with atopic asthma in both cohorts. Immunostaining for PLA2R1 in endobronchial tissue localized to submucosal glandular epithelium and columnar epithelial cells. After OVA sensitization and challenge, Pla2r1−/− mice had increased airway hyperresponsiveness, as well as an increase in cellular trafficking of eosinophils to the peribronchial space and bronchoalveolar lavage fluid, and an increase in airway permeability. In addition, Pla2r1−/− mice had more dendritic cells in the lung, higher levels of OVA-specific IgG, and increased production of both type-1 and type-2 cytokines by lung leukocytes. PLA2R1 is increased in the airway epithelium in asthma, and serves as a regulator of airway hyperresponsiveness, airway permeability, antigen sensitization, and airway inflammation. PMID:27448109

  6. A One Pot Synthesis of Novel Bioactive Tri-Substitute-Condensed-Imidazopyridines that Targets Snake Venom Phospholipase A2

    PubMed Central

    Anilkumar, Nirvanappa C.; Sundaram, Mahalingam S.; Mohan, Chakrabhavi Dhananjaya; Rangappa, Shobith; Bulusu, Krishna C.; Fuchs, Julian E.; Girish, Kesturu S.; Bender, Andreas; Basappa; Rangappa, Kanchugarakoppal S.

    2015-01-01

    Drugs such as necopidem, saripidem, alpidem, zolpidem, and olprinone contain nitrogen-containing bicyclic, condensed-imidazo[1,2-α]pyridines as bioactive scaffolds. In this work, we report a high-yield one pot synthesis of 1-(2-methyl-8-aryl-substitued-imidazo[1,2-α]pyridin-3-yl)ethan-1-onefor the first-time. Subsequently, we performed in silico mode-of-action analysis and predicted that the synthesized imidazopyridines targets Phospholipase A2 (PLA2). In vitro analysis confirmed the predicted target PLA2 for the novel imidazopyridine derivative1-(2-Methyl-8-naphthalen-1-yl-imidazo [1,2-α]pyridine-3-yl)-ethanone (compound 3f) showing significant inhibitory activity towards snake venom PLA2 with an IC50 value of 14.3 μM. Evidently, the molecular docking analysis suggested that imidazopyridine compound was able to bind to the active site of the PLA2 with strong affinity, whose affinity values are comparable to nimesulide. Furthermore, we estimated the potential for oral bioavailability by Lipinski's Rule of Five. Hence, it is concluded that the compound 3f could be a lead molecule against snake venom PLA2. PMID:26196520

  7. The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells.

    PubMed

    Kifor, O; Diaz, R; Butters, R; Brown, E M

    1997-05-01

    The extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR) is a G protein-coupled receptor that activates phospholipase C (PLC). In the present studies, we assessed Ca2+(o)-dependent changes in the generation of inositol phosphates (IP), free arachidonic acid (AA), and phosphatidylbutanol (PtdBtOH) by PLC, phospholipase A2 (PLA2), and phospholipase D (PLD), respectively, in bovine parathyroid cells as well as in wild-type or CaR-transfected human embryonic kidney (HEK293) cells (HEK-WT and HEK-CaR, respectively). Elevated Ca2+(o) increased the formation of IPs in parathyroid cells as well in HEK-CaR but not in HEK-WT cells. High Ca2+(o) also elicited time- and dose-dependent increases in PtdBtOH in parathyroid cells and HEK-CaR but not in HEK-WT cells. Brief treatment of parathyroid and HEK-CaR cells with an activator of protein kinase C (PKC), phorbol 12-myristate,13-acetate (PMA), stimulated PLD activity at both low and high Ca2+(o). Moreover, high Ca2+(o)-stimulated PLD activity was abolished following down-regulation of PKC by overnight phorbol myristate acetate (PMA) pretreatment, suggesting that CaR-mediated activation of PLD depends largely upon stimulation of PKC. High Ca2+(o) likewise increased the release of free AA in parathyroid and HEK-CaR but not in HEK-WT cells. Mepacrine, a general PLA2 inhibitor, and AACOCF3, an inhibitor of cytosolic PLA2, reduced AA release in parathyroid cells at high Ca2+(o), suggesting a major role for PLA2 in high Ca2+(o)-elicited AA release. Pretreatment of parathyroid cells with PMA stimulated release of AA at low and high Ca2+(o), while a PKC inhibitor, chelerythrine, reduced AA release at high Ca2+(o) to the level observed with low Ca2+(o) alone. Thus, PKC contributes importantly to the high Ca2+(o)-evoked, CaR-mediated activation of not only PLD but also PLA2. Finally, high Ca2+(o)-stimulated production of IP, PtdBtOH, and AA all decreased substantially in parathyroid cells cultured for 4 days, in which expression of the Ca

  8. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.

    PubMed

    Rouault, Morgane; Rash, Lachlan D; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H; Lambeau, Gérard

    2006-05-09

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity.

  9. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity

    PubMed Central

    Rouault, Morgane; Rash, Lachlan D.; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H.; Lambeau, Gérard

    2009-01-01

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, an homologous but non toxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1–22) of OS2, but not the central one (residues 58–89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102–119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID:16669624

  10. Biochemical characterization, action on macrophages, and superoxide anion production of four basic phospholipases A2 from Panamanian Bothrops asper snake venom.

    PubMed

    Rueda, Aristides Quintero; Rodríguez, Isela González; Arantes, Eliane C; Setúbal, Sulamita S; Calderon, Leonardo de A; Zuliani, Juliana P; Stábeli, Rodrigo G; Soares, Andreimar M

    2013-01-01

    Bothrops asper (Squamata: Viperidae) is the most important venomous snake in Central America, being responsible for the majority of snakebite accidents. Four basic PLA2s (pMTX-I to -IV) were purified from crude venom by a single-step chromatography using a CM-Sepharose ion-exchange column (1.5 × 15 cm). Analysis of the N-terminal sequence demonstrated that pMTX-I and III belong to the catalytically active Asp49 phospholipase A2 subclass, whereas pMTX-II and IV belong to the enzymatically inactive Lys49 PLA2s-like subclass. The PLA2s isolated from Panama Bothrops asper venom (pMTX-I, II, III, and IV) are able to induce myotoxic activity, inflammatory reaction mainly leukocyte migration to the muscle, and induce J774A.1 macrophages activation to start phagocytic activity and superoxide production.

  11. Biochemical Characterization, Action on Macrophages, and Superoxide Anion Production of Four Basic Phospholipases A2 from Panamanian Bothrops asper Snake Venom

    PubMed Central

    Rueda, Aristides Quintero; Rodríguez, Isela González; Arantes, Eliane C.; Setúbal, Sulamita S.; Calderon, Leonardo de A.; Zuliani, Juliana P.; Stábeli, Rodrigo G.; Soares, Andreimar M.

    2013-01-01

    Bothrops asper (Squamata: Viperidae) is the most important venomous snake in Central America, being responsible for the majority of snakebite accidents. Four basic PLA2s (pMTX-I to -IV) were purified from crude venom by a single-step chromatography using a CM-Sepharose ion-exchange column (1.5 × 15 cm). Analysis of the N-terminal sequence demonstrated that pMTX-I and III belong to the catalytically active Asp49 phospholipase A2 subclass, whereas pMTX-II and IV belong to the enzymatically inactive Lys49 PLA2s-like subclass. The PLA2s isolated from Panama Bothrops asper venom (pMTX-I, II, III, and IV) are able to induce myotoxic activity, inflammatory reaction mainly leukocyte migration to the muscle, and induce J774A.1 macrophages activation to start phagocytic activity and superoxide production. PMID:23509779

  12. 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.

  13. Bee venom phospholipase A2 induces a primary type 2 response that is dependent on the receptor ST2 and confers protective immunity

    PubMed Central

    Palm, Noah W.; Rosenstein, Rachel K.; Yu, Shuang; Schenten, Dominik; Florsheim, Esther; Medzhitov, Ruslan

    2013-01-01

    SUMMARY Venoms consist of toxic components that are delivered to their victims via bites or stings. Venoms also represent a major class of allergens in humans. Phospholipase A2 (PLA2) is a conserved component of venoms from multiple species and is the major allergen in bee venom. Here we examined how bee venom PLA2 is sensed by the innate immune system and induces a type 2 immune response in mice. We found that bee venom PLA2 induced a T helper type 2 (Th2) cell-type response and group 2 innate lymphoid cell activation via the enzymatic cleavage of membrane phospholipids and release of interleukin-33. Furthermore, we showed that the IgE response to PLA2 could protect mice from future challenge with a near-lethal dose of PLA2. These data suggest that the innate immune system can detect the activity of a conserved component of venoms and induce a protective immune response against a venom toxin. PMID:24210353

  14. Bee venom phospholipase A2 induces a primary type 2 response that is dependent on the receptor ST2 and confers protective immunity.

    PubMed

    Palm, Noah W; Rosenstein, Rachel K; Yu, Shuang; Schenten, Dominik D; Florsheim, Esther; Medzhitov, Ruslan

    2013-11-14

    Venoms consist of toxic components that are delivered to their victims via bites or stings. Venoms also represent a major class of allergens in humans. Phospholipase A2 (PLA2) is a conserved component of venoms from multiple species and is the major allergen in bee venom. Here we examined how bee venom PLA2 is sensed by the innate immune system and induces a type 2 immune response in mice. We found that bee venom PLA2 induced a T helper type 2 (Th2) cell-type response and group 2 innate lymphoid cell activation via the enzymatic cleavage of membrane phospholipids and release of interleukin-33. Furthermore, we showed that the IgE response to PLA2 could protect mice from future challenge with a near-lethal dose of PLA2. These data suggest that the innate immune system can detect the activity of a conserved component of venoms and induce a protective immune response against a venom toxin. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Cloning and characterization of a latex allergen (Hev b 7): homology to patatin, a plant PLA2

    PubMed Central

    Kostyal, D A; Hickey, V L; Noti, J D; Sussman, G L; Beezhold, D H

    1998-01-01

    We previously identified a 46-kD protein allergen in latex as having amino acid sequence homology to the patatin gene family. The objective of this study was to characterize this protein by molecular techniques. RNA was isolated from the latex or leaf material from Hevea brasiliensis and from potato tubers. Specific polymerase chain reaction (PCR) primers were designed from the amino acid sequence and reverse transcriptase (RT)-PCR amplified a specific product from latex RNA that was subsequently cloned and sequenced. This product was 1493 bp in length with an 1167 bp open reading frame. The deduced amino acid sequence encodes for a 389 aa protein, pI 4.82 with 43% homology to tobacco patatin. Northern analysis of potato, Hevea leaf, and latex RNA demonstrated the message to be most abundant in latex, weakly present in Hevea leaf, but no hybridization occurred with potato RNA. Patatin has lipid acyl-transferase and PLA2-like activity, suggesting it plays a role as a defence-related protein. Other defence-related proteins in latex such as hevein, glucanase, and hevamine are also allergens. Increased production of defence-related proteins as a result of increased tapping of the rubber trees to meet the demand for latex may explain the increased allergenicity of latex. PMID:9649202

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

    PubMed

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

    2017-04-01

    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.

  17. A peptide derived from bee venom-secreted phospholipase A2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor.

    PubMed

    Fenard, D; Lambeau, G; Maurin, T; Lefebvre, J C; Doglio, A

    2001-08-01

    We have previously shown that secreted phospholipases A2 (sPLA2) from bee and snake venoms have potent anti-human immunodeficiency virus (HIV) activity. These sPLA2s block HIV-1 entry into host cells through a mechanism linked to sPLA2 binding to cells. In this study, 12 synthetic peptides derived from bee venom sPLA2 (bvPLA2) have been tested for inhibition of HIV-1 infection. The p3bv peptide (amino acids 21 to 35 of bvPLA2) was found to inhibit the replication of T-lymphotropic (T-tropic) HIV-1 isolates (ID(50) = 2 microM) but was without effect on monocytotropic (M-tropic) HIV-1 isolates. p3bv was also found capable of preventing the cell-cell fusion process mediated by T-tropic HIV-1 envelope. Finally, p3bv can inhibit the binding of radiolabeled stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and the binding of 12G5, an anti-CXCR4 monoclonal antibody. Taken together, these results indicate that p3bv blocks the replication of T-tropic HIV-1 strains by interacting with CXCR4. Its mechanism of action however appears distinct from that of bvPLA2 because the latter inhibits replication of both T-tropic and M-tropic isolates and does not compete with SDF-1alpha and 12G5 binding to CXCR4.

  18. [Clinical features and expression of PLA(2)R in renal tissue with idiopathic membranous nephropathy in children].

    PubMed

    Dong, Y F; Sun, L W; Zhang, B; Kuang, X Y; Niu, X L; Kang, Y L; Hao, S; Wang, P; Li, Z; Zhu, G H; Huang, W Y; Wu, Y

    2018-03-02

    Objective: To explore the clinical features and expression of PLA(2)R in renal tissue of children with idiopathic membranous nephropathy. Methods: Retrospective study was performed in patients with membranous nephropathy diagnosed through renal biopsy and the follow-up time was at least half a year in Shanghai Children ' s Hospital from January 2010 to February 2017. We compared their clinicopathological and pathological findings of IMN. Indirect immunofluorescence assay was used to detect glomerular PLA(2)R expression. We analyzed the differences of clinical features between the PLA(2)R negative and positive groups. T test, rank-sum test and Fisher exact test were used. Results: Eleven cases had hematuria and proteinuria, 9 cases presented with nephrotic syndrome, and 2 cases showed isolated proteinuria. Of the 22 cases of children with IMN, 16 patients had complete remission (complete remission rate was 72.8%), and 22 patients had partial remission. The renal function of all cases was normal and in all cases the estimated glomerular filtration rate was > 90 ml/(min·1.73m(2)). Of 22 cases with IMN, 7 cases were PLA(2)R-positive in renal tissue and 15 cases were PLA(2)R-negative. The age of positive group (10 years old) was older than the negative group (6 years old)( Z= -2.483, P< 0.05) and the time of positive group (6 months) for urine protein to return to negative was longer than the negative group (2.5 months) through treatment. These differences were significantly different ( Z= -2.072, P< 0.05). Conclusions: Hematuria and proteinuria can be found in most children with idiopathic primary membranous nephropathy. Prednisone combined with immunosuppressant was effective. The positive rate of PLA(2)R in renal tissue of children with IMN was about 32%. The age of PLA(2)R positive group was older than the negative group. And the time of urine protein turning to negative in positive group was longer than that in the negative group.

  19. Antibacterial properties of chicken intestinal phospholipase A2

    PubMed Central

    2011-01-01

    Background The presence of chicken group-IIA PLA2 (ChPLA2-IIA) in the intestinal secretion suggests that this enzyme plays an important role in systemic bactericidal defence. We have analyzed the bactericidal activity of purified ChPLA2-IIA, on several gram-positive and gram-negative bacteria by using the diffusion well and dilution methods. Results ChPLA2-IIA displays potent bactericidal activity against gram-positive bacteria but lacks bactericidal activity against gram negative ones. We have also demonstrated a synergic action of ChPLA2-IIA with lysozyme when added to the bacteria culture prior to ChPLA2-IIA. The bactericidal efficiency of ChPLA2-IIA was shown to be dependent upon the presence of calcium ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Interestingly ChPLA2-IIA displays a higher dependence to Ca2+ ions than to Mg2+ions. Conclusion We conclude that the main physiological role of ChPLA2-IIA could be the defence of the intestine against bacterial invasions. PMID:21226897

  20. Inhibitory effect of a potent and selective cytosolic phospholipase A2alpha inhibitor RSC-3388 on skin inflammation in mice.

    PubMed

    Yamamoto, Mina; Haruna, Takayo; Imura, Kinichi; Hikita, Ichiro; Furue, Yoko; Higashino, Kenichi; Gahara, Yoshinari; Deguchi, Masashi; Yasui, Kiyoshi; Arimura, Akinori

    2008-01-01

    Cytosolic phospholipase A2alpha (cPLA2alpha) preferentially hydrolyzes membrane phospholipids containing arachidonic acid, resulting in the biosynthesis of eicosanoids such as prostaglandins and leukotrienes. To examine the contribution of cPLA2alpha to skin inflammation, we evaluated the effect of (E)-N-[(2S,4R)-4-[N-(biphenyl-2-ylmethyl)-N-2-methylpropylamino]-1-[2-(2,4-difluorobenzoyl)benzoyl]pyrrolidin- 2-yl]methyl-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl) phenyl]acrylamide (RSC-3388), a potent and selective cPLA2alpha inhibitor, on 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced ear inflammation and mite antigen-induced dermatitis in mice. Topical application of RSC-3388 showed a significant inhibitory activity against TNCB-induced ear swelling and eicosanoid production in mice. Comprehensive expression analysis using Gene-Chip technology and subsequent experiments concerning mRNA and protein expression demonstrated that RSC-3388 clearly reduced the levels of interleukin-1beta, macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta in a TNCB-induced mouse model. In addition, RSC-3388 ointment significantly alleviated atopic dermatitis-like skin lesions induced by repeated application of mite antigen. Furthermore, increased expression of cPLA(2)alpha, assessed by anti-phospho-cPLA2alpha antibody, was observed in the skin lesions of mite-antigen-induced dermatitis. These results indicate that cPLA2alpha is involved in the development of skin inflammation in mice, and RSC-3388 is expected to be useful for the treatment of inflammatory skin disorders such as atopic dermatitis. (c) 2008 S. Karger AG, Basel

  1. Evidence for de novo synthesis of lysophosphatidic acid in the spinal cord through phospholipase A2 and autotaxin in nerve injury-induced neuropathic pain.

    PubMed

    Ma, Lin; Uchida, Hitoshi; Nagai, Jun; Inoue, Makoto; Aoki, Junken; Ueda, Hiroshi

    2010-05-01

    We previously reported that lysophosphatidic acid (LPA) initiates nerve injury-induced neuropathic pain and its underlying mechanisms. In addition, we recently demonstrated that intrathecal injection of LPA induces de novo LPA production through the action of autotaxin (ATX), which converts lysophosphatidylcholine to LPA. Here, we examined nerve injury-induced de novo LPA production by using a highly sensitive biological titration assay with B103 cells expressing LPA1 receptors. Nerve injury caused high levels of LPA production in the ipsilateral sides of the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve, or sciatic nerve. Nerve injury-induced LPA production reached its maximum at 3 h after injury, followed by a rapid decline by 6 h. The LPA production was significantly attenuated in ATX heterozygous mutant mice, whereas the concentration and activity of ATX in cerebrospinal fluid were not affected by nerve injury. On the other hand, the activities of cytosolic phospholipase A2 (cPLA2) and calcium-independent phospholipase A2 (iPLA2) were enhanced, with peaks at 1 h after injury. Both de novo LPA production and neuropathic pain-like behaviors were substantially abolished by intrathecal injection of arachidonyl trifluoromethyl ketone, a mixed inhibitor of cPLA2 and iPLA2, or bromoenol lactone, an iPLA2 inhibitor, at 1 h after injury. However, administration of these inhibitors at 6 h after injury had no significant effect on neuropathic pain. These findings provide evidence that PLA2- and ATX-mediated de novo LPA production in the early phase is involved in nerve injury-induced neuropathic pain.

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

    PubMed

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

    2016-08-01

    There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate. © 2016 Society for Laboratory Automation and Screening.

  3. Synergism between Basic Asp49 and Lys49 Phospholipase A2 Myotoxins of Viperid Snake Venom In Vitro and In Vivo

    PubMed Central

    Mora-Obando, Diana; Fernández, Julián; Montecucco, Cesare; Gutiérrez, José María; Lomonte, Bruno

    2014-01-01

    Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 myotoxins acting synergistically in viperid venoms. PMID:25290688

  4. 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

  5. A red yeast rice-olive extract supplement reduces biomarkers of oxidative stress, OxLDL and Lp-PLA2, in subjects with metabolic syndrome: a randomised, double-blind, placebo-controlled trial.

    PubMed

    Hermans, Nina; Van der Auwera, Anastasia; Breynaert, Annelies; Verlaet, Annelies; De Bruyne, Tess; Van Gaal, Luc; Pieters, Luc; Verhoeven, Veronique

    2017-07-03

    Metabolic syndrome (MetS) refers to clustered cardiovascular risk factors (abdominal obesity, pre-diabetes, high blood pressure, dyslipidaemia). Therapies targeting oxidative stress may delay progression to atherosclerosis and diabetes. We investigated the anti-oxidative effect of a supplement combining red yeast rice and olive extract in patients with MetS. A double-blind, placebo-controlled, randomised trial was conducted with 50 patients with MetS as defined by National Cholesterol Education Program Adult Treatment Panel III criteria. Forty-nine subjects randomly assigned to red yeast rice-olive extract (RYR-olive extract; 10.82 mg of monacolins and 9.32 mg of hydroxytyrosol per Cholesfytolplus capsule) or placebo completed the 8-week trial. Whereas effects on cardiovascular risk parameters of MetS have been reported recently, the observed significant 20% increase in oxidised low-density lipoprotein (OxLDL) prompted us to investigate other oxidative stress-related parameters: malondialdehyde (MDA), lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Statistical calculations included univariate quantitative analysis, multivariate linear regression and correlation analysis. The updated results indicate that an RYR-olive extract supplement significantly reduced Lp-PLA 2 by 7% (p < 0.001), but it failed to show a significant decrease in plasma MDA and 8-OHdG (p > 0.05). Reductions in OxLDL (20%) and Lp-PLA 2 (7%) were associated with each other (r = 0.740, p < 0.001). RYR-olive extract significantly reduced Lp-PLA 2 in correlation with the marked reduction in plasma OxLDL, which may lead to a reduced risk for cardiovascular disease in patients with MetS. ClinicalTrials.gov identifier: NCT02065180 . Registered on 13 February 2014.

