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Sample records for a2 pla2 inhibitors

  1. Discovery of Potent and Orally Active Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitors as a Potential Therapy for Diabetic Macular Edema.

    PubMed

    Chen, Xinde; Wang, Kai; Xu, Wenwei; Ma, Quanxin; Chen, Minli; Du, Lili; Mo, Mingguang; Wang, Yiping; Shen, Jianhua

    2016-03-24

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is considered to be a promising therapeutic target for several inflammation-associated diseases. Herein, we describe the discovery of a series of pyrimidone derivatives as Lp-PLA2 inhibitors. Systematic structural modifications led to the identification of several pyrimidone compounds with promising in vitro inhibitory potency and pharmacokinetic properties. Compound 14c, selected for in vivo evaluation, demonstrated decent pharmacokinetic profiles and robust inhibitory potency against Lp-PLA2 in Sprague-Dawley (SD) rats. Furthermore, 14c significantly inhibited retinal thickening in STZ-induced diabetic SD rats as a model of diabetic macular edema (DME) after oral dosing for 4 weeks. Taken together, these results suggested that 14c can serve as a valuable lead in the search for new Lp-PLA2 inhibitors for prevention and/or treatment of DME.

  2. 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

  3. Fragment-Based Approach to the Development of an Orally Bioavailable Lactam Inhibitor of Lipoprotein-Associated Phospholipase A2 (Lp-PLA2).

    PubMed

    Woolford, Alison J-A; Day, Philip J; Bénéton, Véronique; Berdini, Valerio; Coyle, Joseph E; Dudit, Yann; Grondin, Pascal; Huet, Pascal; Lee, Lydia Y W; Manas, Eric S; McMenamin, Rachel L; Murray, Christopher W; Page, Lee W; Patel, Vipulkumar K; Potvain, Florent; Rich, Sharna J; Sang, Yingxia; Somers, Don O; Trottet, Lionel; Wan, Zehong; Zhang, Xiaomin

    2016-12-08

    Lp-PLA2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA2. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile.

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

    PubMed Central

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

    2009-01-01

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

  5. Anti-Phospholipase A2 Receptor (PLA2R) Antibody and Glomerular PLA2R Expression in Japanese Patients with Membranous Nephropathy

    PubMed Central

    Tachibana, Shohei; Iseri, Ken; Saito, Tomohiro; Yamamoto, Yasutaka; Suzuki, Taihei; Wada, Yukihiro; Matsumoto, Kei; Shibata, Takanori

    2016-01-01

    The phospholipase A2 receptor (PLA2R) is the major target antigen (Ag) in idiopathic membranous nephropathy (IMN). Recently, several types of immunoassay systems for anti-PLA2R antibody (Ab) have been developed. However, the correlation of serum anti-PLA2R Abs and glomerular expression of PLA2R Ag, and their association with clinicopathological characteristics have yet to be proven in Japanese patients. We examined serum anti-PLA2R Abs by both ELISA and cell-based indirect immunofluorescence assay (CIIFA), and glomerular PLA2R expression by immunofluorescence (IF) in 59 biopsy-proven MN patients including IMN (n = 38) and secondary MN (SMN) (n = 21). In this study, anti-PLA2R Abs were present in 50% of IMN patients, but was absent in SMN patients. The concordance rate between ELISA and CIIFA was 100%. Serum IgG levels were significantly lower in anti-PLA2R Ab-positive patients. Serum albumin levels correlated inversely with serum anti-PLA2R Ab titers. The prevalence and intensity of glomerular staining for IgG4 by IF were significantly higher in anti-PLA2R Ab-positive patients than in -negative patients. Glomerular PLA2 Ag expression evaluated by IF was positive in 52.6% of IMN patients, but was absent in SMN patients. The concordance rate between the prevalence of glomerular PLA2R Ag expression and anti-PLA2R Ab was 84.2%. The prevalence of anti-PLA2R Abs measured by ELISA/CIIFA was equivalent to previous Japanese studies evaluated using Western blotting. These analyses showed an excellent specificity for the diagnosis of IMN, and anti-PLA2R positivity was associated with some clinicopathological features, especially glomerular IgG4-dominant deposition. PMID:27355365

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-28

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

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

    PubMed

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

    2014-10-22

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

  9. 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 of 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.