  6. Virtual analysis of structurally diverse synthetic analogs as inhibitors of snake venom secretory phospholipase A2.

    PubMed

    Sivaramakrishnan, V; Ilamathi, M; Ghosh, K S; Sathish, S; Gowda, T V; Vishwanath, B S; Rangappa, K S; Dhananjaya, B L

    2016-01-01

    Due to the toxic pathophysiological role of snake venom phospholipase A2 (PLA2 ), its compelling limitations to anti-venom therapy in humans and the need for alternative therapy foster considerable pharmacological interest towards search of PLA2 specific inhibitors. In this study, an integrated approach involving homology modeling, molecular dynamics and molecular docking studies on VRV-PL-V (Vipera russellii venom phospholipase A2 fraction-V) belonging to Group II-B secretory PLA2 from Daboia russelli pulchella is carried out in order to study the structure-based inhibitor design. The accuracy of the model was validated using multiple computational approaches. The molecular docking study of this protein was undertaken using different classes of experimentally proven, structurally diverse synthetic inhibitors of secretory PLA2 whose selection is based on IC50 value that ranges from 25 μM to 100 μM. Estimation of protein-ligand contacts by docking analysis sheds light on the importance of His 47 and Asp 48 within the VRV-PL-V binding pocket as key residue for hydrogen bond interaction with ligands. Our virtual analysis revealed that compounds with different scaffold binds to the same active site region. ADME analysis was also further performed to filter and identify the best potential specific inhibitor against VRV-PL-V. Additionally, the e-pharmacophore was generated for the best potential specific inhibitor against VRV-PL-V and reported here. The present study should therefore play a guiding role in the experimental design of VRV-PL-V inhibitors that may provide better therapeutic molecular models for PLA2 recognition and anti-ophidian activity. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Alkylation of histidine residues of Bothrops jararacussu venom proteins and isolated phospholipases A2: a biotechnological tool to improve the production of antibodies.

    PubMed

    Guimarães, C L S; Andrião-Escarso, S H; Moreira-Dill, L S; Carvalho, B M A; Marchi-Salvador, D P; Santos-Filho, N A; Fernandes, C A H; Fontes, M R M; Giglio, J R; Barraviera, B; Zuliani, J P; Fernandes, C F C; Calderón, L A; Stábeli, R G; Albericio, F; da Silva, S L; Soares, A M

    2014-01-01

    Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

  8. Alkylation of Histidine Residues of Bothrops jararacussu Venom Proteins and Isolated Phospholipases A2: A Biotechnological Tool to Improve the Production of Antibodies

    PubMed Central

    Guimarães, C. L. S.; Andrião-Escarso, S. H.; Moreira-Dill, L. S.; Carvalho, B. M. A.; Marchi-Salvador, D. P.; Santos-Filho, N. A.; Fernandes, C. A. H.; Fontes, M. R. M.; Giglio, J. R.; Barraviera, B.; Zuliani, J. P.; Fernandes, C. F. C.; Calderón, L. A.; Stábeli, R. G.; Albericio, F.; da Silva, S. L.; Soares, A. M.

    2014-01-01

    Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom. PMID:24901004

  9. Polymorphisms in nevus-associated genes MTAP, PLA2G6, and IRF4 and the risk of invasive cutaneous melanoma.

    PubMed

    Kvaskoff, Marina; Whiteman, David C; Zhao, Zhen Z; Montgomery, Grant W; Martin, Nicholas G; Hayward, Nicholas K; Duffy, David L

    2011-10-01

    An evolving hypothesis postulates that melanomas may arise through 'nevus-associated' and 'chronic sun exposure' pathways. We explored this hypothesis by examining associations between nevus-associated loci and melanoma risk across strata of body site and histological subtype. We genotyped 1028 invasive case patients and 1469 controls for variants in methylthioadenosine phosphorylase (MTAP), phospholipase A2, group VI (PLA2G6), and Interferon regulatory factor 4 (IRF4), and compared allelic frequencies globally and by anatomical site and histological subtype of melanoma. Odds-ratios (ORs) and 95% confidence intervals (CIs) were calculated using classical and multinomial logistic regression models. Among controls, MTAP rs10757257, PLA2G6 rs132985 and IRF4 rs12203592 were the variants most significantly associated with number of nevi. In adjusted models, a significant association was found between MTAP rs10757257 and overall melanoma risk (OR = 1.32, 95% CI = 1.14-1.53), with no evidence of heterogeneity across sites (Phomogeneity =.52). In contrast, MTAP rs10757257 was associated with superficial spreading/nodular melanoma (OR = 1.34, 95% CI = 1.15- 1.57), but not with lentigo maligna melanoma (OR = 0.79, 95% CI = 0.46-1.35) (Phomogeneity =.06), the subtype associated with chronic sun exposure. Melanoma was significantly inversely associated with rs12203592 in children (OR = 0.35, 95% CI = 0.16-0.77) and adolescents (OR = 0.61, 95% CI = 0.42-0.91), but not in adults (Phomogeneity =.0008). Our results suggest that the relationship between MTAP and melanoma is subtype-specific, and that the association between IRF4 and melanoma is more evident for cases with a younger age at onset. These findings lend some support to the 'divergent pathways' hypothesis and may provide at least one candidate gene underlying this model. Further studies are warranted to confirm these findings and improve our understanding of these relationships.

  10. Extra-hepatic metabolism of 7-ketocholesterol occurs by esterification to fatty acids via cPLA2α and SOAT1 followed by selective efflux to HDL

    PubMed Central

    Lee, Jung Wha; Huang, Jiahn-Dar; Rodriguez, Ignacio R.

    2015-01-01

    Accumulation of 7-ketocholesterol (7KCh) in tissues has been previously associated with various chronic aging diseases. Orally ingested 7KCh is readily metabolized by the liver and does not pose a toxicity threat. However, 7KCh formed in situ, usually associated with lipoprotein deposits, can adversely affect surrounding tissues by causing inflammation and cytotoxicity. In this study we have investigated various mechanisms for extra-hepatic metabolism of 7KCh (e.g. hydroxylation, sulfation) and found only esterification to fatty acids. The esterification of 7KCh to fatty acids involves the combined action of cytosolic phospholipase A2 alpha (cPLA2α) and sterol O-acyltransferase (SOAT1). Inhibition of either one of these enzymes ablates 7KCh-fatty acid ester (7KFAE) formation. The 7KFAEs are not toxic and do not induce inflammatory responses. However, they can be unstable and re-release 7KCh. The higher the degree of unsaturation, the more unstable the 7KFAE (e.g. 18:0>18:1>18:2>18:3>>20:4). Biochemical inhibition and siRNA knockdown of SOAT1 and cPLA2α ablated the 7KFAE synthesis in cultured ARPE19 cells, but had little effect on the 7KCh-induced inflammatory response. Overexpression of SOAT1 reduced the 7KCh-induced inflammatory response and provided some protection from cell death. This effect is likely due to the increased conversion of 7KCh to 7KFAEs, which reduced the intracellular 7KCh levels. Addition of HDL selectively increased the efflux of 7KFAEs and enhanced the effect of SOAT1 overexpression. Our data suggests an additional function for HDL in aiding extra-hepatic tissues to eliminate 7KCh by returning 7KFAEs to the liver for bile acid formation. PMID:25617738

  11. Systemic treatment of cerebral cortex lesions in rats with a new secreted phospholipase A2 inhibitor.

    PubMed

    Cunningham, Timothy J; Souayah, Nizar; Jameson, Bradford; Mitchell, Jennifer; Yao, Lihua

    2004-11-01

    An internal fragment of the human neuroprotective polypeptide DSEP (Diffusible Survival Evasion Peptide) was delivered at 0.4 mg/kg (subcutaneously) 20-30 min after stab wound lesions in the parietal cortex of anesthetized rats. The peptide, CHEASAAQC or CHEC-9, inhibited the inflammatory response to the lesion and the degeneration of neurons adjacent to the wound. Four days after surgery, peptide-treated animals (n = 6) had 75% fewer reactive ameboid microglia/brain macrophages in the cortical parenchyma surrounding the lesion compared to vehicle-injected control rats (n = 6, p = 0.004). The cortical laminae in area 2 adjacent to the lesion were completely obscured in controls because of the increase in inflammatory cells and frank degeneration of neurons, while there was preservation of the neurons and cytoarchitecture after peptide treatment. In parallel experiments, CHEC-9 was found to inhibit the enzymatic activity of secreted phospholipase A2 (sPLA2), including activity present in the serum of peptide-injected rats. Kinetic analysis revealed the peptide increased the average Km for serum by 318% when tested 45 min after treatment (peptide-treated, n = 6; control-treated, n = 6; p = 0.0087), suggesting the principal effect of the peptide was to lower the affinity of serum sPLA2 for substrate. The sPLA2 inhibition by this particular peptide sequence appeared to be highly specific since inversion of a single pair of amino acids eliminated the inhibitory effect. Phorbol-12-myristate-13-acetate stimulated platelet aggregation, a PLA2-regulated activity, was also inhibited by the peptide. The discovery of CHEC-9 makes it possible to study in vivo the long appreciated contribution made by PLA2-directed inflammation to both acute and chronic neurodegeneration and may be helpful in designing therapies to limit neuron death in these conditions.

  12. Biochemical, pharmacological and structural characterization of a new PLA2 from Crotalus durissus terrificus (South American rattlesnake) venom.

    PubMed

    Hernandez-Oliveira, Saraguaci; Toyama, Marcos Hikari; Toyama, Daniela Oliveira; Marangoni, Sergio; Hyslop, Stephen; Rodrigues-Simioni, Léa

    2005-05-01

    A new PLA2 (F16) was purified from Crotalus durissus terrificus venom by molecular exclusion chromatography followed by analytical reverse phase HPLC. The PLA2 (14.86 kDa by MALDI-TOF mass spectrometry) had an amino acid sequence of SLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGRRRPKDATDRCCFVHDCCYEKVTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNGYMFYPDSRCRGPSETC, and showed highly conserved Ca2+-binding and catalytic sites. F16 showed allosteric behavior with 10 mM Ca2+ and had temperature and pH optima of 25 degrees C and 7.9, respectively. F16 (10 microg/ml) produced neuromuscular blockade in chick biventer cervicis preparations in the absence and presence of crotapotin, indicating that crotapotin was not essential for neuromuscular action in this preparation. In contrast, in mouse phrenic nerve-diaphragm preparations, the neuromuscular blockade produced by the same concentration of toxin was dependent on crotapotin. Pre-incubation with heparin markedly reduced the neurotoxicity of F16. These results show that the biochemical and structural properties of F16 are similar to those of the PLA2 isoforms F15 and F17, but that the neurotoxicity and the requirement for crotapotin to form the crotoxin complex varies according to the neuromuscular preparation.

  13. Serum group II phospholipase A(2) levels during menstrual cycle and pregnancy.

    PubMed

    Hayashi, K; Kawamura, T; Endo, S; Hata, T

    2000-01-01

    The purpose of this study was to evaluate the circulating group II phospholipase A(2) (PLA(2)-II) levels during normal menstrual cycle and to assess alterations in maternal circulating PLA(2)-II concentrations during pregnancy and at puerperium. Circulating serum PLA(2)-II concentrations were compared between 38 nonpregnant women with normal menstrual cycle (15 at menstrual phase, 11 at follicular phase, and 12 at luteal phase), 61 normal pregnant women (13 in the first trimester, 12 in the second trimester, and 36 in the third trimester), and 14 normal postpartum women at 5th puerperal day. Serum PLA(2)-II concentrations were also measured in 11 patients with threatened premature labor. Maternal and fetal serum PLA(2)-II levels before and after delivery were made to determine differences in 11 neonates delivered vaginally and 11 neonates delivered by elective cesarean section. Serum PLA(2)-II level was measured with an immunoradiometric assay. Serum PLA(2)-II concentrations at luteal phase were significantly lower than those at menstrual or follicular phase (p< 0.05). There was no significant difference for PLA(2)-II levels between first trimester and menstrual phase or follicular phase. There were no significant differences among three trimesters during pregnancy. There was no significant difference in serum PLA(2)-II levels between normal pregnant women and patients with threatened premature labor. Labor stress did not affect both maternal and fetal serum PLA(2)-II concentrations. There was also no significant difference for circulating PLA(2)-II levels between maternal and fetal serum. Interestingly, serum PLA(2)-II concentrations in postpartum women were significantly higher than those in normal pregnant women (p<0.05). These results suggest that a regulatory mechanism of PLA(2)-II may exist during the normal menstrual cycle and at puerperium. Copyright 2000 S. Karger AG, Basel.

  14. Retinoids inhibit phospholipase A2 in human synovial fluid and arachidonic acid release from rat peritoneal macrophages.

    PubMed

    Hope, W C; Patel, B J; Fiedler-Nagy, C; Wittreich, B H

    1990-10-01

    Retinoids have demonstrated antiinflammatory activity in certain animal models and human disease states. The mechanism by which retinoids elicit this activity is unknown. Some retinoids are known to inhibit arachidonic acid (AA) release and metabolism in intact cells in vitro. Retinoids may exert their antiinflammatory effects by inhibiting phospholipase A2 (PLA2) and the resultant production of inflammatory AA metabolites. Retinoids were evaluated in vitro as inhibitors of the PLA2 activity in human synovial fluid (HSF-PLA2). Of the naturally occurring, nonaromatic retinoids tested, all-trans-retinal, all-trans-retinoic acid (all-trans-RA) and 13-cis-RA were the most potent inhibitors (IC50 S 6-15 microM), whereas all-trans-retinol was much less potent. Of the synthetic aromatic retinoids and arotinoids examined, the free carboxylic, sulfonic, and sulfinic acid forms were more than 15-fold more potent inhibitors of HSF-PLA2 than their corresponding ethyl esters. These retinoids also were evaluated as inhibitors of calcium ionophore A23187-induced AA release from rat peritoneal macrophages. All-trans-RA and 13-cis-RA were potent inhibitors of AA release from these cells (IC50 S 4 microM), while the other natural retinoids were inactive. Of the aromatic retinoids and arotinoids tested, the free acid forms (IC50 S 2-6 microM) were 5- to 21-fold more potent inhibitors of AA release from the macrophages than their corresponding ethyl esters. The potencies of the arotinoids as inhibitors of HSF-PLA2 appeared to correlate with their potencies as inhibitors of AA release from A23187-stimulated rat peritoneal macrophages. These data support the hypothesis that one possible mechanism for the known antiinflammatory activity of some retinoids may be by inhibition of phospholipase A2.

  15. Cryptogenic multifocal ulcerating stenosing enteritis associated with homozygous deletion mutations in cytosolic phospholipase A2-α.

    PubMed

    Brooke, Matthew A; Longhurst, Hilary J; Plagnol, Vincent; Kirkby, Nicholas S; Mitchell, Jane A; Rüschendorf, Franz; Warner, Timothy D; Kelsell, David P; MacDonald, Thomas T

    2014-01-01

    Cryptogenic multifocal ulcerating stenosing enteritis (CMUSE) is an extremely rare, but devastating, disease of unknown aetiology. We investigated the genetic basis of this autosomal recessive condition in a pair of affected siblings who have 40-year histories of catastrophic gastrointestinal and extraintestinal disease. Genome-wide single-nucleotide polymorphism homozygosity mapping in the two affected family members combined with whole-exome sequencing of one affected sibling. This was followed by confirmatory Sanger sequencing of the likely disease-causing sequence variant and functional studies in affected and unaffected family members. Insertion/deletion variation analysis revealed the presence of a homozygous 4 bp deletion (g.155574_77delGTAA) in the PLA2G4A gene, located in the splice donor site directly after exon 17 (the penultimate exon) of the gene in both affected siblings. This introduces a frameshift of 10 amino acids before a premature stop codon (p.V707fsX10), which is predicted to result in the loss of 43 amino acids (residues 707-749) at the C-terminus of cytosolic phospholipase A2-α (cPLA(2)α). cPLA(2)α protein expression was undetectable in the gut of both siblings, with platelet aggregation and thromboxane A(2) production, as functional assays for cPLA(2activity, grossly impaired. We have identified mutations in PLA2G4A as a cause of CMUSE in two affected siblings. Further studies are needed to determine if mutations in this gene are also responsible for disease of a similar phenotype in other cases.

  16. Biochemical characterization of a phospholipase A2 homologue from the venom of the social wasp Polybia occidentalis.

    PubMed

    Diniz-Sousa, Rafaela; Kayano, Anderson M; Caldeira, Cleópatra A; Simões-Silva, Rodrigo; Monteiro, Marta C; Moreira-Dill, Leandro S; Grabner, Fernando P; Calderon, Leonardo A; Zuliani, Juliana P; Stábeli, Rodrigo G; Soares, Andreimar M

    2018-01-01

    Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A 2 (PLA 2 ) from the venom of the social wasp Polybia occidentalis . P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation. The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896.47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA 2 homologues. This is the first report of an enzymatically inactive PLA 2 isolated from wasp venom, similar to snake PLA 2 homologues.

  17. Leishmania donovani-Induced Prostaglandin E2 Generation Is Critically Dependent on Host Toll-Like Receptor 2-Cytosolic Phospholipase A2 Signaling.

    PubMed

    Bhattacharjee, Amrita; Majumder, Saikat; Das, Shibali; Ghosh, Sweta; Biswas, Satabdi; Majumdar, Subrata

    2016-10-01

    Visceral leishmaniasis (VL) is the second-largest parasitic killer disease after malaria. During VL, the protozoan Leishmania donovani induces prostaglandin E2 (PGE2) generation within host macrophages to aid parasite survival. PGE2 significantly influences leishmanial pathogenesis, as L. donovani proliferation is known to be attenuated in PGE2-inhibited macrophages. Here, we report for the first time that signaling via macrophage Toll-like receptor 2 (TLR2) plays an instrumental role in inducing PGE2 release from L. donovani-infected macrophages. This signaling cascade, mediated via the TLR2-phosphatidylinositol 3-kinase (PI3K)-phospholipase C (PLC) signaling pathway, was found to be indispensable for activation of two major enzymes required for PGE2 generation: cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (Cox2). Inhibition of cPLA2, but not secreted phospholipase A2 (sPLA2) or calcium-independent phospholipase A2 (iPLA2), arrested L. donovani infection. During infection, cPLA2 activity increased >7-fold in a calcium-dependent and extracellular signal-regulated kinase (ERK)-dependent manner, indicating that elevation of intracellular calcium and ERK-mediated phosphorylation was necessary for L. donovani-induced cPLA2 activation. For transcriptional upregulation of cyclooxygenase 2, activation of the calcium-calcineurin-nuclear factor of activated T cells (NFAT) signaling was required in addition to the TLR2-PI3K-PLC pathway. Detailed studies by site-directed mutagenesis of potential NFAT binding sites and chromatin immunoprecipitation (ChIP) analysis revealed that the binding of macrophage NFATc2, at the -73/-77 site on the cox2 promoter, induced L. donovani-driven cox2 transcriptional activation. Collectively, these findings highlight the contribution of TLR2 downstream signaling toward activation of cPLA2 and Cox2 and illustrate how the TLR2-PI3K-PLC pathway acts in a concerted manner with calcium-calcineurin-NFATc2 signaling to modulate PGE2 release

  18. Phospholipase A2 in chamber angle of normal eyes and patients with primary open angle glaucoma and exfoliation glaucoma

    PubMed Central

    Rekonen, Petri; Kaarniranta, Kai; Puustjärvi, Tuomo; Teräsvirta, Markku; Uusitalo, Hannu

    2007-01-01

    Purpose Phospholipase A2 (PLA2) is a growing family of lipolytic enzymes that play a key role in various biological processes including general lipid metabolism, membrane homeostasis, and in diseases such as atherosclerosis, arthritis, and acute pancreatitis. Oxidative stress as well as inflammation may be associated with glaucoma pathogenesis. Therefore, our aim was to examine the expression of group IIA secretory PLA2 (sPLA2-IIA), group V secretory PLA2 (sPLA2-V), calcium-independent PLA2 (iPLA2), and cytosolic PLA2 (cPLA2) type in the trabecular meshwork (TM) and the canal of Schlemm in normal eyes and in juxtacanalicular tissue samples from patients with primary open angle glaucoma (POAG) or exfoliation glaucoma (ExG). Methods TM tissues were isolated from healthy donor eyes for corneal transplantation. Specimens of inner wall of the Schlemm's canal and the juxtacanalicular tissue were collected during deep sclerectomy from the eyes of patients who had POAG or ExG. Antibodies against PLA2s (sPLA2-IIA, sPLA2-V, iPLA2, and cPLA2) and a standard immunohistochemical procedure were used for the analysis. Quantification of immunoreactions was provided using a Photoshop-based image analysis. Double-staining immunofluorescence of macrophages and sPLA2-IIA was performed by using confocal microscopy. Results sPLA2-IIA was not present in normal TM. In contrast, sPLA2-IIA levels were significantly higher in glaucoma patients than in controls. Furthermore, sPLA2-IIA expression was much higher in POAG when compared to ExG. iPLA2 was found to predominate in normal human TM, and it demonstrated strong labeling in the uveal and corneoscleral meshwork. The staining of juxtacanalicular meshwork was only moderate in density. In contrast, expression of the enzyme was significantly decreased in glaucoma patients, especially in ExG, when compared to normal controls or to POAG. In addition, strong regional differences were detected in sPLA2-IIA and iPLA2 levels in POAG, whereas

  19. Substituted thiobenzoic acid S-benzyl esters as potential inhibitors of a snake venom phospholipase A2: Synthesis, spectroscopic and computational studies

    NASA Astrophysics Data System (ADS)

    Henao Castañeda, I. C.; Pereañez, J. A.; Jios, J. L.

    2012-11-01

    4-Chlorothiobenzoic acid S-benzyl ester (I), 3-nitrothiobenzoic acid S-benzyl ester (II), 4-nitrothiobenzoic acid S-benzyl ester (III) and 4-methylthiobenzoic acid S-benzyl ester (IV) were prepared and characterized by 1H and 13C NMR, Mass spectrometry and IR spectroscopy. Quantum chemical calculations were performed with Gaussian 09 to calculate the geometric parameters and vibrational spectra. Phospholipase A2 (PLA2) was purified from Crotalus durissus cumanensis venom by molecular exclusion chromatography, followed by reverse phase-high performance liquid chromatography. Two studies of the inhibition of phospholipase A2 activity were performed using phosphatidilcholine and 4-nitro-3-octanoyloxybenzoic acid as substrates, in both cases compound II showed the best inhibitory ability, with 74.89% and 69.91% of inhibition, respectively. Average percentage of inhibition was 52.49%. Molecular docking was carried out with Autodock Vina using as ligands the minimized structures of compounds (I-IV) and as protein PLA2 (PDB code 2QOG). The results suggest that compounds I-IV could interact with His48 at the active site of PLA2. In addition, all compounds showed Van der Waals interactions with residues from hydrophobic channel of the enzyme. This interaction would impede normal catalysis cycle of the PLA2.

  20. Anti-Phospholipase A2 Receptor Antibody Titer Predicts Post-Rituximab Outcome of Membranous Nephropathy.

    PubMed

    Ruggenenti, Piero; Debiec, Hanna; Ruggiero, Barbara; Chianca, Antonietta; Pellé, Timothee; Gaspari, Flavio; Suardi, Flavio; Gagliardini, Elena; Orisio, Silvia; Benigni, Ariela; Ronco, Pierre; Remuzzi, Giuseppe

    2015-10-01

    Rituximab induces nephrotic syndrome (NS) remission in two-thirds of patients with primary membranous nephropathy (MN), even after other treatments have failed. To assess the relationships among treatment effect, circulating nephritogenic anti-phospholipase A2 receptor (anti-PLA2R) autoantibodies and genetic polymorphisms predisposing to antibody production we serially monitored 24-hour proteinuria and antibody titer in patients with primary MN and long-lasting NS consenting to rituximab (375 mg/m(2)) therapy and genetic analyses. Over a median (range) follow-up of 30.8 (6.0-145.4) months, 84 of 132 rituximab-treated patients achieved complete or partial NS remission (primary end point), and 25 relapsed after remission. Outcomes of patients with or without detectable anti-PLA2R antibodies at baseline were similar. Among the 81 patients with antibodies, lower anti-PLA2R antibody titer at baseline (P=0.001) and full antibody depletion 6 months post-rituximab (hazard ratio [HR], 7.90; 95% confidence interval [95% CI], 2.54 to 24.60; P<0.001) strongly predicted remission. All 25 complete remissions were preceded by complete anti-PLA2R antibody depletion. On average, 50% anti-PLA2R titer reduction preceded equivalent proteinuria reduction by 10 months. Re-emergence of circulating antibodies predicted disease relapse (HR, 6.54; 95% CI, 1.57 to 27.40; P=0.01), whereas initial complete remission protected from the event (HR, 6.63; 95% CI, 2.37 to 18.53; P<0.001). Eighteen patients achieved persistent antibody depletion and complete remission and never relapsed. Outcome was independent of PLA2R1 and HLA-DQA1 polymorphisms and of previous immunosuppressive treatment. Therefore, assessing circulating anti-PLA2R autoantibodies and proteinuria may help in monitoring disease activity and guiding personalized rituximab therapy in nephrotic patients with primary MN. Copyright © 2015 by the American Society of Nephrology.

  1. Convergent and enantioselective syntheses of cytosolic phospholipase A(2)α inhibiting N-(1-indazol-1-ylpropan-2-yl)carbamates.

    PubMed

    Sundermann, Tom; Arnsmann, Martina; Schwarzkopf, Julian; Hanekamp, Walburga; Lehr, Matthias

    2014-06-21

    Cytosolic phospholipase A2α (cPLA2α) is an important enzyme of the inflammation cascade. Therefore, inhibitors of cPLA2α are assumed to be promising drug candidates for the treatment of inflammatory disorders. Recently we have found that indole-5-carboxylic acid with a 3-(4-octylphenoxy)-2-(phenoxycarbonylamino)propyl substituent in position 1 is an inhibitor of cPLA2α. We have now synthesized a corresponding derivative with the indole heterocycle replaced by an indazole (4) employing an analogous reaction sequence as for the synthesis of the indole derivative. Besides, a more convergent synthesis for 4 was established using an aziridine as central intermediate. Furthermore, a chiral-pool based enantioselective synthesis was developed for the synthesis of (R)- and (S)-4. Starting compound for both enantiomers was the (R)-serine derived oxazolidine (R)-25. Compound 4 proved to be a moderate inhibitor of cPLA2α, with the S-enantiomer being twice as active as the R-enantiomer. The racemate 4 and the enantiomers (R)- and (S)-4 showed a high in vitro metabolic stability in rat liver S9 fractions.

  2. 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.

  3. Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities

    PubMed Central

    Nicholson, Benjamin; Leach, Craig A.; Goldenberg, Seth J.; Francis, Dana M.; Kodrasov, Matthew P.; Tian, Xufan; Shanks, John; Sterner, David E.; Bernal, Alejandro; Mattern, Michael R.; Wilkinson, Keith D.; Butt, Tauseef R.

    2008-01-01

    Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A2 (PLA2). Isopeptidase activity releases PLA2, which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA2 assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA2 assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity. PMID:18424514

  4. Epigenetic control of phospholipase A2 receptor expression in mammary cancer cells.

    PubMed

    Menschikowski, Mario; Hagelgans, Albert; Nacke, Brit; Jandeck, Carsten; Sukocheva, Olga; Siegert, Gabriele

    2015-12-16

    It has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. Considering that DNA methylation is an important regulator of gene transcription during carcinogenesis, in the current study we analyzed the PLA2R1 expression, PLA2R1 promoter methylation, and selected micro RNA (miRNA) levels in normal human mammary epithelial cells (HMEC) and cancer cell lines. Levels of PLA2R1 and DNA methyltransferases (DNMT) specific mRNA were determined using real-time RT-PCR. Methylation specific-high resolution melting (MS-HRM) analysis was utilized to quantify the methylation degree of selected CpG sites localized in the promoter region of the PLA2R1 gene. Expression of miRNA was tested using miScript Primer Assay system. Nearly complete methylation of the analyzed PLA2R1 promoter region along with PLA2R1 gene silencing was identified in MDA-MB-453 mammary cancer cells. In MCF-7 and BT-474 mammary cancer cell lines, a higher DNA methylation degree and reduced PLA2R1 expression were found in comparison with those in normal HMEC. Synergistic effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on PLA2R1 transcription in MDA-MB-453 cells confirmed the importance of DNA methylation and histone modification in the regulation of the PLA2R1 gene expression in mammary cells. Furthermore, significant positive correlation between the expression of DNMT1 and PLA2R1 gene methylation and negative correlation between the cellular levels of hsa-mir-141, -181b, and -181d-1 and the expression of PLA2R1 were identified in the analyzed cells. Analysis of combined z-score of miR-23b, -154 and -302d demonstrated a strong and significant positive correlation with PLA2R1 expression. Our data indicate that (i) PLA2R1 expression in breast cancer cells is controlled by DNA methylation and histone modifications, (ii) hypermethylation of the PLA2R1 promoter

  5. Analgesic Effects of Bee Venom Derived Phospholipase A(2) in a Mouse Model of Oxaliplatin-Induced Neuropathic Pain.

    PubMed

    Li, Dongxing; Lee, Younju; Kim, Woojin; Lee, Kyungjin; Bae, Hyunsu; Kim, Sun Kwang

    2015-06-29

    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.

  6. Structural bases for a complete myotoxic mechanism: crystal structures of two non-catalytic phospholipases A2-like from Bothrops brazili venom.

    PubMed

    Fernandes, Carlos A H; Comparetti, Edson J; Borges, Rafael J; Huancahuire-Vega, Salomón; Ponce-Soto, Luis Alberto; Marangoni, Sergio; Soares, Andreimar M; Fontes, Marcos R M

    2013-12-01

    Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A2-like (Lys49-PLA2s) are among the main components from Bothrops genus venoms and are capable of causing drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA2s (BbTX-II and MTX-II) from B. brazili venom and a comprehensive structural comparison with several Lys49-PLA2s. Based on these results, two independent sites of interaction were identified between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA2s composed of five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors to complement the conventional antivenom administration. © 2013.

  7. Synthesis of 2-oxoamides based on sulfonamide analogues of γ-amino acids and their activity on phospholipase A2

    PubMed Central

    Antonopoulou, Georgia; Magrioti, Victoria; Stephens, Daren; Constantinou-Kokotou, Violetta; Dennis, Edward A.; Kokotos, George

    2008-01-01

    A variety of lipophilic 2-oxoamides containing sulfonamide analogues of γ-amino acids as well as acyl sulfonamides of γ-aminobutyric acid were synthesized. Their ability to inhibit intracellular GIVA cPLA2 and GVIA iPLA2 as well as secreted GV sPLA2 was evaluated. The sulfonamide group seems a bioisosteric group suitable to replace the carboxyl group in 2-oxoamide inhibitors of GVIA cPLA2. PMID:18618424

  8. Efficacy of a therapeutic treatment using gas-filled microbubble-associated phospholipase A2 in a mouse model of honeybee venom allergy.

    PubMed

    Corthésy, B; Lassus, A; Terrettaz, J; Tranquart, F; Bioley, G

    2016-07-01

    Venom immunotherapy is efficient to desensitize people suffering from insect sting allergies. However, the numerous injections required over several years and important risks of severe side reactions complicate the widespread use of immunotherapy. In the search for novel approaches to blunt the overwhelming pro-allergic Th2 response, we evaluated the therapeutic efficacy of a treatment based on a denatured form of the major allergen, phospholipase A2, associated with microbubbles (PLA2denat -MB) in a mouse model of honeybee venom allergy. Antibodies measured by ELISA, T-cell responses assessed by CFSE-based proliferation assays and ELISA, and basophil degranulation were examined after PLA2denat -MB-based therapeutic treatment of sensitized mice. Mice were challenged with a lethal dose of PLA2 to evaluate protection against anaphylaxis. Therapeutic subcutaneous administration of two different PLA2denat -MB formulations, in contrast to PLA2denat alone, reduced allergic symptoms and protected all mice from anaphylaxis-mediated death after allergen challenge. At the functional level, the use of PLA2denat decreased IgE-mediated basophil degranulation as compared to the native form of the allergen. In comparison with PLA2denat alone, both PLA2denat -MB formulations decreased allergen-specific Th2 CD4 T-cell reactivity. At the mechanistic level, PLA2denat -MB containing 20% palmitic acid and PEG induced PLA2-specific IgA and increased Foxp3(+) Treg frequencies and TGF-β production, whereas the formulation bearing 80% palmitic acid triggered the production of IFN-γ, IgG2a, and IgG3. In contrast to conventional PLA2 subcutaneous immunotherapy, the therapeutic administration of PLA2-MB treatment to mice that already had established allergy to PLA2 protects all subsequently challenged animals. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Interisland variegation of venom [Lys(49)]phospholipase A2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan.

    PubMed

    Yamaguchi, Kazuaki; Chijiwa, Takahito; Yamamura, Takeshi; Ikeda, Naoki; Yatsui, Takayo; Hayama, Setsuko; Hattori, Shosaku; Oda-Ueda, Naoko; Ohno, Motonori

    2015-12-01

    Protobothrops tokarensis (Pt), a Crotalinae snake, inhabits only Takarajima and Kodakarajima islands of the Tokara Islands located in the immediate north of Amami-Oshima island of Japan. Kodakarajima P. tokarensis venom gland cDNA library gave four types of phospholipase A2 (PLA2) cDNAs encoding neutral [Asp(49)]PLA2, basic [Asp(49)]PLA2, highly basic [Asp(49)]PLA2, and [Lys(49)]PLA2. As the amino acid sequences encoded by their open reading frames (ORFs) were identical to those of PLA2, PLA-B, PLA-N, and BPI (a [Lys(49)]PLA2), respectively, from Amami-Oshima P. flavoviridis (Pf) venom, they were named PtPLA2, PtPLA-B, PtPLA-N, and PtBPI. Chromatography of P. tokarensis venom gave three PLA2 isozymes, PtPLA2, PtPLA-B, and PtBPI. However, BPII and BPIII ([Lys(49)]PLA2s) expressed in Amami-Oshima P. flavoviridis venom were not found in P. tokarensis venom. Genomic polymerase chain reaction (PCR) for P. tokarensis liver DNAs with the unique primers gave PtBPI gene. Notably it was found that LINE (long interspersed nuclear element)-1 fragment is inserted into second intron of PtBPI gene. The LINE-1 fragment may prevent duplication of PtBPI gene and thus formation of plural [Lys(49)]PLA2 genes in P. tokarensis genome. The interisland variegation of venom [Lys(49)]PLA2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan is discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. 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

  11. Characterization and structural analysis of a potent anticoagulant phospholipase A2 from Pseudechis australis snake venom.

    PubMed

    Du, Qianyun Sharon; Trabi, Manuela; Richards, Renée Stirling; Mirtschin, Peter; Madaras, Frank; Nouwens, Amanda; Zhao, Kong-Nan; de Jersey, John; Lavin, Martin F; Guddat, Luke W; Masci, Paul P

    2016-03-01

    Pseudechis australis is one of the most venomous and lethal snakes in Australia. Numerous phospholipase A2 (PLA2) isoforms constitute a major portion of its venom, some of which have previously been shown to exhibit not only enzymatic, but also haemolytic, neurotoxic and anticoagulant activities. Here, we have purified a potent anticoagulant PLA2 (identified as PA11) from P. australis venom to investigate its phospholipase, anticoagulant, haemolytic and cytotoxic activities and shown that addition of 11 nM PA11 resulted in a doubling of the clotting time of recalcified whole blood. We have also demonstrated that PA11 has high PLA2 enzymatic activity (10.9 × 10(4) Units/mg), but low haemolytic activity (0.6% of red blood cells hydrolysed in the presence of 1 nM PA11). PA11 at a concentration lower than 600 nM is not cytotoxic towards human cultured cells. Chemical modification experiments using p-bromophenacyl bromide have provided evidence that the catalytic histidine of PA11 is critical for the anticoagulant activity of this PLA2. PA11 that was subjected to trypsin digestion without previous reduction and alkylation of the disulfide bonds maintained enzymatic and anticoagulant activity, suggesting that proteolysis alone cannot abolish these properties. Consistent with these results, administration of PA11 by gavage in a rabbit stasis thrombosis model increased the clotting time of recalcified citrated whole blood by a factor of four. These data suggest that PA11 has potential to be developed as an anticoagulant in a clinical setting. Copyright © 2015. Published by Elsevier Ltd.

  12. Association study of genetic variants in PLA2G4A, PLCG1, LAT, SYK, and TNFRS11A genes in NSAIDs-induced urticaria and/or angioedema patients.

    PubMed

    Ayuso, Pedro; Plaza-Serón, María del Carmen; Doña, Inmaculada; Blanca-López, Natalia; Campo, Paloma; Cornejo-García, José A; Perkins, James R; Torres, Maria J; Blanca, Miguel; Canto, Gabriela

    2015-12-01

    NSAIDs-induced urticaria and/or angioedema (NIUA) is the most frequent entity of hypersensitivity reactions to NSAIDs. The underlying cause is considered to be because of a nonspecific immunological mechanism in which mast cells are key players. We studied the association of nine single nucleotide polymorphisms in five genes involved in mast cell activation (SYK, LAT1, PLCG1, PLA2G4A, and TNFRSF11A) in 450 NIUA patients and 500 controls. We identified several statistically significant associations when stratifying patients by symptoms: PLA2G4A rs12746200 (urticaria vs. controls, Pc=0.005). PLCG1 rs2228246 (angioedema vs. controls; Pc=0.044), and TNFRS11A rs1805034 (urticaria+angioedema vs. controls; Pc=0.041). The frequency of haplotype PLCG1 rs753381-rs2228246 (C-G) in angioedema-NIUA patients was lower than that in controls (Pc=0.040). In addition, the haplotype frequency of TNFRS11A rs1805034-rs35211496 (C-T) was higher among urticaria-NIUA and urticaria+angioedema-NIUA patients than the controls (Pc=0.045 and 0.046). Our results shed light on the involvement of variants in genes related to non-immunological mast cell activation in NIUA.

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

    PubMed

    Lee, Gihyun; Bae, Hyunsu

    2016-02-22

    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.

  14. 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 the experiments were done in vitro.

  15. The Group VIA Calcium-independent Phospholipase A2 and NFATc4 Pathway Mediates IL-1β-induced Expression of Chemokines CCL2 and CXCL10 in Rat Fibroblasts.

    PubMed

    Kuwata, Hiroshi; Yuzurihara, Chihiro; Kinoshita, Natsumi; Taki, Yuki; Ikegami, Yuki; Washio, Sana; Hirakawa, Yushi; Yoda, Emiko; Aiuchi, Toshihiro; Itabe, Hiroyuki; Nakatani, Yoshihito; Hara, Shuntaro

    2018-04-10

    Chemokines are secreted proteins that regulate cell migration and are involved in inflammatory and immune responses. Here we sought to define the functional crosstalk between the lipid signaling and chemokine signaling. We obtained evidence that the induction of some chemokines is regulated by group VIA calcium-independent phospholipase A 2 β (iPLA 2 β) in IL-1β-stimulated rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with IL-1β elicited an increased release of chemotactic factor(s) for monocytic THP-1 cells into culture medium in a time-dependent manner. Inhibitor studies revealed that an intracellular PLA 2 inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF 3 ), but not the cyclooxygenase inhibitor indomethacin, attenuated the release of chemotactic factor(s). The chemotactic activity was inactivated by treatment with either heat or proteinase K, suggesting this chemotactic factor(s) is a proteinaceous factor(s). We purified the chemotactic factor(s) from the conditioned medium of IL-1β-stimulated 3Y1 cells using a heparin column and identified several chemokines, including CCL2 and CXCL10. The inducible expressions of CCL2 and CXCL10 were significantly attenuated by pretreatment with AACOCF 3 . Gene silencing using siRNA revealed that the inductions of CCL2 and CXCL10 were attenuated by iPLA 2 β knockdown. Additionally, the transcriptional activation of nuclear factor of activated T-cell proteins (NFATs), but not NF-κB, by IL-1β stimulation was markedly attenuated by the iPLA 2 inhibitor bromoenol lactone, and NFATc4 knockdown markedly attenuated the IL-1β-induced expression of both CCL2 and CXCL10. Collectively, these results indicated that iPLA 2 β plays roles in IL-1β-induced chemokine expression, in part via NFATc4 signaling. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Lipoprotein phospholipase A2 in patients with isolated coronary artery ectasia.

    PubMed

    Korkmaz, Levent; Erkuş, Emre; Kırış, Abdulkadir; Ağaç, Mustafa Tarık; Acar, Zeydin; Turan, Turhan; Erkan, Hakan; Dursun, Ihsan; Celik, Sükrü

    2011-06-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is among the multiple cardiovascular biomarkers that have been associated with increased cardiovascular disease. Lp-PLA2 appears, however, to be relatively unique in its high specificity for vascular inflammation as opposed to systemic inflammation, its low biologic variability, and its direct role in the causal pathway of plaque inflammation. Nevertheless, the relation between LP-PLA2 and isolated coronary artery ectasia (CAE) has not been investigated yet. The aim of our study was to assess this relation. Twenty-five patients with isolated CAE without stenosis and 25 control subjects with angiographically normal coronary arteries were included in this study. Lp-PLA2 mass was determined in serum by a dual monoclonal antibody immunoassay. Patients with isolated CAE had significantly higher level of Lp-PLA2 compared to the control subjects with angiographically normal coronary arteries (284 ± 102 ng/ml in ectasia and 199 ± 62 ng/ml in control group, respectively, p 0.001). Also we detected a significant positive correlation between the presence of CAE and Lp-PLA2 (r = 0.452, p 0.001). We have demonstrated for the first time increased Lp-PLA2 level in patients with isolated CAE, suggesting that Lp-PLA2 may be involved in the pathogenesis of CAE.

  17. Expression of a mutated phospholipase A2 in transgenic Aedes fluviatilis mosquitoes impacts Plasmodium gallinaceum development

    PubMed Central

    Rodrigues, F. G.; Santos, M. N.; de Carvalho, T. X. T.; Rocha, B. C.; Riehle, M. A.; Pimenta, P. F. P.; Abraham, E. G.; Jacobs-Lorena, M.; Alves de Brito, C. F.; Moreira, L. A.

    2014-01-01

    The genetic manipulation of mosquito vectors is an alternative strategy in the fight against malaria. It was previously shown that bee venom phospholipase A2 (PLA2) inhibits ookinete invasion of the mosquito midgut although mosquito fitness was reduced. To maintain the PLA2 blocking ability without compromising mosquito biology, we mutated the protein-coding sequence to inactivate the enzyme while maintaining the protein’s structure. DNA encoding the mutated PLA2 (mPLA2) was placed downstream of a mosquito midgut-specific promoter (Anopheles gambiae peritrophin protein 1 promoter, AgPer1) and this construct used to transform Aedes fluviatilis mosquitoes. Four different transgenic lines were obtained and characterized and all lines significantly inhibited Plasmodium gallinaceum oocyst development (up to 68% fewer oocysts). No fitness cost was observed when this mosquito species expressed the mPLA2. PMID:18353106

  18. Expression of a mutated phospholipase A2 in transgenic Aedes fluviatilis mosquitoes impacts Plasmodium gallinaceum development.