  10. Platelet PlA2 Polymorphism and the risk for thrombosis in heparin-induced thrombocytopenia.

    PubMed

    Harris, Kenneth; Nguyen, Phan; Van Cott, Elizabeth M

    2008-02-01

    Platelet glycoprotein (GP) IIb/IIIa has an important role in platelet aggregation. A polymorphism of platelet GPIIIa (PlA2, also called HPA1b) has been associated with a higher risk of thrombosis, but its implication in heparin-induced thrombocytopenia (HIT) is unclear. To investigate the hypothesis that the PlA2 polymorphism influences the prothrombotic effects of HIT, we conducted a prospective study of 66 consecutive patients with a laboratory diagnosis of HIT. The end point of the study was the diagnosis of a thrombus within 30 days of the positive HIT test result. The Diagnostica Stago (Asnières, France) enzyme-linked immunosorbent assay was used to detect HIT antibodies, and a polymerase chain reaction assay was used to detect the PlA2 polymorphism. Of the 66 patients, thrombotic complications developed in 27 (41%). Patients with the PlA2 allele demonstrated a significantly higher thrombosis risk than did patients without (69% vs 32%; P = .0088; odds ratio, 4.68; 95% confidence interval, 1.39-15.72). The risk was stronger for arterial thrombosis and for patients 60 years or older. There was a significant association between the PlA2 polymorphism of GPIIIa and the risk of thrombosis in patients with HIT antibodies.

  11. Differential hydrolysis of erythrocyte and mitochondrial membrane phospholipids by two phospholipase A2 isoenzymes (NK-PLA2-I and NK-PLA2-II) from the venom of the Indian monocled cobra Naja kaouthia.

    PubMed

    Doley, Robin; King, Glenn F; Mukherjee, Ashis K

    2004-05-01

    We previously demonstrated that venom from the Indian monocled cobra Naja kaouthia is a rich source of phospholipase A2 enzymes, and we purified and characterized a major PLA2 isoenzyme (NK-PLA2-I) from N. kaouthia venom. In the present study, we report the purification and biochemical characterization of a second PLA2 isoenzyme (NK-PLA2-II) from the same venom. A comparison of the membrane phospholipid hydrolysis patterns by these two PLA2s has revealed that they cause significantly more damage to mitochondrial membranes (NK-PLA2-I > NK-PLA2-II) as compared to erythrocyte membranes due to more efficient binding of the enzymes to mitochondrial membranes. Fatty acid release patterns by these PLA2s from the membrane phospholipid PC-pools indicate that NK-PLA2-I does not discriminate between saturated and unsaturated fatty acids whereas NK-PLA2-II shows a preference for unsaturated fatty acids during the initial phase of attack. The current investigation provides new insight into the molecular arrangement of NK-PLA2-sensitive domains in erythrocyte and mitochondrial membranes and highlights the contribution of polar, but uncharged, amino acids such as serine and cysteine in NK-PLA2 induced membrane damage.

  12. Activation of mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) by divalent cations mediating arachidonate release and production of downstream eicosanoids.

    PubMed

    Moon, Sung Ho; Jenkins, Christopher M; Liu, Xinping; Guan, Shaoping; Mancuso, David J; Gross, Richard W

    2012-04-27

    Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)β selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ∼20 μm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl

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

    PubMed

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

    2016-06-09

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

  14. Polymorphisms in PLA2G6 and PLA2G4C genes for calcium-independent phospholipase A2 do not contribute to attenuated niacin skin flush response in schizophrenia patients.