    PubMed

    Rodrigues, F G; Santos, M N; de Carvalho, T X T; Rocha, B C; Riehle, M A; Pimenta, P F P; Abraham, E G; Jacobs-Lorena, M; Alves de Brito, C F; Moreira, L A

    2008-04-01

    The genetic manipulation of mosquito vectors is an alternative strategy in the fight against malaria. It was previously shown that bee venom phospholipase A2 (PLA2) inhibits ookinete invasion of the mosquito midgut although mosquito fitness was reduced. To maintain the PLA2 blocking ability without compromising mosquito biology, we mutated the protein-coding sequence to inactivate the enzyme while maintaining the protein's structure. DNA encoding the mutated PLA2 (mPLA2) was placed downstream of a mosquito midgut-specific promoter (Anopheles gambiae peritrophin protein 1 promoter, AgPer1) and this construct used to transform Aedes fluviatilis mosquitoes. Four different transgenic lines were obtained and characterized and all lines significantly inhibited Plasmodium gallinaceum oocyst development (up to 68% fewer oocysts). No fitness cost was observed when this mosquito species expressed the mPLA2.

  19. Simvastatin and a Plant Galactolipid Protect Animals from Septic Shock by Regulating Oxylipin Mediator Dynamics through the MAPK-cPLA2 Signaling Pathway

    PubMed Central

    Apaya, Maria Karmella; Lin, Chih-Yu; Chiou, Ching-Yi; Yang, Chung-Chih; Ting, Chen-Yun; Shyur, Lie-Fen

    2015-01-01

    Sepsis remains a major medical issue despite decades of research. Identification of important inflammatory cascades and key molecular mediators are crucial for developing intervention and prevention strategies. In this study, we conducted a comparative oxylipin metabolomics study to gain a comprehensive picture of lipid mediator dynamics during the initial hyperinflammatory phase of sepsis, and demonstrated, in parallel, the efficacy of simvastatin and plant galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-­galactopyranosyl-sn-glycerol (dLGG) in the homeostatic regulation of the oxylipin metabolome using a lipopolysaccharide (LPS)-induced sepsis C57BL/6J mouse model. LPS increased the systemic and organ levels of proinflammatory metabolites of linoleic acid including leukotoxin diols (9-,10-DHOME, 12-,13-DHOME) and octadecadienoic acids (9-HODE and 13-HODE) and arachidonic acid-derived prostanoid, PGE2, and hydroxyeicosatetraenoic acids (8-, 12- and 15-HETE). Treatment with either compound decreased the levels of proinflammatory metabolites and elevated proresolution lipoxin A4, 5(6)-EET, 11(12)-EET and 15-deoxy-PGJ2. dLGG and simvastatin ameliorated the effects of LPS-induced mitogen-activated protein kinase (MAPK)-dependent activation of cPLA2, cyclooxygenase-2, lipoxygenase, cytochrome P450 and/or epoxide hydrolase lowered systemic TNF-α and IL-6 levels and aminotransferase activities and decreased organ-specific infiltration of inflammatory leukocytes and macrophages, and septic shock-induced multiple organ damage. Furthermore, both dLGG and simvastatin increased the survival rates in the cecal ligation and puncture (CLP) sepsis model. This study provides new insights into the role of oxylipins in sepsis pathogenesis and highlights the potential of simvastatin and dLGG in sepsis therapy and prevention. PMID:26701313

  20. Simvastatin and a plant galactolipid protect animals from septic shock by regulating oxylipin mediator dynamics through the MAPK-cPLA2 signaling pathway.

    PubMed

    Apaya, Maria Karmella; Lin, Chih-Yu; Chiou, Ching-Yi; Yang, Chung-Chih; Ting, Chen-Yun; Shyur, Lie-Fen

    2015-12-14

    Sepsis remains a major medical issue despite decades of research. Identification of important inflammatory cascades and key molecular mediators are crucial for developing intervention and prevention strategies. In this study, we conducted a comparative oxylipin metabolomics study to gain a comprehensive picture of lipid mediator dynamics during the initial hyper-inflammatory phase of sepsis, and demonstrated, in parallel, the efficacy of simvastatin and plant galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG) in the homeostatic regulation of the oxylipin metabolome using a lipopolysaccharide (LPS)-induced sepsis C57BL/6J mouse model. LPS increased the systemic and organ levels of pro-inflammatory metabolites of linoleic acid including leukotoxin diols, i.e., 9,10-DHOME, 12,13-DHOME, and octadecadienoic acids, i.e., 9-HODE and 13-HODE; and arachidonic acid-derived prostanoid, PGE 2 , and hydroxyeicosatetraenoic acids, i.e., 8, 12- and 15-HETE. Treatment with either compound decreased the levels of pro-inflammatory metabolites and elevated pro-resolution lipoxin A 4 , 5(6)-EET, 11(12)-EET and 15-deoxy-PGJ 2 . dLGG and simvastatin ameliorated the effects of LPS-induced MAPK-dependent activation of cPLA 2 , cyclooxygenase-2, lipoxygenase, cytochrome P450, and/or epoxide hydrolase, lowered systemic TNF-α and IL-6 levels and aminotransferase activities and decreased organ-specific infiltration of inflammatory leukocytes and macrophages, and septic shock-induced multiple organ damage. Furthermore, both dLGG and simvastatin increased the survival rates in the cecal ligation and puncture (CLP) sepsis model. This study provides new insights into the role of oxylipins in sepsis pathogenesis and highlights the potential of simvastatin and dLGG in sepsis therapy and prevention.

  1. 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

    2017-12-01

    Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A 2 (PLA 2 ), 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 LTB 4 production, cytosolic PLA 2 s activity, myeloperoxidase (MPO) and superoxide anion (O 2 - ) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB 4 , but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O 2 - release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O 2 - . However, it induces LTB 4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB 4 production, and on neutrophils, by stimulating chemotaxis and LTB 4 production, via cytosolic PLA 2 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.

  2. Regulatory T Cells Contribute to the Inhibition of Radiation-Induced Acute Lung Inflammation via Bee Venom Phospholipase A2 in Mice

    PubMed Central

    Shin, Dasom; Lee, Gihyun; Sohn, Sung-Hwa; Park, Soojin; Jung, Kyung-Hwa; Lee, Ji Min; Yang, Jieun; Cho, Jaeho; Bae, Hyunsu

    2016-01-01

    Bee venom has long been used to treat various inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Previously, we reported that bee venom phospholipase A2 (bvPLA2) has an anti-inflammatory effect through the induction of regulatory T cells. Radiotherapy is a common anti-cancer method, but often causes adverse effects, such as inflammation. This study was conducted to evaluate the protective effects of bvPLA2 in radiation-induced acute lung inflammation. Mice were focally irradiated with 75 Gy of X-rays in the lung and administered bvPLA2 six times after radiation. To evaluate the level of inflammation, the number of immune cells, mRNA level of inflammatory cytokine, and histological changes in the lung were measured. BvPLA2 treatment reduced the accumulation of immune cells, such as macrophages, neutrophils, lymphocytes, and eosinophils. In addition, bvPLA2 treatment decreased inflammasome-, chemokine-, cytokine- and fibrosis-related genes’ mRNA expression. The histological results also demonstrated the attenuating effect of bvPLA2 on radiation-induced lung inflammation. Furthermore, regulatory T cell depletion abolished the therapeutic effects of bvPLA2 in radiation-induced pneumonitis, implicating the anti-inflammatory effects of bvPLA2 are dependent upon regulatory T cells. These results support the therapeutic potential of bvPLA2 in radiation pneumonitis and fibrosis treatments. PMID:27144583

  3. L-type voltage-dependent calcium channel is involved in the snake venom group IA secretory phospholipase A2-induced neuronal apoptosis.

    PubMed

    Yagami, Tatsurou; Yamamoto, Yasuhiro; Kohma, Hiromi; Nakamura, Tsutomu; Takasu, Nobuo; Okamura, Noboru

    2013-03-01

    Snake venom group IA secretory phospholipase A2 (sPLA2-IA) is known as a neurotoxin. Snake venom sPLA2s are neurotoxic in vivo and in vitro, causing synergistic neurotoxicity to cortical cultures when applied with toxic concentrations of glutamate. However, it has not yet been cleared sufficiently how sPLA2-IA exerts neurotoxicity. Here, we found sPLA2-IA induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6h. sPLA2-IA-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. NMDA receptor blockers suppressed the neurotoxicity of sPLA2-IA, but an AMPA receptor blocker did not. Interestingly, L-type voltage-dependent Ca(2+) channel (L-VDCC) blocker significantly protected neurons from the sPLA2-IA-induced apoptosis. On the other hand, neither N-VDCC blockers nor P/Q-VDCC blocker did. In conclusion, we demonstrated that sPLA2-IA induced neuronal cell death via apoptosis. Furthermore, the present study suggests that not only NMDA receptor but also L-VDCC contributed to the neurotoxicity of snake venom sPLA2-IA. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Inflammatory events induced by Lys-49 and Asp-49 phospholipases A2 isolated from Bothrops asper snake venom: role of catalytic activity.

    PubMed

    Zuliani, Juliana P; Fernandes, Cristina M; Zamuner, Stella R; Gutiérrez, José M; Teixeira, Catarina F P

    2005-03-01

    The inflammatory events induced in the peritoneal cavity of mice by two PLA2s isolated from Bothrops asper snake venom were investigated. MT-III, an Asp-49 catalytically active enzyme and MT-II, a catalytically inactive Lys-49 variant induced increase in vascular permeability. Inhibition of enzymatic activity of MT-III with p-bromophenacyl bromide reduced this effect. MT-III induced a larger neutrophil infiltrate than MT-II. This activity was significantly reduced after inhibition of catalytic activity. Reduction in the number of neutrophils was observed when antibodies against L-selectin, CD18 or LFA-1 were used, suggesting the involvement of these adhesion molecules in the effects of both PLA2s. There was no effect with antibodies against ICAM-1 and PECAM-1. Increase in the levels of LTB4 and TXA2, as well as of IL-1, IL-6 and TNF-alpha, were observed in the peritoneal exudates induced by MT-III. MT-II did not enhance levels of eicosanoids but increased those of cytokines. It is concluded that both PLA2s induce inflammatory events in this model. Since MT-III exerts a stronger proinflammatory effect, the enzymatic phospholipid hydrolysis may be relevant for these phenomena. However, the fact that MT-II induced inflammation suggests that molecular regions distinct from the catalytic site elicit inflammatory events perhaps by interacting with specific cell membrane acceptors.

  5. Unmasking Snake Venom of Bothrops leucurus: Purification and Pharmacological and Structural Characterization of New PLA2 Bleu TX-III

    PubMed Central

    Marangoni, Fábio André; Ponce-Soto, Luis Alberto; Marangoni, Sergio; Landucci, Elen Cristina Teizem

    2013-01-01

    Bleu TX-III was isolated from Bothrops leucurus snake venom on one-step analytical chromatography reverse phase HPLC, was homogeneous on SDS-PAGE, and was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry in 14243.8 Da. Multiple alignments of Bleu TX-III show high degree of homology with basic PLA2 myotoxins from other Bothrops venoms. Our studies on local and systemic myotoxicity “in vivo” reveal that Bleu TX-III is myotoxin with local but not systemic action due to the decrease in the plasmatic CK levels when Bleu TX-III is administrated by intravenous route in mice (dose 1 and 5 μg). And at a dose of 20 μg myotoxin behaves like a local and systemic action. Bleu TX-III induced moderate marked paw edema, evidencing the local increase in vascular permeability. The inflammatory events induced in the mice (I.M.) were investigated. The increase in the levels of IL-1, IL-6, and TNF-α was observed in the plasma. It is concluded that Bleu TX-III induces inflammatory events in this model. The enzymatic phospholipid hydrolysis may be relevant to these phenomena. Bothrops leucurus venom is still not extensively explored, and the knowledge of its toxins separately through the study of structure/function will contribute for a better understanding of its action mechanism. PMID:23509815

  6. Preparation of the Full Set of Recombinant Mouse- and Human-Secreted Phospholipases A2.

    PubMed

    Ghomashchi, F; Brglez, V; Payré, C; Jeammet, L; Bezzine, S; Gelb, M H; Lambeau, G

    2017-01-01

    A family of 14-20kDa, disulfide-rich, calcium-dependent secreted phospholipases A 2 (sPLA 2 s) that release fatty acids from the sn-2 position of glycerophospholipids can be found in mammals. They have a diverse array of tissue distribution and biological functions. In this chapter we provide detailed protocols for production of nearly all of the mouse and human sPLA 2 s mainly by expression in bacteria and in vitro refolding or by expression in insect cells. High-resolution mass spectrometry and enzymatic assays were, respectively, used to show that all disulfides are formed and that the enzymes are active, strongly suggesting that each sPLA 2 was prepared in the structurally native form. The availability of these proteins has allowed kinetic studies to be carried out, to prepare highly selective antisera, to screen for selective inhibitors, to study receptor binding, and to study the action of each enzyme on mammalian cell membranes and their in vivo biological roles. © 2017 Elsevier Inc. All rights reserved.

  7. Role of enzymatic activity in muscle damage and cytotoxicity induced by Bothrops asper Asp49 phospholipase A2 myotoxins: are there additional effector mechanisms involved?

    PubMed

    Mora-Obando, Diana; Díaz, Cecilia; Angulo, Yamileth; Gutiérrez, José María; Lomonte, Bruno

    2014-01-01

    Viperid venoms often contain mixtures of Asp49 and Lys49 PLA2 myotoxin isoforms, relevant to development of myonecrosis. Given their difference in catalytic activity, mechanistic studies on each type require highly purified samples. Studies on Asp49 PLA2s have shown that enzyme inactivation using p-bromophenacyl bromide (p-BPB) drastically affects toxicity. However, based on the variable levels of residual toxicity observed in some studies, it has been suggested that effector mechanisms independent of catalysis may additionally be involved in the toxicity of these enzymes, possibly resembling those of the enzymatically inactive Lys49 myotoxins. A possibility that Lys49 isoforms could be present in Asp49 PLA2 preparations exists and, if undetected in previous studies, could explain the variable residual toxicity. This question is here addressed by using an enzyme preparation ascertained to be free of Lys49 myotoxins. In agreement with previous reports, inactivation of the catalytic activity of an Asp49 myotoxin preparation led to major inhibition of toxic effects in vitro and in vivo. The very low residual levels of myotoxicity (7%) and cytotoxicity (4%) observed can be attributed to the low, although detectable, enzyme remaining active after p-BPB treatment (2.7%), and would be difficult to reconcile with the proposed existence of additional catalytic-independent toxic mechanisms. These findings favor the concept that the effector mechanism of toxicity of Asp49 PLA2 myotoxins from viperids fundamentally relies on their ability to hydrolyze phospholipids, arguing against the proposal that membrane disruption may also be caused by additional mechanisms that are independent of catalysis.

  8. Inhibition of cyclooxygenase-2 aggravates secretory phospholipase A2-mediated progression of acute liver injury.

    PubMed

    Bhave, Vishakha S; Donthamsetty, Shashikiran; Latendresse, John R; Mehendale, Harihara M

    2008-04-15

    Our previous study [Bhave, V. S., Donthamsetty, S., Latendresse, J. R., Muskhelishvili, L., and Mehendale, H. M. 2008-this issue. Secretory phospholipase A(2) mediates progression of acute liver injury in the absence of sufficient COX-2. Toxicol Appl Pharmacol] showed that in the absence of sufficient induction and co-presence of cyclooxygenase-2 (COX-2), secretory phospholipase A(2) (sPLA(2)) appearing in the intercellular spaces for cleanup of post-necrotic debris seems to contribute to the progression of toxicant-initiated liver injury, possibly by hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas. To further test our hypothesis on the protective role of COX-2, male Fisher-344 rats were administered a selective COX-2 inhibitor, NS-398, and then challenged with a moderately toxic dose of CCl(4). This led to a 5-fold increase in the susceptibility of the COX-2 inhibited rats to CCl(4) hepatotoxicity and mortality. The CCl(4) bioactivating enzyme CYP2E1 protein, CYP2E1 enzyme activity, and the (14)CCl(4)-derived radiolabel covalently bound to the liver proteins were unaffected by the COX-2 inhibitor suggesting that the increased hepatotoxic sensitivity of the COX-2 inhibited rats was not due to higher bioactivation of CCl(4). Further investigation showed that this increased mortality was due to higher plasma and hepatic sPLA(2) activities, inhibited PGE(2) production, and progression of liver injury as compared to the non-intervened rats(.) In conclusion, inhibition of COX-2 mitigates the tissue protective mechanisms associated with COX-2 induction, which promotes sPLA(2)-mediated progression of liver injury in an acute liver toxicity model. Because increased sPLA(2) activity in the intercellular space is associated with increased progression of injury, and induced COX-2 is associated with hepatoprotection, ratios of hepatic COX-2 and sPLA(2) activities may turn out to be a useful tool in predicting the extent of hepatotoxicities.

  9. Inhibitor Binding of Group IVA Phospholipase A2 Probed by Molecular Dynamics and Deuterium Exchange Mass Spectrometry

    PubMed Central

    Burke, John E.; Babakhani, Arneh; Gorfe, Alemayehu A.; Kokotos, George; Li, Sheng; Woods, Virgil L.; McCammon, J. Andrew; Dennis, Edward A.

    2009-01-01

    An analysis of Group IVA (GIVA) phospholipase A2 (PLA2) inhibitor binding was conducted using a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models of the GIVA PLA2 inhibitors pyrrophenone and the 2-oxoamide AX007 docked into the protein were designed based on deuterium exchange results, and extensive molecular dynamics simulations were run to determine protein-inhibitor contacts. The models show that both inhibitors interact with key residues that also exhibit changes in deuterium exchange upon inhibitor binding. Pyrrophenone is bound to the protein through numerous hydrophobic residues located distal from the active site, while the oxoamide is bound mainly through contacts near the active site. We also show differences in protein dynamics around the active site between the two inhibitor-bound complexes. This combination of computational and experimental methods is useful in defining more accurate inhibitor binding sites, and can be used in the generation of better inhibitors against GIVA PLA2. PMID:19459633

  10. IgG4 anti-phospholipase A2 receptor might activate lectin and alternative complement pathway meanwhile in idiopathic membranous nephropathy: an inspiration from a cross-sectional study.

    PubMed

    Yang, Yang; Wang, Chao; Jin, Liping; He, Fagui; Li, Changchun; Gao, Qingman; Chen, Guanglei; He, Zhijun; Song, Minghui; Zhou, Zhuliang; Shan, Fujun; Qi, Ka; Ma, Lu

    2016-08-01

    The deposition of IgG4 of antibodies against phospholipase A2 receptor (anti-PLA2R) is predominating in the kidneys of patients with idiopathic membranous nephropathy, while its predictive value has not been determined. It was a retrospective study, and 438 patients were included. Serum samples of two time points [before intervention (baseline) and after 1.5-year treatment (endpoint)] were detected for total and IgG4 anti-PLA2R. IgG4 <0.26 RU/mL or total <20 RU/mL was considered as seronegativity. Bi-positivity/bi-negativity was defined when patients'antibodies were found positive or negative both at the baseline and endpoint. Completed remission (CR) was a major clinical outcome. A series of complement ingredients (MASP-1/2, MBL, C3a, C5a, Factor B, Ba, Bb and C5b-9) were measured in the patients of bi-positivity and bi-negativity: (1) meta-analysis based on six papers conducted seropositivity of anti-PLA2R was a useful predictor for achieving CR, but there was a high heterogeneity; (2) there was significant correlation between the baseline and decrease in IgG4 subclass and the achievement of CR; (3) bi-negativity of IgG4 has a high accuracy of predicting CR compared with total antibodies; (4) in patients of bi-positivity, those achieving CR showed lower MASP-1/2, MBL, C3a, C5a, FB, Ba and Bb than patients failing to achieve CR; (5) the titers of endpoint and decrease in Ba and Bb were associated with improvement of 24 h-UP in those of bi-positivity; and (6) the decrease in Ba was a significant factor for achieving CR in those of bi-positivity. Continuous IgG4 negativity was a useful tool to predict the achievement of CR; however, in patients of continuous IgG4 positivity, those with lower activation of lectin and alternative pathways would still more probably achieve CR.

  11. Extending David Horrobin's membrane phospholipid theory of schizophrenia: overactivity of cytosolic phospholipase A(2) in the brain is caused by overdrive of coupled serotonergic 5HT(2A/2C) receptors in response to stress.