    PubMed

    Nadalin, S; Radović, I; Buretić-Tomljanović, A

    2015-09-01

    We hypothesized that attenuated niacin skin flushing in schizophrenia patients might be associated with polymorphic variants in PLA2G6 and PLA2G4C genes (rs4375 and rs1549637 variations) which encode calcium-independent phospholipase A2 beta (iPLA2β) and cytosolic phospholipase A2 gamma (cPLA2γ) enzymes. The iPLA2β and cPLA2γ may play an important role in niacin-mediated signaling; in addition to their major role - mediating phospholipids remodeling, which alters membrane receptors and signal transduction, they regulate the reservoir of arachidonic acid for prostaglandins synthesis. Skin response to topical niacin of 0.1M, 0.01M, 0.001M and 0.0001M concentrations in 75 schizophrenia patients was rated using the method of volumetric niacin response (VNR). Neither PLA2G6 nor PLA2G4C gene polymorphisms were significantly associated with VNR values. Furthermore, polymorphisms׳ synergy on niacin skin flushing was also not detected.

  15. Assembly of an Intact Golgi Complex Requires Phospholipase A2 (PLA2) Activity, Membrane Tubules, and Dynein-Mediated Microtubule Transport

    PubMed Central

    Judson, Bret L.; Brown, William J.

    2009-01-01

    Previous studies have shown that treatment of mammalian cells with phospholipase A2 (PLA2) antagonists cause the normally interconnected Golgi ribbon to break up into large fragments of stacked Golgi cisternae (“mini-stacks”) that remain located in the juxtanuclear region. Using the reversible PLA2 antagonist, ONO-RS-082 (ONO) and live-cell, time-lapse microscopy to image the Golgi reassembly process, we found that Golgi mini-stacks underwent a burst of membrane tubule formation following washout of ONO: before washout only 4.3 ± 3.8 tubules/cell/10 min were formed, whereas after washout 29.9 ± 11.9 tubules/cell/10 min formed. These membranes tubules formed bridges between physically separate mini-stacks, thus mediating their coalescence into intact Golgi ribbons. Formation of inter-stack tubules and an intact Golgi ribbon was also facilitated by microtubules because treatment with nocodazole significantly inhibited both processes. This microtubule-dependent process was also dependent on dynein because the dynein inhibitor nordihydroguaiaretic acid (NDGA) inhibited reassembly. These studies show that a late stage of Golgi assembly occurs via membrane tubules, whose formation is dependent on PLA2 activity and microtubules. Considering these results together, we concluded that the maintenance and assembly of normal Golgi architecture is dependent on the PLA2-mediated, dynamic formation of inter-Golgi membrane tubules. PMID:19747452

  16. Expression of the PlA2 allele of glycoprotein IIIa and its impact on platelet function

    PubMed Central

    Ferro, Albert; Warner, Timothy D

    2015-01-01

    Background The platelet fibrinogen receptor represents the final common pathway of platelet activation, and is formed from two glycoprotein (GP) subunits (GPIIb/IIIa). Carriage of the mutant PlA2 allele of GPIIIa has been shown to confer an increased risk of cardiovascular events, but published studies have disagreed as to the mechanism for this association. Objectives To assess whether carriage of the PlA2 allele conforms to Mendelian patterns of expression and to identify whether carriage of the mutant allele modulates platelet function. Methods Expression of the PlA2 allele was assessed in both healthy subjects (n = 25) and patients with known coronary artery disease (n = 90) through the development and validation of a liquid chromatography, tandem mass spectrometry (LC-MS/MS) assay. Platelet function was assessed in the patient cohort in response to multiple agonists, and these data were analysed in the context of the proteomic data. Results Expression of the wild-type PlA1 allele and mutant PlA2 alleles was readily quantifiable and conformed to Mendelian patterns in both healthy and patient cohorts. Patients who were homozygous for the mutant PlA2 allele had an increased aggregatory response to adenosine diphosphate, collagen, adrenaline, ristocetin, thrombin receptor-activating peptide 6 and U46619, when assessed using agonist-concentration response curves. Conclusions These findings support the hypothesis that carriage of the mutant PlA2 allele mediates an increased risk of cardiovascular events through the modulation of platelet reactivity. PMID:26858830