    PubMed

    Eggers, Arnold E

    2012-12-01

    David Horrobin's membrane phospholipid theory of schizophrenia has held up well over time because his therapeutic prediction that dietary supplementation with eicosapentaenoic acid (EPA) would have a therapeutic effect has been partially verified and undergoes continued testing. In the final version of his theory, he hypothesized that there was hyperactivity of phosphoslipase A(2) (PLA(2)) or a related enzyme but did not explain how the hyperactivity came about. It is known that serotonergic 5HT(2A/2C) receptors are coupled to PLA(2), which hydrolyzes both arachidonic acid (AA) and EPA from diacylglycerides at the sn-2 position. In this paper, Horrobin's theory is combined with a previously published theory of chronic stress in which it was hypothesized that a disinhibited dorsal raphe nucleus, the principal nucleus of the serotonergic system, can organize the neuropathology of diseases such as migraine, hypertension, and the metabolic syndrome. The new or combined theory is that schizophrenia is a disease of chronic stress in which a disinhibited DRN causes widespread serotonergic overdrive in the cerebral cortex. This in turn causes overdrive of cPLA(2) and both central and peripheral depletion of AA and EPA. Because EPA is present in smaller amounts, it falls below threshold for maintaining an intracellular balance between AA-derived and EPA-derived second messenger cascades, which leads to abnormal patterns of neuronal firing. There are two causes of neuronal dysfunction: the disinhibited DRN and EPA depletion. Schizophrenia is statistically associated with metabolic syndrome, hypertension, and migraine because they form a cluster of diseases with similar pathophysiology. The theory provides an explanation for both the central and peripheral phospholipid abnormalities in schizophrenia. It also explains the role of stress in schizophrenia, elevated serum PLA(2) activity in schizophrenia, the relationship between untreated schizophrenia and metabolic syndrome

  12. Pla2g12b and Hpn Are Genes Identified by Mouse ENU Mutagenesis That Affect HDL Cholesterol

    PubMed Central

    Aljakna, Aleksandra; Choi, Seungbum; Savage, Holly; Hageman Blair, Rachael; Gu, Tongjun; Svenson, Karen L.; Churchill, Gary A.; Hibbs, Matt; Korstanje, Ron

    2012-01-01

    Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: for line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 21 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3′ splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism. PMID:22912808

  13. Signaling pathways involved in zymosan phagocytosis induced by two secreted phospholipases A2isolated from Bothrops asper snake venom in macrophages.

    PubMed

    Zuliani, Juliana Pavan; Gutiérrez, José María; Teixeira, Catarina

    2018-03-03

    Phagocytosis, a process involved in host defense, requires coordination of a variety of signaling reactions. MT-II, a catalytically-inactive Lys49-PLA 2 ¸ and MT-III, an active Asp49-PLA 2 isolated from Bothrops asper snake venom, activate phagocytosis in macrophages. In this study the signal pathways mediating zymosan phagocytosis, focusing in lipidic second messengers, were investigated. Macrophages collected from male Swiss mouse peritoneum were obtained 96h after i.p. injection of thioglycollate. Phagocytosis was evaluated with non-opsonized zymosan in the presence or absence of specific inhibitors. Data showed that both venom PLA 2 s increased phagocytosis. Zileuton, Etoricoxib, PACOCF 3 (5-LO, COX-2 and iPLA 2 inhibitors, respectively), as well as WEB2170 (PAF receptor antagonist) significantly reduced phagocytosis induced by both venom PLA 2 s. However, Indomethacin (COX-1/COX-2 inhibitor) and Montelukast (CysL receptor antagonist) did not affect the toxins-induced phagocytosis. Moreover, while PACOCF3 (iPLA 2 inhibitor), reduced the phagocytosis induced by MT-II and MT-III, AACOCF 3 (cPLA 2 inhibitor) significantly reduced the MT-II, but not MT-III-induced phagocytosis. These data suggest the effect of both sPLA 2 s depends on iPLA 2 and that the effect of MT-II depends on activation of cPLA 2 . COX-2 and 5-LO-derived metabolites as well as PAF are involved in the signaling events required for phagocytosis induced by both venom sPLA 2 s. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Crystal structure of a phospholipase A2from 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 A 2 (PLA 2 s), 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. PLA 2 s from these venoms are generally divided into two classes: (i) catalytic PLA 2 s (or Asp49-PLA 2 s) and (ii) non-catalytic PLA 2 -like toxins (or Lys49-PLA 2 s). In many Viperidae venoms, a subset of the basic Asp49-PLA 2 s displays some functional and structural characteristics of PLA 2 -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-PLA 2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA 2 s and Lys49-PLA 2 s 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-PLA 2 s, 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-PLA 2 s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA 2 s found in viperid snake venoms. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Delineating Metabolic Signatures of Head and Neck Squamous Cell Carcinoma: Phospholipase A2, a Potential Therapeutic Target

    PubMed Central

    Tripathi, Pratima; Kamarajan, Pachiyappan; Somashekar, Bagganahalli S.; MacKinnon, Neil; Chinnaiyan, Arul M.; Kapila, Yvonne L.; Rajendiran, Thekkelnaycke M.; Ramamoorthy, Ayyalusamy

    2012-01-01

    A better understanding of molecular pathways involved in malignant transformation of head and neck squamous cell carcinoma (HNSCC) is essential for the development of novel and efficient anti-cancer drugs. To delineate the global metabolism of HNSCC, we report 1H NMR-based metabolic profiling of HNSCC cells from five different patients that were derived from various sites of the upper aerodigestive tract, including the floor of mouth, tongue and larynx. Primary cultures of normal human oral keratinocytes (NHOK) from three different donors were used for comparison. 1H NMR spectra of polar and non-polar extracts of cells were used to identify more than thirty-five metabolites. Principal component analysis performed on the NMR data revealed a clear classification of NHOK and HNSCC cells. HNSCC cells exhibited significantly altered levels of various metabolites that clearly revealed dysregulation in multiple metabolic events, including Warburg effect, oxidative phosphorylation, energy metabolism, TCA cycle anaplerotic flux, glutaminolysis, hexosamine pathway, osmo-regulatory and anti-oxidant mechanism. In addition, significant alterations in the ratios of phosphatidylcholine/lysophosphatidylcholine and phosphocholine/glycerophosphocholine, and elevated arachidonic acid observed in HNSCC cells reveal an altered membrane choline phospholipid metabolism (MCPM). Furthermore, significantly increased activity of phospholipase A2 (PLA2), particularly cytosolic PLA2 (cPLA2) observed in all the HNSCC cells confirm an altered MCPM. In summary, the metabolomic findings presented here can be useful to further elucidate the biological aspects that lead to HNSCC, and also provide a rational basis for monitoring molecular mechanisms in response to chemotherapy. Moreover, cPLA2 may serve as a potential therapeutic target for anti-cancer therapy of HNSCC. PMID:22743333

  16. Computational and in vitro insights on snake venom phospholipase A2 inhibitor of phytocompound ikshusterol3-O-glucoside of Clematis gouriana Roxb. ex DC.

    PubMed

    Muthusamy, Karthikeyan; Chinnasamy, Sathishkumar; Nagarajan, Subbiah; Sivaraman, Thirunavukkarasu

    2017-12-14

    Ikshusterol3-O-glucoside was isolated from Clematis gouriana Roxb. ex DC. root. A structure of the isolated compound was determined on the basis of various spectroscopic interpretations (UV, NMR, FTIR, and GC-MS-EI). This structure was submitted in the PubChem compound database (SID 249494133). SID 249494133 was carried out by density functional theory calculation to observe the chemical stability and electrostatic potential of this compound. The absorption, distribution, metabolism, and excretion property of this compound was predicted to evaluate the drug likeness and toxicity. In addition, molecular docking, quantum polarized ligand docking, prime MMGBSA calculation, and induced fit docking were performed to predict the binding status of SID 249494133 with the active site of phospholipase A 2 (PLA 2 ) (PDB ID: 1A3D). The stability of the compound in the active site of PLA 2 was carried out using molecular dynamics simulation. Further, the anti-venom activity of the compound was assessed using the PLA 2 assay against Naja naja (Indian cobra) crude venom. The results strongly show that Ikshusterol3-O-glucoside has a potent snake-venom neutralizing capacity and it might be a potential molecule for the therapeutic treatment for snakebites.

  17. Pharmacological study of phospholipase A2-induced histamine release from rat peritoneal mast cells.

    PubMed

    Choi, S H; Sakamoto, T; Fukutomi, O; Inagaki, N; Matsuura, N; Nagai, H; Koda, A

    1989-09-01

    The effect of some drugs on phospholipase A2 (PLA2)-induced histamine release from rat peritoneal mast cells was investigated. 1. PLA2 (Naja naja) caused the release of histamine from rat peritoneal mast cells in a dose related fashion. 2. The release of histamine by PLA2 was decreased by the removal of each of calcium and glucose from the reaction medium (Tyrode's solution). 3. p-Bromophenacyl bromide and mepacrine, PLA2 inhibitors, inhibited the release of histamine caused by PLA2. 4. Cinnarizine and nifedipine, calcium channel blockers, and oxatomide, KC-404, tranilast and disodium cromoglycate, anti-allergic agents, also inhibited the PLA2-induced histamine release. 5. When prednisolone (0.5 mg/kg) was administered i.p. 2, 3 and 4 h prior to the harvest of the mast cells, the amount of histamine released by PLA2 ex vivo was significantly decreased. Hydrocortisone, prednisolone and dexamethasone clearly inhibited the release of histamine from mast cells when they were administered 4 h prior to harvest of the cells.

  18. 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

  19. Snake inhibitors of phospholipase A(2) enzymes.

    PubMed

    Dunn, R D; Broady, K W

    2001-08-29

    Phospholipase A(2) (PLA(2)) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA(2)s and they are nontoxic. In comparison, venom PLA(2)s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA(2) inhibitors (PLIalpha), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA(2)s and appeared to be restricted to the serum of this snake family. Analysis of PLA(2) binding to recombinant fragments of PLIalpha indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIbeta, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIgamma, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIgamma inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA(2)s, and bee venom group III PLA(2). However, differences in the binding affinities indicated specificity for particular PLA(2)s. A different representation has emerged for crotalid and viperid snakes. Their PLIgammas did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the gamma-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid beta-neurotoxins and did not inhibit beta-neurotoxins from elapids [1]. Further studies are required to determine if

  20. Membrane cholesterol modulates the cytolytic mechanism of myotoxin II, a Lys49 phospholipase A2 homologue from the venom of Bothrops asper.

    PubMed

    Rangel, José; Quesada, Orestes; Gutiérrez, José María; Angulo, Yamileth; Lomonte, Bruno

    2011-07-01

    Lys49 phospholipase A2 (PLA2) homologues present in crotalid snake venoms lack enzymatic activity, yet they induce skeletal muscle necrosis by a membrane permeabilizing mechanism whose details are only partially understood. The present study evaluated the effect of altering the membrane cholesterol content on the cytolytic activity of myotoxin II, a Lys49 PLA2 isolated from the venom of Bothrops asper, using the myogenic cell line C2C12 as a model target. Cell membrane cholesterol depletion by methyl-β-cyclodextrin (MβCD) treatment enhanced the cytolytic action of myotoxin II, as well as of its bioactive C-terminal synthetic peptide p(115-129) . Conversely, cell membrane cholesterol enrichment by preformed cholesterol-MβCD complexes reduced the cytolytic effect of myotoxin II. The toxic actions of myotoxin I, a catalytically active PLA2 from the same venom, as well as of the cytolytic peptide melittin from bee venom, also increased in cholesterol-depleted cells. Although physical and functional changes resulting from variations in membrane cholesterol are complex, these findings suggest that membrane fluidity could be a relevant parameter to explain the observed modulation of the cytolytic mechanism of myotoxin II, possibly influencing bilayer penetration. In concordance, the cytolytic effect of myotoxin II decreased in direct proportion to lower temperature, a physical factor that affects membrane fluidity. In conclusion, physicochemical properties that depend on membrane cholesterol content significantly influence the cytolytic mechanism of myotoxin II, reinforcing the concept that the primary site of action of Lys49 PLA2 myotoxins is the plasma membrane. Copyright © 2011 John Wiley & Sons, Ltd.

  1. Phospholipase Activities in Clinical and Environmental Isolates of Acanthamoeba

    PubMed Central

    Matin, Abdul

    2011-01-01

    The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phos-pholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A2 (PLA2) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA2. Acanthamoeba exhibited optimal phospholipase activities at 37℃ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA2-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC. PMID:21461262

  2. 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

  3. Crystallization and preliminary X-ray diffraction analysis of suramin, a highly charged polysulfonated napthylurea, complexed with a myotoxic PLA2 from Bothrops asper venom.

    PubMed

    Murakami, Mário T; Gava, Lisandra M; Zela, Sandro P; Arruda, Emerson Z; Melo, Paulo A; Gutierrez, José M; Arni, Raghuvir K

    2004-12-01

    Suramin is a highly charged polysulfonated napthylurea that interferes in a number of physiologically relevant processes such as myotoxicity, blood coagulation and several kinds of cancers. This synthetic compound was complexed with a myotoxic Lys49 PLA(2) from Bothrops asper venom and crystallized by the hanging-drop vapor diffusion method at 18 degrees C. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a=49.05, b=63.84 and c=85.67 angstroms. Diffraction data was collected to 1.78 angstroms.

  4. Restoration of On-Time Embryo Implantation Corrects the Timing of Parturition in Cytosolic Phospholipase A2 Group IVA Deficient Mice1

    PubMed Central

    Brown, Naoko; Morrow, Jason D.; Slaughter, James C.; Paria, Bibhash C.; Reese, Jeff

    2009-01-01

    Cytosolic phospholipase A2 (cPLA2, PLA2G4A) catalyzes the release of arachidonic acid for prostaglandin synthesis by cyclooxygenase 1 (PTGS1) and cyclooxygenase 2 (PTGS2). Mice with Pla2g4a deficiency have parturition delay and other reproductive deficits, including deferred onset of implantation, crowding of implantation sites, and small litters. In this study, we examined the contribution of PLA2G4A to parturition in mice. Pla2g4a mRNA and protein expression were discretely localized in the term and preterm uterine luminal epithelium and colocalized with Ptgs1, but not Ptgs2, expression. The levels of PGE2, PGF2alpha, 6-keto-PGF1alpha, and TxB2 were significantly decreased in Pla2g4a-null uterine tissues, similar to Ptgs1-null uteri, consistent with predominance of PLA2G4A-PTGS1-mediated prostaglandin synthesis in preparation for murine parturition. Litter size was strongly associated with the timing of parturition in Pla2g4a-null mice but could not fully account for the parturition delay. Pla2g4a-null females that received PGE2 + carbaprostacyclin at the time of implantation delivered earlier (20.5 ± 0.2 days vs. 21.6 ± 0.2 days, P < 0.01), although litter size was not improved (4.6 vs. 4.4 pups per litter, P = 0.6). After correction for small litter size, multivariate analysis indicated that Pla2g4a-null mice given prostaglandin treatment to improve implantation timing had gestational length that was similar to wild-type and Pla2g4a heterozygous mice. These results indicate that, despite specific Pla2g4a expression and function in term gestation uteri, the delayed parturition phenotype in Pla2g4a-null mice is primarily due to deferral of implantation. The role of PLA2G4A in timely parturition appears to be critically related to its actions in early pregnancy. PMID:19684335

  5. Fluoroketone inhibition of Ca(2+)-independent phospholipase A2 through binding pocket association defined by hydrogen/deuterium exchange and molecular dynamics.

    PubMed

    Hsu, Yuan-Hao; Bucher, Denis; Cao, Jian; Li, Sheng; Yang, Sheng-Wei; Kokotos, George; Woods, Virgil L; McCammon, J Andrew; Dennis, Edward A

    2013-01-30

    The mechanism of inhibition of group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA(2) were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA(2) should greatly improve our ability to design new inhibitors with higher potency and selectivity.

  6. Exploring and understanding the functional role, and biochemical and structural characteristics of an acidic phospholipase A2, AplTx-I, purified from Agkistrodon piscivorus leucostoma snake venom.

    PubMed

    Resende, L M; Almeida, J R; Schezaro-Ramos, R; Collaço, R C O; Simioni, L R; Ramírez, D; González, W; Soares, A M; Calderon, L A; Marangoni, S; da Silva, S L

    2017-03-01

    Phospholipases A 2 (PLA 2 s) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA 2 isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLA 2 s in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA 2 from this snake venom, denominated AplTx-I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx-I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI-TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLA 2 s, a theoretical and experimental isoelectric point of ≈5.12, and a calcium-dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx-I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve-diaphragm preparations or antibacterial activities. Interestingly, AplTx-I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification

  7. Lipoprotein-associated phospholipase A2 as a biomarker of coronary heart disease and a therapeutic target.

    PubMed

    Packard, Chris J

    2009-07-01

    Lipoprotein-associated phospholipase A2 (Lp PLA2) is postulated to occupy a key position in the pathogenic sequence leading to formation of complex atherosclerotic lesions. This study reviews evidence supporting its role as a biomarker of vascular disease and as a possible therapeutic target. Evidence continues to build supporting the usefulness of Lp PLA2 as a predictor of coronary events in the general population and in those with established coronary heart disease. Elevated Lp PLA2 is also associated with stroke and heart failure. The crystal structure of Lp PLA2 is now available and offers insight into the links between structure, function and atherogenic properties. Recently completed studies on the efficacy of darapladib, a specific Lp PLA2 inhibitor, show beneficial changes in plaque morphology in animal models and in humans. Lp PLA2 is gaining acceptance as a useful biomarker of chronic inflammation and as a predictor of vascular disease. Early results with darapladib offer promise, but not definitive proof, of a potential role for Lp PLA2 inhibition in coronary heart disease prevention.

  8. 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

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

    PubMed

    Sudarshan, S; Dhananjaya, B L

    2015-08-01

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

  10. Association of lipoprotein-associated phospholipase A2 mass with asymptomatic cerebral artery stenosis.

    PubMed

    Wang, Youxin; Zhou, Bin; Zhou, Pingan; Yao, Yan; Cui, Qinghua; Liu, Yingping; Yang, Jichun; Wu, Shouling; Zhao, Xingquan; Zhou, Yong

    2018-04-01

    Cerebral artery stenosis (CAS) is the most important causes of ischaemic stroke. Lipoprotein-associated phospholipase A2 (Lp-PLA2) plays 2 diverse roles in atherosclerosis (pro-inflammatory and anti-inflammatory), and the association between Lp-PLA2 mass and cardiovascular or cerebrovascular events is inconsistent among previous studies. A cross-sectional study including 2012 North Chinese adults aged ≥40 years was performed in 2010-2011 to investigate whether Lp-PLA2 mass is associated with asymptomatic cerebral artery stenosis (ACAS). Serum Lp-PLA2 mass was determined by enzyme-linked immunosorbent assay (ELISA). All participants underwent transcranial Doppler (TCD) and bilateral carotid duplex ultrasound to evaluate intracranial artery stenosis (ICAS) and extracranial arterial stenosis (ECAS). The median serum Lp-PLA2 mass of the participants was 140.74 ng/mL (interquartile range: 131.79-158.07 ng/mL). The adjusted odds ratio (OR) when comparing the 4th quartile to the 1st quartile of Lp-PLA2 was 1.98 (95% confidence interval (CI): 1.42-2.78), 1.79 (95% CI: 1.08-2.94) and 1.87 (95% CI: 1.28-2.73) for the occurrence of ACAS, asymptomatic ECAS and asymptomatic ICAS, respectively, after controlling for vascular risk factors. These independently significant associations remained statistically significant in the male or elderly subgroups, but not in females or middle-aged participants. Lp-PLA2 mass is positively correlated with subclinical atherosclerosis determined by ACAS, ICAS and ECAS in North Chinese, particularly in male and older participants, suggesting that serum Lp-PLA2 mass might be potential biomarker for the detection of ACAS in the adults. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  11. Group 1B Phospholipase A2–Mediated Lysophospholipid Absorption Directly Contributes to Postprandial Hyperglycemia

    PubMed Central

    Labonté, Eric D.; Kirby, R. Jason; Schildmeyer, Nicholas M.; Cannon, April M.; Huggins, Kevin W.; Hui, David Y.

    2007-01-01

    Postprandial hyperglycemia is an early indicator of abnormality in glucose metabolism leading to type 2 diabetes. However, mechanisms that contribute to postprandial hyperglycemia have not been identified. This study showed that mice with targeted inactivation of the group 1B phospholipase A2 (Pla2g1b) gene displayed lower postprandial glycemia than that observed in wild-type mice after being fed a glucose-rich meal. The difference was caused by enhanced postprandial glucose uptake by the liver, heart, and muscle tissues as well as altered postprandial hepatic glucose metabolism in the Pla2g1b−/ − mice. These differences were attributed to a fivefold decrease in the amount of dietary phospholipids absorbed as lysophospholipids in Pla2g1b−/− mice compared with that observed in Pla2g1b+/+ mice. Elevating plasma lysophospholipid levels in Pla2g1b−/− mice via intraperitoneal injection resulted in glucose intolerance similar to that exhibited by Pla2g1b+/+mice. Studies with cultured hepatoma cells revealed that lysophospholipids dose-dependently suppressed insulin-stimulated glycogen synthesis. These results demonstrated that reduction of lysophospholipid absorption enhances insulin-mediated glucose metabolism and is protective against postprandial hyperglycemia. PMID:16567514

  12. Immunogenicity of phospholipase A2 toxins and their role in Streptococcus equi pathogenicity.

    PubMed

    López-Álvarez, M R; Salze, M; Cenier, A; Robinson, C; Paillot, R; Waller, A S

    2017-05-01

    Streptococcus equi subsp. equi (S. equi) is the causative agent of strangles, one of the most frequently diagnosed infectious diseases of horses worldwide. Phospholipase A 2 toxins (PLA 2 ) cleave phospholipid molecules at position sn-2 contributing to the production of leukotrienes that are important inflammatory mediators. Two homologous phospholipases, SlaA and SlaB are encoded by the S. equi genome suggesting that PLA 2 toxins may contribute to its pathogenicity. Here we report the immunogenicity and role of PLA 2 toxins during natural and experimental infection of horses with S. equi. The levels of anti-PLA 2 specific antibodies in serum from horses naturally exposed to S. equi or without exposure were measured by indirect ELISA. Furthermore, the importance of PLA 2 was determined during experimental infection of Welsh Mountain ponies with a mutant strain of S. equi lacking slaA and slaB. Our results show that PLA 2 toxins are immunogenic, which supports their production during natural S. equi infection, but that these toxins are not essential for the development of strangles in a susceptible natural host. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Ketogenic diet change cPLA2/clusterin and autophagy related gene expression and correlate with cognitive deficits and hippocampal MFs sprouting following neonatal seizures.