  17. 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

  18. Investigation of a Bicyclo[1.1.1]pentane as a Phenyl Replacement within an LpPLA2 Inhibitor.

    PubMed

    Measom, Nicholas D; Down, Kenneth D; Hirst, David J; Jamieson, Craig; Manas, Eric S; Patel, Vipulkumar K; Somers, Don O

    2017-01-12

    We describe the incorporation of a bicyclo[1.1.1]pentane moiety within two known LpPLA2 inhibitors to act as bioisosteric phenyl replacements. An efficient synthesis to the target compounds was enabled with a dichlorocarbene insertion into a bicyclo[1.1.0]butane system being the key transformation. Potency, physicochemical, and X-ray crystallographic data were obtained to compare the known inhibitors to their bioisosteric counterparts, which showed the isostere was well tolerated and positively impacted on the physicochemical profile.

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

    PubMed

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

    2004-11-27

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

  20. Mice deficient in Group VIB phospholipase A2 (iPLA2γ) exhibit relative resistance to obesity and metabolic abnormalities induced by a Western diet

    PubMed Central

    Song, Haowei; Wohltmann, Mary; Bao, Shunzhong; Ladenson, Jack H.; Semenkovich, Clay F.

    2010-01-01

    Phospholipases A2 (PLA2) play important roles in metabolic processes, and the Group VI PLA2 family is comprised of intracellular enzymes that do not require Ca2+ for catalysis. Mice deficient in Group VIA PLA2 (iPLA2β) develop more severe glucose intolerance than wild-type (WT) mice in response to dietary stress. Group VIB PLA2 (iPLA2γ) is a related enzyme distributed in membranous organelles, including mitochondria, and iPLA2γ knockout (KO) mice exhibit altered mitochondrial morphology and function. We have compared metabolic responses of iPLA2γ-KO and WT mice fed a Western diet (WD) with a high fat content. We find that KO mice are resistant to WD-induced increases in body weight and adiposity and in blood levels of cholesterol, glucose, and insulin, even though WT and KO mice exhibit similar food consumption and dietary fat digestion and absorption. KO mice are also relatively resistant to WD-induced insulin resistance, glucose intolerance, and altered patterns of fat vs. carbohydrate fuel utilization. KO skeletal muscle exhibits impaired mitochondrial β-oxidation of fatty acids, as reflected by accumulation of larger amounts of long-chain acylcarnitine (LCAC) species in KO muscle and liver compared with WT in response to WD feeding. This is associated with increased urinary excretion of LCAC and much reduced deposition of triacylglycerols in liver by WD-fed KO compared with WT mice. The iPLA2γ-deficient genotype thus results in a phenotype characterized by impaired mitochondrial oxidation of fatty acids and relative resistance to the metabolic abnormalities induced by WD. PMID:20179248

  1. A previously unreported impact of a PLA2G7 gene polymorphism on the plasma levels of lipoprotein-associated phospholipase A2 activity and mass

    PubMed Central

    Qi, Yue; Zhao, Dong; Jia, Zhangrong; Wang, Wei; Wang, Miao; Sun, Jiayi; Liu, Jun; Li, Yan; Xie, Wuxiang; Liu, Jing

    2016-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) levels are associated with the development of atherosclerosis. We aimed to assess the genetic determinants of Lp-PLA2 activity and mass by genotyping multiple polymorphisms in PLA2G7, the gene encoding Lp-PLA2, among 1258 participants from the Chinese Multi-provincial Cohort Study-Beijing Project. The Sequenom MassARRAY system, Taqman assay and direct sequencing were adopted. For the first time, the rs13218408 polymorphism was found to be significantly associated with reduced Lp-PLA2 levels. We also confirmed the significant association of previously validated polymorphisms (rs1421378, rs1805018, rs16874954 and rs2216465), even after adjusting for traditional cardiovascular risk factors and for Bonferroni correction. Percentages of variance attributable to rs13218408 were 7.2% for activity and 13.3% for mass, and were secondary to those of rs16874954 (8.1% for activity and 16.9% for mass). A significant joint effect of rs13218408 and rs16874954 was observed on Lp-PLA2 activity (P = 0.058) and mass (P = 0.003), with their minor alleles together linking to the largest reduction in Lp-PLA2 levels (37.8% reduction in activity and 41.6% reduction in mass). Taken together, our findings show a significant association of a PLA2G7 polymorphism with Lp-PLA2 levels, which was previously unreported in any population. The functionality of this genetic variation deserves further investigations. PMID:27905470