    PubMed

    Ni, Hong; Zhao, Dong-Jing; Tian, Tian

    2016-02-01

    Because the ketogenic diet (KD) was affecting expression of energy metabolism- related genes in hippocampus and because lipid membrane peroxidation and its associated autophagy stress were also found to be involved in energy depletion, we hypothesized that KD might exert its neuroprotective action via lipid membrane peroxidation and autophagic signaling. Here, we tested this hypothesis by examining the long-term expression of lipid membrane peroxidation-related cPLA2 and clusterin, its downstream autophagy marker Beclin-1, LC3 and p62, as well as its execution molecule Cathepsin-E following neonatal seizures and chronic KD treatment. On postnatal day 9 (P9), 48 Sprague-Dawley rats were randomly assigned to two groups: flurothyl-induced recurrent seizures group and control group. On P28, they were further randomly divided into the seizure group without ketogenic diet (RS+ND), seizure plus ketogenic diet (RS+KD), the control group without ketogenic diet (NS+ND), and the control plus ketogenic diet (NS+KD). Morris water maze test was performed during P37-P43. Then mossy fiber sprouting and the protein levels were detected by Timm staining and Western blot analysis, respectively. Flurothyl-induced RS+ND rats show a long-term lower amount of cPLA2 and LC3II/I, and higher amount of clusterin, Beclin-1, p62 and Cathepsin-E which are in parallel with hippocampal mossy fiber sprouting and cognitive deficits. Furthermore, chronic KD treatment (RS+KD) is effective in restoring these molecular, neuropathological and cognitive changes. The results imply that a lipid membrane peroxidation and autophagy-associated pathway is involved in the aberrant hippocampal mossy fiber sprouting and cognitive deficits following neonatal seizures, which might be a potential target of KD for the treatment of neonatal seizure-induced brain damage. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Effects of 1-H-indole-3-glyoxamide (A-002) on concentration of secretory phospholipase A2 (PLASMA study): a phase II double-blind, randomised, placebo-controlled trial.

    PubMed

    Rosenson, Robert S; Hislop, Colin; McConnell, Daniel; Elliott, Michael; Stasiv, Yuri; Wang, Nan; Waters, David D

    2009-02-21

    Secretory phospholipase A(2) (sPLA(2)) enzymes, produced and secreted in human blood vessels and hepatocytes, contribute to the development of atherosclerosis through mechanisms that are both dependent and independent of lipoprotein. We examined the effects of an sPLA(2) inhibitor on enzyme concentration and on plasma lipoproteins and inflammatory biomarkers in patients with coronary heart disease. Patients aged 18 years and older with stable coronary heart disease from the USA and Ukraine were eligible for enrolment in this phase II, randomised, double-blind, placebo-controlled, parallel-arm, dose-response study. 393 patients were randomly assigned by computer-generated sequence to receive either placebo (n=79) or one of four doses of an sPLA(2) inhibitor, A-002 (1-H-indole-3-glyoxamide; 50 mg [n=79], 100 mg [n=80], 250 mg [n=78], or 500 mg [n=77] twice daily), for 8 weeks. The primary endpoint was the change in sPLA(2) group IIA (sPLA(2)-IIA) concentration or activity from baseline to week 8. Analysis was by modified intention to treat (ITT). The ITT population consisted of all patients who received one dose of study treatment; data for patients who dropped out before the end of the study were carried forward from last observation. This trial is registered with ClinicalTrials.gov, number NCT00455546. All randomised patients received at least one dose and were included in the ITT population. Data for 45 patients were carried forward from last observation (36 in the A-002 group and nine in the placebo group); the main reason for dropout before completion was because of adverse events. 348 patients reached the primary endpoint (A-002 n=278, placebo n=70). Mean sPLA(2)-IIA concentration fell by 86.7%, from 157 pmol/L to 21 [corrected] pmol/L, in the overall active treatment group, and by 4.8%, from 157 pmol/L to 143 [corrected] pmol/L, in the placebo group (p<0.0001 treatment vs placebo). The reductions in sPLA(2)-IIA concentration in the A-002 groups were dose

  15. Ultrastructural analysis of early toxic effects produced by bee venom phospholipase A2 and melittin in Sertoli cells in rats.

    PubMed

    Tilinca, Mariana; Florea, Adrian

    2018-01-01

    In this study, we aimed to investigate the testicular toxicity of two molecules derived from bee venom (BV): phospholipase A2 (PlA2) and melittin (Mlt). Ultrastructural effects of purified BV PlA2 and Mlt were assessed consecutive to repeated dose (30 days) and acute toxicity studies. For the subchronic treatment, PlA2 and Mlt were injected in daily doses equivalent to those released by a bee sting (105 μg PlA2/kg/day and 350 μg Mlt/kg/day), while in the acute treatment their doses corresponded to those released by 100 bee stings (9.3 mg PlA2/kg and 31 mg Mlt/kg). Both PlA2 and Mlt affected the Leydig cells and the cells in seminiferous tubules, the Sertoli cells first of all. PlA2 injection resulted in detachment of the Sertoli cells from the surrounding cells, and extracellular vacuolations, cytoplasmic vacuolations in their basal region and in branches as well, detachment of spermatids, residual bodies and sometimes even spermatocytes into the lumen, changes that had a higher magnitude after the acute treatment. Mlt injection induced similar ultrastructural alterations, but more severe, including degeneration of cellular organelles and cellular necrosis, resulting into rarefaction of the seminiferous epithelium; the ultrastructural changes had a higher magnitude after the 30 repeated dose treatment. We concluded that either of the two molecules tested here, PlA2 and Mlt, were Sertoli cells toxicants at the used doses, and they participated both in the BV testicular toxicity. We consider the observed changes as part of a preceding mechanism of the more severe alterations produced by the BV. It also remains possible that these early unspecific changes reported here could represent the response of the SCs not only to the components of bee venom, but to molecules of other venoms as well. The Sertoli cells were the primary target of PlA2 and Mlt in the spermatogenic epithelium, and their alteration led to further degenerative changes of the germ cells. Since

  16. Antitumoral Activity of Snake Venom Proteins: New Trends in Cancer Therapy

    PubMed Central

    Calderon, Leonardo A.; Sobrinho, Juliana C.; Zaqueo, Kayena D.; de Moura, Andrea A.; Grabner, Amy N.; Mazzi, Maurício V.; Marcussi, Silvana; Fernandes, Carla F. C.; Zuliani, Juliana P.; Carvalho, Bruna M. A.; da Silva, Saulo L.; Stábeli, Rodrigo G.; Soares, Andreimar M.

    2014-01-01

    For more than half a century, cytotoxic agents have been investigated as a possible treatment for cancer. Research on animal venoms has revealed their high toxicity on tissues and cell cultures, both normal and tumoral. Snake venoms show the highest cytotoxic potential, since ophidian accidents cause a large amount of tissue damage, suggesting a promising utilization of these venoms or their components as antitumoral agents. Over the last few years, we have studied the effects of snake venoms and their isolated enzymes on tumor cell cultures. Some in vivo assays showed antineoplastic activity against induced tumors in mice. In human beings, both the crude venom and isolated enzymes revealed antitumor activities in preliminary assays, with measurable clinical responses in the advanced treatment phase. These enzymes include metalloproteases (MP), disintegrins, L-amino acid oxidases (LAAOs), C-type lectins, and phospholipases A2 (PLA2s). Their mechanisms of action include direct toxic action (PLA2s), free radical generation (LAAOs), apoptosis induction (PLA2s, MP, and LAAOs), and antiangiogenesis (disintegrins and lectins). Higher cytotoxic and cytostatic activities upon tumor cells than normal cells suggest the possibility for clinical applications. Further studies should be conducted to ensure the efficacy and safety of different snake venom compounds for cancer drug development. PMID:24683541

  17. Antiviral Activity of Glycyrrhizin against Hepatitis C Virus In Vitro

    PubMed Central

    Matsumoto, Yoshihiro; Matsuura, Tomokazu; Aoyagi, Haruyo; Matsuda, Mami; Hmwe, Su Su; Date, Tomoko; Watanabe, Noriyuki; Watashi, Koichi; Suzuki, Ryosuke; Ichinose, Shizuko; Wake, Kenjiro; Suzuki, Tetsuro; Miyamura, Tatsuo; Wakita, Takaji; Aizaki, Hideki

    2013-01-01

    Glycyrrhizin (GL) has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV) effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc). To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp), replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD), respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2). We found that group 1B PLA2 (PLA2G1B) inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release. PMID:23874843

  18. Combining rosuvastatin with angiotensin-receptor blockers of different PPARγ-activating capacity: effects on high-density lipoprotein subfractions and associated enzymes.

    PubMed

    Rizos, Christos V; Liberopoulos, Evangelos N; Tellis, Kostas; DiNicolantonio, James J; Tselepis, Alexandros D; Elisaf, Moses S

    2015-01-01

    The effects of combining angiotensin-receptor blockers of different peroxisome proliferator-activated receptor γ-activating capacity with rosuvastatin on high-density lipoprotein (HDL) subfractions and associated enzymes in patients with mixed dyslipidemia, hypertension, and prediabetes were assessed. Patients (n = 151) were randomly allocated to rosuvastatin (10 mg/d) plus telmisartan 80 mg/d (RT group, n = 52) or irbesartan 300 mg/d (RI group, n = 48) or olmesartan 20 mg/d (RO group, n = 51). Total and intermediate HDL cholesterol (HDL-C) levels did not change in any group. Large HDL-C increased, while small HDL-C decreased significantly in all the groups (P = not significant between the groups). The mass of HDL lipoprotein-associated phospholipase A2 (HDL-Lp-PLA2) and the activities of paraoxonase 1 remained unchanged in all the groups. However, HDL-Lp-PLA2 activity increased only in the RT group (+21.4%; P < .01 vs baseline) and did not change in the RI (-4.3%; P = .005 vs RT group) and RO (+3.2%; P = .01 vs RT) groups. In conclusion, only the combination of rosuvastatin with telmisartan increased the possibly antiatherosclerotic HDL-Lp-PLA2 activity. © The Author(s) 2013.

  19. Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

    PubMed Central

    Seo, Ho Seong; Kim, Je Hak; Nahm, Moon H.

    2006-01-01

    Bacterial lipoteichoic acid (LTA) shares a structural motif with platelet-activating factor (PAF). Both molecules are strong inflammatory agents and have a glycerol backbone with two lipid chains at the sn-1 and sn-2 positions. PAF is normally inactivated by PAF-acetylhydrolase (PAF-AH), a phospholipase A2 (PLA2), which removes a short acyl group at the sn-2 position. To investigate whether PAF-AH can similarly degrade LTA, we studied the effects of porcine PLA2, bee venom PLA2, and recombinant human PAF-AH on pneumococcal LTA (PnLTA) and staphylococcal LTA (StLTA). After incubation with a porcine or bee venom PLA2, a large fraction of PnLTA lost 264 Da, which corresponds to the mass of the oleic acid group at the sn-2 position. After incubation with recombinant human PAF-AH, PnLTA lost 264 Da; the reduction did not occur when PAF-AH was exposed to Pefabloc SC, an irreversible inhibitor of the PAF-AH active site. Following PAF-AH treatment, PnLTA and StLTA were not able to stimulate mouse RAW 264.7 cells to produce tumor necrosis factor alpha but could stimulate CHO cells expressing human TLR2. This stimulation pattern has been observed with monoacyl PnLTA prepared by mild alkali hydrolysis (22). Taking these data together, we conclude that PAF-AH can remove one acyl chain at the sn-2 position of LTA and produce a monoacyl-LTA that is inactive against mouse cells. PMID:16603612

  20. Phospholipase A2 Receptor 1 Epitope Spreading at Baseline Predicts Reduced Likelihood of Remission of Membranous Nephropathy.

    PubMed

    Seitz-Polski, Barbara; Debiec, Hanna; Rousseau, Alexandra; Dahan, Karine; Zaghrini, Christelle; Payré, Christine; Esnault, Vincent L M; Lambeau, Gérard; Ronco, Pierre

    2018-02-01

    The phospholipase A2 receptor (PLA2R1) is the major autoantigen in primary membranous nephropathy. Several PLA2R1 epitopes have been characterized, and a retrospective study identified PLA2R1 epitope spreading as a potential indicator of poor prognosis. Here, we analyzed the predictive value of anti-PLA2R1 antibody (PLA2R1-Ab) titers and epitope spreading in a prospective cohort of 58 patients positive for PLA2R1-Ab randomly allocated to rituximab ( n =29) or antiproteinuric therapy alone ( n =29). At baseline, the epitope profile (CysR, CysRC1, CysRC7, or CysRC1C7) did not correlate with age, sex, time from diagnosis, proteinuria, or serum albumin, but epitope spreading strongly correlated with PLA2R1-Ab titer ( P <0.001). Ten (58.8%) of the 17 patients who had epitope spreading at baseline and were treated with rituximab showed reversal of epitope spreading at month 6. In adjusted analysis, epitope spreading at baseline was associated with a decreased remission rate at month 6 (odds ratio, 0.16; 95% confidence interval, 0.04 to 0.72; P =0.02) and last follow-up (median, 23 months; odds ratio, 0.14; 95% confidence interval, 0.03 to 0.64; P =0.01), independently from age, sex, baseline PLA2R1-Ab level, and treatment group. We propose that epitope spreading at baseline be considered in the decision for early therapeutic intervention in patients with primary membranous nephropathy. Copyright © 2018 by the American Society of Nephrology.

  1. Interfacial binding of bee venom secreted phospholipase A2 to membranes occurs predominantly by a nonelectrostatic mechanism.

    PubMed

    Bollinger, James G; Diraviyam, Karthikeyan; Ghomashchi, Farideh; Murray, Diana; Gelb, Michael H

    2004-10-26

    The secreted phospholipase A(2) from bee venom (bvPLA(2)) contains a membrane binding surface composed mainly of hydrophobic residues and two basic residues that come in close contact with the membrane. Previous studies have shown that the mutant in which these two basic residues (K14 and R23) as well as three other nearby basic residues were collectively changed to glutamate (charge reversal), like wild-type enzyme, binds with high affinity to anionic phospholipid vesicles. In the present study, we have measured the equilibrium constants for the interaction of wild-type bvPLA(2), the charge-reversal mutant (bvPLA(2)-E5), and the mutant in which the five basic residues were changed to neutral glutamine (bvPLA(2)-Q5) with phosphatidylcholine (PC) vesicles containing various amounts of the anionic phosphatidylserine (PS). Remarkably, bvPLA(2)-E5 with an anionic membrane binding surface binds more tightly to vesicles as the mole percent of PS is increased. Computational studies predict that this is due to a significant upward shift in the pK(a) of E14 (and to some extent E23) when the enzyme binds to PC/PS vesicles such that the carboxylate of the glutamate side chain near the membrane surface undergoes protonation. The experimental pH dependence of vesicle binding supports this prediction. bvPLA(2)-E5 binds more weakly to PS/PC vesicles than does wild-type enzyme due to electrostatic protein-vesicle repulsion coupled with the similar energetics of desolvation of basic residues and glutamates that accompanies enzyme-vesicle contact. Studies with bvPLA(2)-Q5 show that only a small fraction of the total bvPLA(2) interfacial binding energy ( approximately 10%) is due to electrostatics.

  2. Comparative protection against rat intestinal reperfusion injury by a new inhibitor of sPLA2, COX-1 and COX-2 selective inhibitors, and an LTC4 receptor antagonist

    PubMed Central

    Arumugam, Thiruma V; Arnold, Naomi; Proctor, Lavinia M; Newman, Michelle; Reid, Robert C; Hansford, Karl A; Fairlie, David P; Shiels, Ian A; Taylor, Stephen M

    2003-01-01

    A new group IIa sPLA2 inhibitor was compared with selective inhibitors of COX-1, COX-2 and an LTC4 antagonist for effects on local and remote tissue injuries following ischaemia and reperfusion (I/R) of the small intestine in rats. In an acute model of ischaemia (30 min) and reperfusion (150 min) injury in the absence of inhibitors, there was significant intestinal haemorrhage, oedema and mucosal damage, neutropenia, elevated serum levels of aspartate aminotransferase (AST) and hypotension. Preischaemic treatment with the inhibitor of sPLA2 (Group IIa), at 5 mg kg−1 i.v. or 10 mg kg−1 p.o. significantly inhibited I/R-induced neutropenia, the elevation of serum levels of AST, intestinal oedema and hypotension. Pretreatment with the COX-2 inhibitor celebrex (10 mg kg−1 i.v.) and the LTC4 antagonist zafirlukast (1 mg kg−1 i.v.) also showed marked improvement with I/R-induced AST, oedema and neutropenia. Hypotension was only reduced by the LTC4 antagonist. The COX-1 inhibitor flunixin (1 mg kg−1 i.v.) did not effect improvement in the markers of tissue injury. Histological examination of rat I/R injury showed that all of the drugs offered some protection to the mucosal layer damage compared to no drug treatment. Given i.v., the sPLA2 inhibitor was more effective than either the COX-1 or COX-2 inhibitors in preventing rat I/R injury. These results indicate that a potent new inhibitor of sPLA2 (group IIa) protects the rat small intestine from I/R injury after oral or intravenous administration. COX-2 and LTC4 inhibitors also showed some beneficial effects against intestinal I/R injury. Our study suggests that sPLA2 (Group IIa) may have a pathogenic role in intestinal I/R in rats. PMID:12967936

  3. Sumoylation-deficient Prdx6 gains protective function by amplifying enzymatic activity and stability and escapes oxidative stress-induced aberrant Sumoylation.

    PubMed

    Chhunchha, Bhavana; Kubo, Eri; Fatma, Nigar; Singh, Dhirendra P

    2017-01-05

    Aberrant Sumoylation of protein(s) in response to oxidative stress or during aging is known to be involved in etiopathogenesis of many diseases. Upon oxidative stress, Peroxiredoxin (Prdx) 6 is aberrantly Sumoylated by Sumo1, resulting in loss of functions and cell death. We identified lysines (K) 122 and 142 as the major Sumo1 conjugation sites in Prdx6. Intriguingly, the mutant Prdx6 K122/142 R (arginine) gained protective efficacy, increasing in abundance and promoting glutathione (GSH) peroxidase and acidic calcium-independent phospholipase A 2 (aiPLA 2 ) activities. Using lens epithelial cells derived from targeted inactivation of Prdx6 -/- gene and relative enzymatic and stability assays, we discovered dramatic increases in GSH-peroxidase (30%) and aiPLA 2 (37%) activities and stability in the K122/142 R mutant, suggesting Sumo1 destabilized Prdx6 integrity. Prdx6 -/- LECs with EGFP-Sumo1 transduced or co-expressed with mutant TAT-HA-Prdx6K122/142 R or pGFP-Prdx6K122/142 R were highly resistant to oxidative stress, demonstrating mutant protein escaped and interrupted the Prdx6 aberrant Sumoylation-mediated cell death pathway. Mutational analysis of functional sites showed that both peroxidase and PLA 2 active sites were necessary for mutant Prdx6 function, and that Prdx6 phosphorylation (at T177 residue) was essential for optimum PLA 2 activity. Our work reveals the involvement of oxidative stress-induced aberrant Sumoylation in dysregulation of Prdx6 function. Mutant Prdx6 at its Sumo1 sites escapes and abates this adverse process by maintaining its integrity and gaining function. We propose that the K122/142R mutant of Prdx6 in the form of a TAT-fusion protein may be an easily applicable intervention for pathobiology of cells related to aberrant Sumoylation signaling in aging or oxidative stress.

  4. Mechanical and Impact Properties of PLA/2 × 2 Twill and 4 × 4 Hopsack Weave Flax Textile Composites Produced by the Interval Hot Pressing Technique

    NASA Astrophysics Data System (ADS)

    Siengchin, S.; Wongmanee, S.

    2014-07-01

    Polylactide (PLA)/2 × 2 twill and 4 × 4 hopsack weave flax textile composites were produced by the interval hot pressing technique. The dispersion of structural elements of the composites was inspected by the scanning electron microscopy (SEM). The composites were subjected to instrumented falling-weight impact tests. Their mechanical properties (tensile strength and stiffness) were determined in tensile and dynamic mechanical thermal tests. The SEM showed that the interfacial gaps around pulled-out fibers diminished when the interval hot pressing was used. It was also found that both the types of flax composites had a higher impact resistance than the neat PLA. The tensile strength and stiffness of the PLA/flax composites were also markedly higher than those of PLA and reflected the effect of their structures. The storage creep compliance was determined by using the relations of linear viscoelastic materials and the time-temperature superposition principle. The calculated creep response of the flax composites was much lower than that of the neat PLA.

  5. Identification of the PLA2G6 c.1579G>A Missense Mutation in Papillon Dog Neuroaxonal Dystrophy Using Whole Exome Sequencing Analysis

    PubMed Central

    Tsuboi, Masaya; Watanabe, Manabu; Nibe, Kazumi; Yoshimi, Natsuko; Kato, Akihisa; Sakaguchi, Masahiro; Yamato, Osamu; Tanaka, Miyuu; Kuwamura, Mitsuru; Kushida, Kazuya; Harada, Tomoyuki; Chambers, James Kenn; Sugano, Sumio; Uchida, Kazuyuki; Nakayama, Hiroyuki

    2017-01-01

    Whole exome sequencing (WES) has become a common tool for identifying genetic causes of human inherited disorders, and it has also recently been applied to canine genome research. We conducted WES analysis of neuroaxonal dystrophy (NAD), a neurodegenerative disease that sporadically occurs worldwide in Papillon dogs. The disease is considered an autosomal recessive monogenic disease, which is histopathologically characterized by severe axonal swelling, known as “spheroids,” throughout the nervous system. By sequencing all eleven DNA samples from one NAD-affected Papillon dog and her parents, two unrelated NAD-affected Papillon dogs, and six unaffected control Papillon dogs, we identified 10 candidate mutations. Among them, three candidates were determined to be “deleterious” by in silico pathogenesis evaluation. By subsequent massive screening by TaqMan genotyping analysis, only the PLA2G6 c.1579G>A mutation had an association with the presence or absence of the disease, suggesting that it may be a causal mutation of canine NAD. As a human homologue of this gene is a causative gene for infantile neuroaxonal dystrophy, this canine phenotype may serve as a good animal model for human disease. The results of this study also indicate that WES analysis is a powerful tool for exploring canine hereditary diseases, especially in rare monogenic hereditary diseases. PMID:28107443

  6. Bee Venom Phospholipase A2 Alleviate House Dust Mite-Induced Atopic Dermatitis-Like Skin Lesions by the CD206 Mannose Receptor.

    PubMed

    Shin, Dasom; Choi, Won; Bae, Hyunsu

    2018-04-02

    Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by highly pruritic, erythematous, and eczematous skin plaques. We previously reported that phospholipase A2 (PLA2) derived from bee venom alleviates AD-like skin lesions induced by 2,4-dinitrochlorobenzene (DNCB) and house dust mite extract ( Dermatophagoides farinae extract, DFE) in a murine model. However, the underlying mechanisms of PLA2 action in actopic dermatitis remain unclear. In this study, we showed that PLA2 treatment inhibited epidermal thickness, serum immunoglobulin E (IgE) and cytokine levels, macrophage and mast cell infiltration in the ear of an AD model induced by DFE and DNCB. In contrast, these effects were abrogated in CD206 mannose receptor-deficient mice exposed to DFE and DNCB in the ear. These data suggest that bvPLA2 alleviates atopic skin inflammation via interaction with CD206.

  7. 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

  8. 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. © 2014 by The American Society of Hematology.

  9. Antivenomics of Atropoides mexicanus and Atropoides picadoi snake venoms: Relationship to the neutralization of toxic and enzymatic activities

    PubMed Central

    Antúnez, José; Fernández, Julián; Lomonte, Bruno; Angulo, Yamileth; Sanz, Libia; Pérez, Alicia; Calvete, Juan José; Gutiérrez, José María

    2010-01-01

    Viperid snakes of the genus Atropoides are distributed in Mexico and Central America and, owing to their size and venom yield, are capable of provoking severe envenomings in humans. This study evaluated, using an ‘antivenomics’ approach, the ability of a polyspecific (polyvalent) antivenom manufactured in Costa Rica to recognize the proteins of Atropoides mexicanus and A. picadoi venoms, which are not included in the immunization mixture. In addition, the neutralization of lethal, hemorrhagic, myotoxic, coagulant, proteinase and phospholipase A2 (PLA2) activities of these venoms by the antivenom was assessed. The antivenom was highly-effective in immunodepleting many venom components, particularly high molecular mass P-III metalloproteinases (SVMPs), L-amino acid oxidases, and some serine proteinases and P-I SVMPs. In contrast, PLA2s, certain serine proteinases and P-I SVMPs, and a C type lectin-like protein were only partially immunodepleted, and two PLA2 molecules were not depleted at all. The antivenom was able to neutralize all toxic and enzymatic activities tested, although neutralization of lethality by A. nummifer venom was achieved when a challenge dose of 3 LD50s of venom was used, but was iffective when 4 LD50s were used. These results, and previously obtained evidence on the immunoreactivity of this antivenom towards homologous and heterologous venoms, revealed the low immunogenicity of a number of venom components (PLA2s, CRISPs, P-I SVMPs, and some serine proteinases), underscoring the need to search for innovative immunization protocols to improve the immune response to these antigens. PMID:21544177

  10. PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry

    PubMed Central

    Huancahuire-Vega, Salomón; Ponce-Soto, Luis Alberto; Marangoni, Sergio

    2014-01-01

    A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom. PMID:25365526

  11. Antiparasitic effects induced by polyclonal IgY antibodies anti-phospholipase A2 from Bothrops pauloensis venom.

    PubMed

    Borges, Isabela Pacheco; Silva, Mariana Ferreira; Santiago, Fernanda Maria; de Faria, Lucas Silva; Júnior, Álvaro Ferreira; da Silva, Rafaela José; Costa, Mônica Soares; de Freitas, Vitor; Yoneyama, Kelly Aparecida Geraldo; Ferro, Eloísa Amália Vieira; Lopes, Daiana Silva; Rodrigues, Renata Santos; de Melo Rodrigues, Veridiana

    2018-06-01

    Activities of phospholipases (PLAs) have been linked to pathogenesis in various microorganisms, and implicated in cell invasion and so the interest in these enzymes as potential targets that could contribute to the control of parasite survival and proliferation. Chicken eggs immunized with BnSP-7, a Lys49 phospholipase A 2 (PLA 2 ) homologue from Bothrops pauloensis snake venom, represent an excellent source of polyclonal antibodies with potential inhibitory activity on parasite PLA s. Herein, we report the production, characterization and anti-parasitic effect of IgY antibodies from egg yolks of hens immunized with BnSP-7. Produced antibodies presented increasing avidity and affinity for antigenic toxin epitopes throughout immunization, attaining a plateau after 4weeks. Pooled egg yolks-purified anti-BnSP-7 IgY antibodies were able to specifically recognize different PLA 2 s from Bothrops pauloensis and Bothrops jararacussu venom. Antibodies also neutralized BnSP-7 cytotoxic activity in C2C12 cells. Also, the antibodies recognized targets in Leishmania (Leishmania) amazonensis and Toxoplasma gondii extracts by ELISA and immunofluorescence assays. Anti-BnSP-7 IgY antibodies were cytotoxic to T. gondii tachyzoite and L. (L.) amazonensis promastigotes, and were able to decrease proliferation of both parasites treated before infection. These data suggest that the anti-BnSP-7 IgY is an important tool for discovering new parasite targets and blocking parasitic effects. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Crystal structure of Natratoxin, a novel snake secreted phospholipaseA2 neurotoxin from Naja atra venom inhibiting A-type K+ currents.

    PubMed

    Hu, Pu; Sun, Lei; Zhu, Zhi-Qiang; Hou, Xiao-Wei; Wang, Shu; Yu, Shan-Shan; Wang, Hui-Li; Zhang, Ping; Wang, Ming; Niu, Li-Wen; Teng, Mai-Kun; Ruan, Di-Yun

    2008-08-01

    Snake secreted phospholipasesA2 (sPLA2s) are widely used as pharmacological tools to investigate their role in diverse pathophysiological processes. Some members of snake venom sPLA2s have been found to block voltage-activated K(+) channels (K(v) channels). However, most studies involved in their effects on ion channels were indirectly performed on motor nerve terminals while few studies were directly done on native neurons. Here, a novel snake sPLA2 peptide neurotoxin, Natratoxin, composed of 119 amino acid residues and purified from Naja atra venom was reported. It was characterized using whole-cell patch-clamp in acutely dissociated rat dorsal root ganglion (DRG) neurons. It was found to effectively inhibit A-type K(+) currents and cause alterations of channel gating characters, such as the shifts of steady-state activation and inactivation curves to hyperpolarization direction and changes of V(1/2) and slope factor. Therefore, Natratoxin was suggested to be a gating modifier of K(v) channel. In addition, this inhibitory effect was found to be independent of its enzymatic activity. These results suggested that the toxin enacted its inhibitory effect by binding to K(v) channel. To further elucidate the structural basis for this electrophysiological phenomenon, we determined the crystal structure of Natratoxin at 2.2 A resolution by molecular replacement method and refined to an R-factor of 0.190. The observed overall fold has a different structural organization from other K(+) channel inhibitors in animal toxins. Compared with other K(v) channel inhibitors, a similar putative functional surface in its C-terminal was revealed to contribute to protein-protein interaction in such a blocking effect. Our results demonstrated that the spatial distribution of key amino acid residues matters most in the recognition of this toxin towards its channel target rather than its type of fold. (c) 2008 Wiley-Liss, Inc.

  13. A mechanistic approach to the in vivo anti-inflammatory activity of sesquiterpenoid compounds isolated from Inula viscosa.

    PubMed

    Hernández, V; del Carmen Recio, M; Máñez, S; Prieto, J M; Giner, R M; Ríos, J L

    2001-11-01

    The present study was designed to examine the anti-inflammatory activity of the sesquiterpenoids ilicic acid and inuviscolide, isolated from Inula viscosa, on cell degranulation, leukotriene biosynthesis, neurogenic drive and glucocorticoid-like interactions. Swiss female mice were used to measure the ear oedema induced by phorbol esters or ethyl phenylpropiolate (EPP), and the paw oedema induced by phospholipase A(2) (PLA(2)) or serotonin. Drug treatment consisted of one topically-applied dose in the ear models and a subcutaneous or intraperitoneal injection in the paw models. Quantitative analysis of leukotriene B(4) (LTB(4)) formation was performed on rat peritoneal neutrophils by high performance liquid chromatography (HPLC). The lactone inuviscolide reduced the PLA(2)-induced oedema (ID(50): 98 micromol/kg). The effect on serotonin-induced oedema was not changed by modifiers of the glucocorticoid response. Ilicic acid showed minor in vivo effects, but was slightly more potent than inuviscolide on the 12-O-tetradecanoylphorbol 13-acetate (TPA) acute oedema test (ID(50): 0.650 micromol per ear). Inuviscolide reduced LTB(4) generation in intact cells, with an IC(50) value of 94 microM. On the basis of the reported results, inuviscolide is the main anti-inflammatory sesquiterpenoid from Inula viscosa, and may act by interfering with leukotriene synthesis and PLA(2)-induced mastocyte release of inflammatory mediators.

  14. Bee Venom Phospholipase A2 Protects against Acetaminophen-Induced Acute Liver Injury by Modulating Regulatory T Cells and IL-10 in Mice

    PubMed Central

    Kim, Hyunseong; Keum, Dong June; Kwak, Jung won; Chung, Hwan-Suck; Bae, Hyunsu

    2014-01-01

    The aim of this study was to investigate the protective effects of phospholipase A2 (PLA2) from bee venom against acetaminophen-induced hepatotoxicity through CD4+CD25+Foxp3+ T cells (Treg) in mice. Acetaminophen (APAP) is a widely used antipyretic and analgesic, but an acute or cumulative overdose of acetaminophen can cause severe hepatic failure. Tregs have been reported to possess protective effects in various liver diseases and kidney toxicity. We previously found that bee venom strongly increased the Treg population in splenocytes and subsequently suppressed immune disorders. More recently, we found that the effective component of bee venom is PLA2. Thus, we hypothesized that PLA2 could protect against liver injury induced by acetaminophen. To evaluate the hepatoprotective effects of PLA2, C57BL/6 mice or interleukin-10-deficient (IL-10−/−) mice were injected with PLA2 once a day for five days and sacrificed 24 h (h) after acetaminophen injection. The blood sera were collected 0, 6, and 24 h after acetaminophen injection for the analysis of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PLA2-injected mice showed reduced levels of serum AST, ALT, proinflammatory cytokines, and nitric oxide (NO) compared with the PBS-injected control mice. However, IL-10 was significantly increased in the PLA2-injected mice. These hepatic protective effects were abolished in Treg-depleted mice by antibody treatment and in IL-10−/− mice. Based on these findings, it can be concluded that the protective effects of PLA2 against acetaminophen-induced hepatotoxicity can be mediated by modulating the Treg and IL-10 production. PMID:25478691

  15. Bee venom phospholipase A2 protects against acetaminophen-induced acute liver injury by modulating regulatory T cells and IL-10 in mice.

    PubMed

    Kim, Hyunseong; Keum, Dong June; Kwak, Jung won; Chung, Hwan-Suck; Bae, Hyunsu

    2014-01-01

    The aim of this study was to investigate the protective effects of phospholipase A2 (PLA2) from bee venom against acetaminophen-induced hepatotoxicity through CD4+CD25+Foxp3+ T cells (Treg) in mice. Acetaminophen (APAP) is a widely used antipyretic and analgesic, but an acute or cumulative overdose of acetaminophen can cause severe hepatic failure. Tregs have been reported to possess protective effects in various liver diseases and kidney toxicity. We previously found that bee venom strongly increased the Treg population in splenocytes and subsequently suppressed immune disorders. More recently, we found that the effective component of bee venom is PLA2. Thus, we hypothesized that PLA2 could protect against liver injury induced by acetaminophen. To evaluate the hepatoprotective effects of PLA2, C57BL/6 mice or interleukin-10-deficient (IL-10-/-) mice were injected with PLA2 once a day for five days and sacrificed 24 h (h) after acetaminophen injection. The blood sera were collected 0, 6, and 24 h after acetaminophen injection for the analysis of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PLA2-injected mice showed reduced levels of serum AST, ALT, proinflammatory cytokines, and nitric oxide (NO) compared with the PBS-injected control mice. However, IL-10 was significantly increased in the PLA2-injected mice. These hepatic protective effects were abolished in Treg-depleted mice by antibody treatment and in IL-10-/- mice. Based on these findings, it can be concluded that the protective effects of PLA2 against acetaminophen-induced hepatotoxicity can be mediated by modulating the Treg and IL-10 production.

  16. Multiphysics of bone remodeling: A 2D mesoscale activation simulation.

    PubMed

    Spingarn, C; Wagner, D; Rémond, Y; George, D

    2017-01-01

    In this work, we present an evolutive trabecular model for bone remodeling based on a boundary detection algorithm accounting for both biology and applied mechanical forces, known to be an important factor in bone evolution. A finite element (FE) numerical model using the Abaqus/Standard® software was used with a UMAT subroutine to solve the governing coupled mechanical-biological non-linear differential equations of the bone evolution model. The simulations present cell activation on a simplified trabeculae configuration organization with trabecular thickness of 200µm. For this activation process, the results confirm that the trabeculae are mainly oriented in the active direction of the principal mechanical stresses and according to the principal applied mechanical load directions. The trabeculae surface activation is clearly identified and can provide understanding of the different bone cell activations in more complex geometries and load conditions.

  17. The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation.

    PubMed

    Lappas, Courtney M

    2015-01-01

    Cytokinins are plant hormones that play an integral role in multiple aspects of plant growth and development. The biological functions of cytokinins in mammalian systems are, however, largely uncharacterized. The naturally occurring cytokinin zeatin riboside has recently been demonstrated to activate the mammalian adenosine A(2A) receptor, which is broadly expressed by various cell types including immune system cells, with the activation of the A(2A)R playing a role in the regulation of cells involved in both innate and adaptive immunity. We show for the first time that zeatin riboside modulates mammalian immune system activity via an A(2A)R-dependent mechanism. Specifically, zeatin riboside treatment induces the production of cyclic adenosine monophosphate (cAMP) by T lymphocytes and inhibits the production by CD3(+)CD4(+) T cells of interferon (IFN)-γ, IL-2, tumor-necrosis factor (TNF)-α, IL-4 and IL-13, and the production by CD3(+)CD8(+) T cells of IFN-γ, IL-2 and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by zeatin riboside. Zeatin riboside treatment also potently inhibits thioglycollate-induced peritoneal leukocytosis. The immunomodulatory activities of zeatin riboside are blocked by co-treatment with the selective A(2A)R antagonist ZM241385. These data suggest that zeatin riboside possesses therapeutic potential as a mammalian immunomodulatory agent.

  18. A novel synthetic peptide inspired on Lys49 phospholipase A2 from Crotalus oreganus abyssus snake venom active against multidrug-resistant clinical isolates.

    PubMed

    Almeida, José R; Mendes, Bruno; Lancellotti, Marcelo; Marangoni, Sergio; Vale, Nuno; Passos, Óscar; Ramos, Maria J; Fernandes, Pedro A; Gomes, Paula; Da Silva, Saulo L

    2018-04-10

    Currently, the evolving and complex mechanisms of bacterial resistance to conventional antibiotics are increasing, while alternative medicines are drying up, which urges the need to discover novel agents able to kill antibiotic-resistant bacteria. Lys49 phospholipase A 2 s (PLA 2 s) from snake venoms are multifunctional toxins able to induce a huge variety of therapeutic effects and consequently serve as templates for new drug leads. Hence, the present study was aimed at the synthesis of oligopeptides mimicking regions of the antibacterial Lys49 PLA 2 toxin (CoaTx-II), recently isolated from Crotalus oreganus abyssus snake venom, to identify small peptides able to reproduce the therapeutic action of the toxin. Five peptides, representing major regions of interest within CoaTx-II, were synthesized and screened for their antibacterial properties. The 13-mer peptide pC-CoaTxII, corresponding to residues 115-129 of CoaTx-II, was able to reproduce the promising bactericidal effect of the toxin against multi-resistant clinical isolates. Peptide pC-CoaTxII is mainly composed by positively charged and hydrophobic amino acids, a typical trait in most antimicrobial peptides, and presented no defined secondary structure in aqueous environment. The physicochemical properties of pC-CoaTxII are favorable towards a strong interaction with anionic lipid membranes as those in bacteria. Additional in silico studies suggest formation of a water channel across the membrane upon peptide insertion, eventually leading to bacterial cell disruption and death. Overall, our findings confirm the valuable potential of snake venom toxins towards design and synthesis of novel antimicrobials, thus representing key insights towards development of alternative efficient antimicrobials to fight bacterial resistance to current antibiotics. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  19. Large Particles in Active Asteroid P/2010 A2

    NASA Astrophysics Data System (ADS)

    Jewitt, David; Ishiguro, Masateru; Agarwal, Jessica

    2013-02-01

    The previously unknown asteroid P/2010 A2 rose to prominence in 2010 by forming a transient, comet-like tail consisting of ejected dust. The observed dust production was interpreted as the result of either a hypervelocity impact with a smaller body or a rotational disruption. We have re-observed this object, finding that large particles remain a full orbital period after the initial outburst. In the intervening years, particles smaller than ~3 mm in radius have been dispersed by radiation pressure, leaving only larger particles in the trail. Since the total mass is dominated by the largest particles, the radiation pressure filtering allows us to obtain a more reliable estimate of the debris mass than was previously possible. We find that the mass contained in the debris is ~5 × 108 kg (assumed density 3000 kg m-3), the ratio of the total debris mass to the nucleus mass is ~0.1, and that events like P/2010 A2 contribute <3% to the Zodiacal dust production rate. Physical properties of the nucleus and debris are also determined. The data presented herein were obtained at the W. M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California, and the National Aeronautics and Space Administration. The Observatory was made possible by the generous financial support of the W. M. Keck Foundation.

  20. LARGE PARTICLES IN ACTIVE ASTEROID P/2010 A2

    SciTech Connect

    Jewitt, David; Ishiguro, Masateru; Agarwal, Jessica, E-mail: jewitt@ucla.edu

    2013-02-10

    The previously unknown asteroid P/2010 A2 rose to prominence in 2010 by forming a transient, comet-like tail consisting of ejected dust. The observed dust production was interpreted as the result of either a hypervelocity impact with a smaller body or a rotational disruption. We have re-observed this object, finding that large particles remain a full orbital period after the initial outburst. In the intervening years, particles smaller than {approx}3 mm in radius have been dispersed by radiation pressure, leaving only larger particles in the trail. Since the total mass is dominated by the largest particles, the radiation pressure filtering allowsmore » us to obtain a more reliable estimate of the debris mass than was previously possible. We find that the mass contained in the debris is {approx}5 Multiplication-Sign 10{sup 8} kg (assumed density 3000 kg m{sup -3}), the ratio of the total debris mass to the nucleus mass is {approx}0.1, and that events like P/2010 A2 contribute <3% to the Zodiacal dust production rate. Physical properties of the nucleus and debris are also determined.« less

  1. In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity.

    PubMed

    Pan, Yan; Tiong, Kai Hung; Abd-Rashid, Badrul Amini; Ismail, Zakiah; Ismail, Rusli; Mak, Joon Wah; Ong, Chin Eng

    2014-10-15

    This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC50 values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC50 values of 50.8 and 40.2 μM, while the other active compounds did not significantly affect CYP1A2 activity with IC50 values more than 100 μM. Ki values further determined for eupatorin and sinensetin were 46.4 and 35.2 μM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations. Copyright © 2014 Elsevier GmbH. All rights reserved.