  2. A previously unreported impact of a PLA2G7 gene polymorphism on the plasma levels of lipoprotein-associated phospholipase A2 activity and mass.

    PubMed

    Qi, Yue; Zhao, Dong; Jia, Zhangrong; Wang, Wei; Wang, Miao; Sun, Jiayi; Liu, Jun; Li, Yan; Xie, Wuxiang; Liu, Jing

    2016-12-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) levels are associated with the development of atherosclerosis. We aimed to assess the genetic determinants of Lp-PLA2 activity and mass by genotyping multiple polymorphisms in PLA2G7, the gene encoding Lp-PLA2, among 1258 participants from the Chinese Multi-provincial Cohort Study-Beijing Project. The Sequenom MassARRAY system, Taqman assay and direct sequencing were adopted. For the first time, the rs13218408 polymorphism was found to be significantly associated with reduced Lp-PLA2 levels. We also confirmed the significant association of previously validated polymorphisms (rs1421378, rs1805018, rs16874954 and rs2216465), even after adjusting for traditional cardiovascular risk factors and for Bonferroni correction. Percentages of variance attributable to rs13218408 were 7.2% for activity and 13.3% for mass, and were secondary to those of rs16874954 (8.1% for activity and 16.9% for mass). A significant joint effect of rs13218408 and rs16874954 was observed on Lp-PLA2 activity (P = 0.058) and mass (P = 0.003), with their minor alleles together linking to the largest reduction in Lp-PLA2 levels (37.8% reduction in activity and 41.6% reduction in mass). Taken together, our findings show a significant association of a PLA2G7 polymorphism with Lp-PLA2 levels, which was previously unreported in any population. The functionality of this genetic variation deserves further investigations.

  3. Neuroprotective effects of a nanocrystal formulation of sPLA(2) inhibitor PX-18 in cerebral ischemia/reperfusion in gerbils.

    PubMed

    Wang, Qun; Sun, Albert Y; Pardeike, Jana; Müller, Rainer H; Simonyi, Agnes; Sun, Grace Y

    2009-08-18

    The group IIA secretory phospholipase A2 (sPLA(2)-IIA) has been studied extensively because of its involvement in inflammatory processes. Up-regulation of this enzyme has been shown in a number of neurodegenerative diseases including cerebral ischemia and Alzheimer's disease. PX-18 is a selective sPLA(2) inhibitor effective in reducing tissue damage resulting from myocardial infarction. However, its use as a neuroprotective agent has been hampered due to its low solubility. In this study, we test the possible neuroprotective effects of PX-18 formulated as a suspension of nanocrystals. Transient global cerebral ischemia was induced in gerbils by occlusion of both common carotid arteries for 5 min. Four days after ischemia/reperfusion (I/R), extensive delayed neuronal death, DNA damage, and increases in reactive astrocytes and microglial cells were observed in the hippocampal CA1 region. PX-18 nanocrystals (30 and 60 mg/kg body wt) and vehicle controls were injected i.p. immediately after I/R. PX-18 nanocrystal injection significantly reduced delayed neuronal death, DNA damage, as well as glial cell activation. These findings demonstrated the effective neuroprotection of PX-18 in the form of nanocrystal against I/R-induced neuronal damage. The results also suggest that nanocrystals hold promise as an effective strategy for the delivery of compounds with poor solubility that would otherwise be precluded from preclinical development.

  4. Evaluation of the safety, pharmacokinetics, pharmacodynamics, and drug-drug interaction potential of a selective Lp-PLA2 inhibitor (GSK2647544) in healthy volunteers

    PubMed Central

    Wu, Kai; Xu, Jianfeng; Fong, Regan; Yao, Xiaozhou; Xu, Yanmei; Guiney, William; Gray, Frank; Lockhart, Andrew

    2016-01-01

    Abstract. Objective: To evaluate in healthy volunteers the safety, pharmacokinetics (PK), pharmacodynamics (PD), and drug-drug interaction (DDI) potential of GSK2647544, (a selective lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor). Methods: Study 1 was a single-blind, randomized, placebo-controlled, crossover study with healthy male volunteers randomized to receive single escalating oral doses (0.5 – 750 mg) of GSK2647544. Study 2 was a single-blind, randomized, placebo-controlled study with healthy volunteers randomized to receive repeat doses (80 mg) of GSK2647544. The drug-drug interaction of GSK2647544 with simvastatin was also evaluated in study 2. Results: Across both studies GSK2647544 doses were generally well tolerated with no GSK2647544-related clinically significant findings. GSK2647544 was readily absorbed and its plasma concentration declined bi-exponentially with a terminal half-life ranging from 8 to 16 hours. Plasma exposure of GSK2647544 increased approximately dose-proportionally. There was GSK2647544 dose-dependent inhibition of plasma Lp-PLA2 activity, with a trough inhibition (12 hours after dose) of 85.6% after 7-day twice daily dosing. The administration of simvastatin concomitantly with GSK2647544 increased the overall exposure (area under the plasma concentration-time curve and maximum plasma concentration) of simvastatin and simvastatin acid by 3.6- to 4.3-fold and 1.5- to 3.1-fold, respectively. Conclusions: GSK2647544 was generally well tolerated and had a reasonable PK-PD profile. The clinically significant drug-drug interaction led to an early termination of study 2. PMID:27719741

  5. Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, platelet-activating factor acetylhydrolase (PAF-AH) in leukocytes and body composition in healthy adults

    PubMed Central

    Detopoulou, Paraskevi; Nomikos, Tzortzis; Fragopoulou, Elizabeth; Panagiotakos, Demosthenis B; Pitsavos, Christos; Stefanadis, Christodoulos; Antonopoulou, Smaragdi

    2009-01-01

    Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) also known as serum platelet activating factor acetylhydrolase (PAF-AH) activity constitutes a novel risk marker for cardiovascular disease. Leukocytes constitute one main cellular source of circulating Lp-PLA2. The aim of the present study was to evaluate the association of both serum and leukocyte PAF-AH activities with fat distribution and lean tissue. One hundred healthy volunteers without cardiovascular disease history participated in this study (n = 52 men, 44 ± 13 years and n = 48 women, 43 ± 13 years). Body composition was assessed with dual-energy X-ray absorptiometry, while anthropometrical indices were also measured. The activity of Lp-PLA2 and levels of lipid and glycemic parameters were determined in fasting samples. Results Mean Lp-PLA2 activity was 24.8 ± 4.5 and 19.6 ± 5.0 nmol/min/mL in men and women, respectively (P < 0.001). Mean activity of PAF-AH in leukocyte homogenates was 386 ± 127 pmol/min/mg and 292 ± 92 pmol/min/mg in men and women, correspondingly (P < 0.001). In multiple regression models upper and total adiposity measures were positively associated with Lp-PLA2 activity in men after adjusting for LDL-cholesterol, age, smoking, hs-CRP and physical activity, whereas no associations were found with PAF-AH leukocyte homogenates activity. Hierarchical analysis revealed that the variables with the highest explanatory ability of Lp-PLA2 activity in men, were DXA deriving L1–L4 region of interest and arms fat (increase in R2 = 0.136, P = 0.005 and increase in R2 = 0.118, P = 0.009, respectively), followed by trunk fat and total fat. In women, no association of body composition variables with Lp-PLA2 nor PAF-AH leukocyte homogenates activity was found. Conclusion Lp-PLA2 activity is differentiated across levels of adiposity and topology of adipose tissue, whereas no association was found regarding PAF-AH leukocyte homogenates activity. Our findings suggest that Lp-PLA2 may