  2. A Lys49 Phospholipase A2, Isolated from Bothrops asper Snake Venom, Induces Lipid Droplet Formation in Macrophages Which Depends on Distinct Signaling Pathways and the C-Terminal Region

    PubMed Central

    Cristina Giannotti, Karina; Leiguez, Elbio; Moreira, Vanessa; Nascimento, Neide Galvão; Lomonte, Bruno; Gutiérrez, José Maria; Lopes de Melo, Robson; Teixeira, Catarina

    2013-01-01

    MT-II, a Lys49PLA2 homologue devoid of catalytic activity from B. asper venom, stimulates inflammatory events in macrophages. We investigated the ability of MT-II to induce formation of lipid droplets (LDs), key elements of inflammatory responses, in isolated macrophages and participation of protein kinases and intracellular PLA2s in this effect. Influence of MT-II on PLIN2 recruitment and expression was assessed, and the effects of some synthetic peptides on LD formation were further evaluated. At noncytotoxic concentrations, MT-II directly activated macrophages to form LDs. This effect was reproduced by a synthetic peptide corresponding to the C-terminal sequence 115–129 of MT-II, evidencing the critical role of C-terminus for MT-II-induced effect. Moreover, MT-II induced expression and recruitment of PLIN2. Pharmacological interventions with specific inhibitors showed that PKC, PI3K, ERK1/2, and iPLA2, but not P38MAPK or cPLA2, signaling pathways are involved in LD formation induced by MT-II. This sPLA2 homologue also induced synthesis of PGE2 that colocalized to LDs. In conclusion, MT-II is able to induce formation of LDs committed to PGE2 formation in a process dependent on C-terminal loop engagement and regulated by distinct protein kinases and iPLA2. LDs may constitute an important inflammatory mechanism triggered by MT-II in macrophages. PMID:23509782

  3. A Lys49 phospholipase A2, isolated from Bothrops asper snake venom, induces lipid droplet formation in macrophages which depends on distinct signaling pathways and the C-terminal region.

    PubMed

    Giannotti, Karina Cristina; Leiguez, Elbio; Moreira, Vanessa; Nascimento, Neide Galvão; Lomonte, Bruno; Gutiérrez, José Maria; Lopes de Melo, Robson; Teixeira, Catarina

    2013-01-01

    MT-II, a Lys49PLA2 homologue devoid of catalytic activity from B. asper venom, stimulates inflammatory events in macrophages. We investigated the ability of MT-II to induce formation of lipid droplets (LDs), key elements of inflammatory responses, in isolated macrophages and participation of protein kinases and intracellular PLA2s in this effect. Influence of MT-II on PLIN2 recruitment and expression was assessed, and the effects of some synthetic peptides on LD formation were further evaluated. At noncytotoxic concentrations, MT-II directly activated macrophages to form LDs. This effect was reproduced by a synthetic peptide corresponding to the C-terminal sequence 115-129 of MT-II, evidencing the critical role of C-terminus for MT-II-induced effect. Moreover, MT-II induced expression and recruitment of PLIN2. Pharmacological interventions with specific inhibitors showed that PKC, PI3K, ERK1/2, and iPLA2, but not P38(MAPK) or cPLA2, signaling pathways are involved in LD formation induced by MT-II. This sPLA2 homologue also induced synthesis of PGE2 that colocalized to LDs. In conclusion, MT-II is able to induce formation of LDs committed to PGE2 formation in a process dependent on C-terminal loop engagement and regulated by distinct protein kinases and iPLA2. LDs may constitute an important inflammatory mechanism triggered by MT-II in macrophages.

  4. 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

  5. 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.

  6. Mechanisms involved in hemoglobin-mediated oxidation of lipids in washed fish muscle and inhibitory effects of phospholipase A2.

    PubMed

    Tatiyaborworntham, Nantawat; Richards, Mark P

    2017-11-13

    Hemoglobin (Hb) is a lipid oxidation promoter in fish muscle. Phospholipase A2 (PLA2; EC 3.1.1.4) is linked to an increased resistance to lipid oxidation of frozen-thawed cod fillets via an unknown mechanism. The present study aimed to investigate the mechanism of Hb-mediated lipid oxidation with a focus on ferryl Hb and methemoglobin (metHb), the pro-oxidative Hb species, and to examine how porcine pancreatic PLA2 inhibits Hb-mediated lipid oxidation in washed cod muscle (WCM). Lipid hydroperoxides (LOOHs) and thiobarbituric acid reactive substances (TBARS) were measured as primary and secondary lipid oxidation products, respectively. The formation of metHb and ferryl Hb was also monitored. Ferryl Hb and metHb formed during the Hb-mediated lipid oxidation. PLA2 inhibited the formation of LOOHs and TBARS and suppressed the formation of metHb and ferryl Hb. WCM was pre-oxidized by hemin to increase the amount of LOOHs. PLA2 promoted the depletion of LOOHs in the pre-oxidized WCM with limited TBARS formation at the expense of the heme moiety of Hb. The results of the present study suggest that ferryl Hb may play a role in Hb-mediated lipid oxidation and that PLA2 from pig pancreas may work together with Hb as a novel antioxidant with an ability to remove pre-formed LOOHs from a lipid substrate. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  7. [Application of Immunohistochemistry and Immunofluorescence Staining in Detection of Phospholipase A2 Receptor on Paraffin Section of Renal Biopsy Tissue].

    PubMed

    Dong, Hong-rui; Wang, Yan-yan; Wang, Guo-qin; Sun, Li-jun; Cheng, Hong; Chen, Yi-pu

    2015-10-01

    To evaluate the application of immunohistochemistry and fluorescence staining method in the detection of phospholipase A2 receptor (PLA2R) on paraffin section of renal biopsy tissue,and to find an accurate and fast method for the detection of PLA2R in renal tissue. The PLA2R of 193 cases were detected by immunohistochemical staining,and the antigen was repaired by the method of high pressure cooker (HPC) hot repair plus trypsin repair. The 193 samples including 139 cases of idiopathic membranous nephropathy (IMN), 15 cases of membranous lupus nephritis, 8 cases of hepatitis B virus associated membranous nephropathy, 18 cases of IgA nephropathy, and 13 cases of minimal change diseases. To compare the dyeing effects, 22 paraffin sections of renal biopsy tissue of IMN cases with positive PLA2R were stained by using 4 different. of antigen repairing,which included HPC hot repair, HPC hot repair plus trypsin repair, water bath heat repair, and water bath heat repair plus trypsin repair. To compare the dyeing effects, 15 paraffin sections of renal biopsy tissue of IMN cases with positive PLA2R were stained by using 3 different. of antigen repairing,which included water bath heat repair plus trypsin repair, protease K digestion repair, and pepsin digestion repair. In 193 cases, the positive rate of PLA2R in IMN cases was 90.6% (126/139), and the other 54 patients without IMN were negative. Twenty-two IMN patients were positive for PLA2R by using the HPC heat repair plus trypsin repaire or the water bath heat repair plus trypsin repair;while only a few cases of 22 IMN cases were positive by using the HPC hot repair alone or water bath heat repair alone. Fifteen IMN patients were positive for PLA2R by using water bath heat repair plus trypsin repair,protease K digestion repair,and pepsin digestion repair, but the distribution of positive deposits and the background were different. PLA2R immunohistochemical staining can effectively identify IMN and secondary MN. For

  8. 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...

  9. PLA2 polymorphism of platelet glycoprotein IIb/IIIa but not Factor V Leiden and prothrombin G20210A polymorphisms is associated with venous thromboembolism and more recurrent events in central Iran.

    PubMed

    Pourgheysari, Batoul; Boroujeni, Hamid Rouhi; Hasheminia, Ali Mohammad; Drees, Fatima

    2013-07-01

    Inherited thrombophilic gene polymorphisms have been linked to the pathogenesis of venous thromboembolism (VTE). As there are very limited data of these polymorphisms in the Iranian population, we aimed to investigate the correlation between them and VTE in central Iran. Seventy-two unrelated VTE patients and 306 healthy control individuals were recruited for the study. Genotyping from venous blood with EDTA for the factor V Leiden (FVL), prothrombin (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and PLA2 polymorphism of platelet glycoprotein IIb/IIIa were undertaken by PCR-restriction fragment length polymorphism (PCR-RFLP). A total of 57 investigated polymorphisms with a mean of 0.79 per individual and 151 with a mean of 0.49 were found in patients and controls, respectively (P<0.001). FVL and FII G20210A were found, respectively, in 5.6 and 1.4% of the patients compared with 2.3 and 1% of the controls (P=NS). PLA2 polymorphism of GPIIb/IIIa was seen in 27.8 and 10.1% in patients and controls, respectively [odds ratio (OR), 3.4; 95% confidence interval (CI), 1.08-6.44, P<0.001]. Approximately 15.3% of VTE patients compared with 5.9% of controls had coinheritance of more than one genetic risk factor (P=0.007) and more recurrent events occurred in such patients. Patients with PLA2 polymorphism had more recurrent events than the other patients (P=0.02). Patients with more than one genetic risk factor and recurrent events were younger. The prevalence of these polymorphisms is different from some previously published data in other populations, but is consistent with some others. Higher prevalence of PLA2 polymorphism of GPIIa/IIIb in VTE patients is indicative of the impact of this polymorphism in the pathogenesis of VTE in this population. Because of the impact of coinheritance on the recurrence and the age of occurrence, such patients may need to be managed differently.

  10. Neuromuscular paralysis by the basic phospholipase A2 subunit of crotoxin from Crotalus durissus terrificus snake venom needs its acid chaperone to concurrently inhibit acetylcholine release and produce muscle blockage.

    PubMed

    Cavalcante, Walter L G; Noronha-Matos, José B; Timóteo, Maria A; Fontes, Marcos R M; Gallacci, Márcia; Correia-de-Sá, Paulo

    2017-11-01

    Crotoxin (CTX), a heterodimeric phospholipase A 2 (PLA 2 ) neurotoxin from Crotalus durissus terrificus snake venom, promotes irreversible blockade of neuromuscular transmission. Indirect electrophysiological evidence suggests that CTX exerts a primary inhibitory action on transmitter exocytosis, yet contribution of a postsynaptic action of the toxin resulting from nicotinic receptor desensitization cannot be excluded. Here, we examined the blocking effect of CTX on nerve-evoked transmitter release measured directly using radioisotope neurochemistry and video microscopy with the FM4-64 fluorescent dye. Experiments were conducted using mice phrenic-diaphragm preparations. Real-time fluorescence video microscopy and liquid scintillation spectrometry techniques were used to detect transmitter exocytosis and nerve-evoked [ 3 H]-acetylcholine ([ 3 H]ACh) release, respectively. Nerve-evoked myographic recordings were also carried out for comparison purposes. Both CTX (5μg/mL) and its basic PLA 2 subunit (CB, 20μg/mL) had biphasic effects on nerve-evoked transmitter exocytosis characterized by a transient initial facilitation followed by a sustained decay. CTX and CB reduced nerve-evoked [ 3 H]ACh release by 60% and 69%, respectively, but only the heterodimer, CTX, decreased the amplitude of nerve-evoked muscle twitches. Data show that CTX exerts a presynaptic inhibitory action on ACh release that is highly dependent on its intrinsic PLA 2 activity. Given the high safety margin of the neuromuscular transmission, one may argue that the presynaptic block caused by the toxin is not enough to produce muscle paralysis unless a concurrent postsynaptic inhibitory action is also exerted by the CTX heterodimer. Copyright © 2017. Published by Elsevier Inc.

  11. Group X secretory phospholipase A2 augments Angiotensin II-induced inflammatory responses and abdominal aortic aneurysm formation in apoE-deficient mice

    PubMed Central

    Zack, Melissa; Boyanovsky, Boris B.; Shridas, Preetha; Bailey, William; Forrest, Kathy; Howatt, Deborah A.; Gelb, Michael H.; de Beer, Frederick C.; Daugherty, Alan; Webb, Nancy R.

    2010-01-01

    Objective Abdominal aortic aneurysm (AAA) is a complex vascular disease characterized by matrix degradation and inflammation and is a major cause of mortality in older men. Specific interventions that prevent AAA progression remain to be identified. In this study, we tested the hypothesis that Group X secretory phospholipase A2 (GX sPLA2), an enzyme implicated in inflammatory processes, mediates AAA. Methods and Results GX sPLA2 was detected by immunostaining in human aneurysmal tissue and in angiotensin II (Ang II)-induced AAAs in apolipoprotein E-deficient (apoE−/−) mice. GX sPLA2 mRNA was increased significantly (11-fold) in abdominal aortas of apoE−/− mice in response to Ang II infusion. To define the role of GX sPLA2 in experimental AAAs, apoE−/− and apoE−/− × GX sPLA2−/− (GX DKO) mice were infused with Ang II for either 10 (n=7) or 28 (n=24–26) days. Deficiency of GX sPLA2 significantly reduced the incidence and severity of AAAs, as assessed by ultrasound measurements in vivo of aortic lumens and by computer-assisted morphometric analyses ex vivo of external diameter. Results from gene expression profiling indicated that the expression of specific matrix metalloproteinases and inflammatory mediators was blunted in aortas from GX DKO mice compared to apoE−/− mice after 10-day Ang II infusion. Ang II induction of cyclooxygenase-2, interleukin-6, matrix metalloproteinase (MMP)-2, MMP-13 and MMP-14 was reduced significantly in GX DKO mice compared to apoE−/− mice. Conclusion GX sPLA2 promotes Ang II-induced pathological responses leading to AAA formation. PMID:20833395

  12. Lipoprotein-associated phospholipase A2 and oxidized low-density lipoprotein in young patients with acute coronary syndrome in China.

    PubMed

    Huang, Yuli; Wu, Yu; Yang, You; Li, Wensheng; Lu, Jianhua; Hu, Yunzhao

    2017-11-23

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is considered to be a risk factor for acute coronary syndrome (ACS), but this remains controversial. This study investigated the role of Lp-PLA2 in young Chinese patients with ACS. 228 young patients (aged ≤55 years) with ACS and 237 age-matched controls were included. Lp-PLA2 and oxidized low-density lipoprotein (ox-LDL) levels were measured by sandwich enzyme-linked immunosorbent assay. Lp-PLA2 levels were significantly correlated with smoking, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and ox-LDL levels (all P < 0.05). Multivariate logistic regression analysis showed that male sex (OR = 3.25, 95%CI = 1.26-8.38), smoking (OR = 3.50, 95%CI = 1.75-7.0), triglyceride (OR = 1.76, 95%CI = 1.08-2.87), high sensitivity C-reactive protein (hs-CRP) (OR = 2.11, 95%CI = 1.14-3.90) and ox-LDL (OR = 2.98, 95%CI = 1.72-5.1) were independently associated with ACS risk in young patients. Lp-PLA2 was associated with risk of ACS in young patients when adjusted for traditional risk factors, including age, sex, diabetes, hypertension, smoking, TC, LDL-C, triglyceride and hs-CRP (OR = 1.98, 95%CI = 1.10-3.56). When further adjusted for ox-LDL levels, the association between Lp-PLA2 and ACS became insignificant (OR = 1.69, 95%CI = 0.90-3.17). Lp-PLA2 was a marker of oxidative stress and inflammation, rather than an independent risk factor for ACS in young Chinese patients.

  13. Measuring temperature-dependent activation energy in thermally activated processes: a 2D Arrhenius plot method.

    PubMed

    Li, Jian V; Johnston, Steven W; Yan, Yanfa; Levi, Dean H

    2010-03-01

    Thermally activated processes are characterized by two key quantities, activation energy (E(a)) and pre-exponential factor (nu(0)), which may be temperature dependent. The accurate measurement of E(a), nu(0), and their temperature dependence is critical for understanding the thermal activation mechanisms of non-Arrhenius processes. However, the classic 1D Arrhenius plot-based methods cannot unambiguously measure E(a), nu(0), and their temperature dependence due to the mathematical impossibility of resolving two unknown 1D arrays from one 1D experimental data array. Here, we propose a 2D Arrhenius plot method to solve this fundamental problem. Our approach measures E(a) at any temperature from matching the first and second moments of the data calculated with respect to temperature and rate in the 2D temperature-rate plane, and therefore is able to unambiguously solve E(a), nu(0), and their temperature dependence. The case study of deep level emission in a Cu(In,Ga)Se(2) solar cell using the 2D Arrhenius plot method reveals clear temperature dependent behavior of E(a) and nu(0), which has not been observable by its 1D predecessors.

  14. Effect of different alcohols on stratum corneum kallikrein 5 and phospholipase A2 together with epidermal keratinocytes and skin irritation.

    PubMed

    Cartner, T; Brand, N; Tian, K; Saud, A; Carr, T; Stapleton, P; Lane, M E; Rawlings, A V

    2017-04-01

    The aim of this exploratory study was to investigate the effect of ethanol, isopropanol and n-propanol on stratum corneum (SC) enzymes and keratinocytes in vitro together with their effects on skin condition and function. Activities of kallikrein 5 (KLK5) and phospholipase A2 (PLA2) as well as keratinocyte metabolic activity, interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α) were measured in vitro in the presence and absence of the different alcohols. We also measured transepidermal water loss (TEWL), skin capacitance, visual dryness and visual redness on the volar forearms of 25 Caucasian women following application of the alcohols 20 and 100 times per day over a period of 14 days in a clinical study. Reduced activities of KLK5 and PLA2 were observed in the presence of the alcohols. The greatest denaturing effect was always observed for n-propanol (P < 0.001), and in the case of PLA2, the effect of isopropanol was greater than ethanol (P < 0.001). Equally, ethanol had the mildest effects on keratinocyte metabolic activity and cytokine secretion (P < 0.001) and n-propanol always produced the most severe changes in normal and differentiated keratinocytes. These in vitro findings supported the clinical results where the major effects were on the induction of skin irritation (increased dropout rates) and ranked the intolerance of the different alcohols as follows: n-propanol > isopropanol > ethanol. At the high application frequencies, the effect of the different alcohols on transepidermal water loss (TEWL) and skin capacitance was similar, but at the low application frequencies, n-propanol had a significant effect on TEWL and capacitance values (P < 0.05). Equally, n-propanol and isopropanol produced significantly more skin redness at the low application frequencies. Clearly, isopropanol and n-propanol caused significant SC and keratinocyte perturbation in vitro together with damage to skin condition and function in vivo whereas ethanol

  15. Role for calcium signaling and arachidonic acid metabolites in the activity-dependent increase of AHP amplitude in leech T sensory neurons.

    PubMed

    Scuri, Rossana; Mozzachiodi, Riccardo; Brunelli, Marcello

    2005-08-01

    Previous studies have revealed a new form of activity-dependent modulation of the afterhyperpolarization (AHP) in tactile (T) neurons of the leech Hirudo medicinalis. The firing of T cells is characterized by an AHP, which is mainly due to the activity of the Na+/K+ ATPase. Low-frequency repetitive stimulation of T neurons leads to a robust increment of the AHP amplitude, which is correlated with a synaptic depression between T neuron and follower cells. In the present study, we explored the molecular cascades underlying the AHP increase. We tested the hypothesis that this activity-dependent phenomenon was triggered by calcium influx during neural activity by applying blockers of voltage-dependent Ca2+ channels. We report that AHP increase requires calcium influx that, in turn, induces release of calcium from intracellular stores so sustaining the enhancement of AHP. An elevation of the intracellular calcium can activate the cytosolic isoforms of the phosholipase A2 (PLA2). Therefore we analyzed the role of PLA2 in the increase of the AHP, and we provide evidence that not only PLA2 but also the recruitment of arachidonic acid metabolites generated by the 5-lipoxygenase pathway are necessary for the induction of AHP increase. These data indicate that a sophisticated cascade of intracellular signals links the repetitive discharge of T neurons to the activation of molecular pathways, which finally may alter the activity of critical enzymes such as the Na+/K+ ATPase, that sustains the generation of the AHP and its increase during repetitive stimulation. These results also suggest the potential importance of the poorly studied 5-lipoxygenase pathway in forms of neuronal plasticity.

  16. Cell adhesion and EGFR activation regulate EphA2 expression in cancer.

    PubMed

    Larsen, Alice Bjerregaard; Stockhausen, Marie-Thérése; Poulsen, Hans Skovgaard

    2010-04-01

    EphA2 is frequently overexpressed in cancer, and increasing amounts of evidence show that EphA2 contributes to multiple aspects of the malignant character including angiogenesis and metastasis. Several aspects of the regulation and functional significance of EphA2 expression in cancer are still largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src family kinases (SRC). Moreover, the results show that adhesion-induced EGFR activation and EphA2 expression is affected by interactions with extracellular matrix (ECM) proteins working as integrin ligands. Stimulation with the EphA2 ligand, ephrinA1 inhibited ERK phosphorylation and cancer cell viability. These effects were however abolished by activation of the EGF-receptor ligand system favoring Ras/MAPK signaling and cell proliferation. Based on our results, we propose a regulatory mechanism where cell adhesion induces EGFR kinase activation and EphA2 expression; and where the effect of ephrinA1 mediated reduction in cell viability by inhibiting EphA2 expression is overruled by activated EGFR in human cancer cells. Copyright 2009 Elsevier Inc. All rights reserved.

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

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  18. A role of the SAM domain in EphA2 receptor activation

    PubMed Central

    Shi, Xiaojun; Hapiak, Vera; Zheng, Ji; Muller-Greven, Jeannine; Bowman, Deanna; Lingerak, Ryan; Buck, Matthias; Wang, Bing-Cheng; Smith, Adam W.

    2017-01-01

    Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization. PMID:28338017

  19. A role of the SAM domain in EphA2 receptor activation.

    PubMed

    Shi, Xiaojun; Hapiak, Vera; Zheng, Ji; Muller-Greven, Jeannine; Bowman, Deanna; Lingerak, Ryan; Buck, Matthias; Wang, Bing-Cheng; Smith, Adam W

    2017-03-24

    Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.

  20. Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants.

    PubMed

    Berg, O G; Yu, B Z; Rogers, J; Jain, M K

    1991-07-23

    Hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) by pig pancreatic phospholipase A2 (PLA2) occurs in a highly processive "scooting" mode, and the rate is comparable to or exceeds the rates observed with detergent-dispersed mixed micelles under optimal conditions. A complete kinetic description of the steady-state time course of the hydrolysis is developed. The analysis covers the whole Michaelis-Menten space: it emphasizes the key features of interfacial catalysis by a detailed theoretical analysis, describes t