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

    PubMed Central

    Hains, P G; Broady, K W

    2000-01-01

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

  7. Antioxidant and inflammatory aspects of lipoprotein-associated phospholipase A2 (Lp-PLA2 ): a review

    PubMed Central

    2011-01-01

    The association of cardiovascular events with Lp-PLA2 has been studied continuously today. The enzyme has been strongly associated with several cardiovascular risk markers and events. Its discovery was directly related to the hydrolysis of the platelet-activating factor and oxidized phospholipids, which are considered protective functions. However, the hydrolysis of bioactive lipids generates lysophospholipids, compounds that have a pro-inflammatory function. Therefore, the evaluation of the distribution of Lp-PLA2 in the lipid fractions emphasized the dual role of the enzyme in the inflammatory process, since the HDL-Lp-PLA2 enzyme contributes to the reduction of atherosclerosis, while LDL-Lp-PLA2 stimulates this process. Recently, it has been verified that diet components and drugs can influence the enzyme activity and concentration. Thus, the effects of these treatments on Lp-PLA2 may represent a new kind of prevention of cardiovascular disease. Therefore, the association of the enzyme with the traditional assessment of cardiovascular risk may help to predict more accurately these diseases. PMID:21955667

  8. A novel calcium-independent cellular PLA2 acts in insect immunity and larval growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phospholipase A2 (PLA2) catalyzes the position-specific hydrolysis of fatty acids linked to the sn-2 position of phospholipids (PLs). PLA2s make up a very large superfamily, with more than known 15 groups, classified into secretory PLA2 (sPLA2), Ca2+-dependent cellular PLA2 (sPLA2), and Ca2+-indepen...

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

    PubMed

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

    2016-05-26

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

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

    PubMed Central

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

    2013-01-01

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

  11. Secreted phospholipase A2 inhibitors are also potent blockers of binding to the M-type receptor.

    PubMed

    Boilard, Eric; Rouault, Morgane; Surrel, Fanny; Le Calvez, Catherine; Bezzine, Sofiane; Singer, Alan; Gelb, Michael H; Lambeau, Gérard

    2006-11-07

    Mammalian secreted phospholipases A(2) (sPLA(2)s) constitute a family of structurally related enzymes that are likely to play numerous biological roles because of their phospholipid hydrolyzing activity and binding to soluble and membrane-bound proteins, including the M-type receptor. Over the past decade, a number of competitive inhibitors have been developed against the inflammatory-type human group IIA (hGIIA) sPLA(2) with the aim of specifically blocking its catalytic activity and pathophysiological functions. The fact that many of these inhibitors, including the indole analogue Me-Indoxam, inhibit several other sPLA(2)s that bind to the M-type receptor prompted us to investigate the impact of Me-Indoxam and other inhibitors on the sPLA(2)-receptor interaction. By using a Ca(2+) loop mutant derived from a venom sPLA(2) which is insensitive to hGIIA inhibitors but still binds to the M-type receptor, we demonstrate that Me-Indoxam dramatically decreases the affinity of various sPLA(2)s for the receptor, yet an sPLA(2)-Me-Indoxam-receptor complex can form at very high sPLA(2) concentrations. Me-Indoxam inhibits the binding of iodinated mouse sPLA(2)s to the mouse M-type receptor expressed on live cells but also enhances binding of sPLA(2) to phospholipids. Because Me-Indoxam and other competitive inhibitors protrude out of the sPLA(2) catalytic groove, it is likely that the inhibitors interfere with the sPLA(2)-receptor interaction by steric hindrance and to different extents that depend on the type of sPLA(2) and inhibitor. Our finding suggests that the various anti-inflammatory therapeutic effects of sPLA(2) inhibitors may be due not only to inhibition of enzymatic activity but also to modulation of binding of sPLA(2) to the M-type receptor or other as yet unknown protein targets.

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

    PubMed Central

    Seo, Samyeol; Lee, Sunghong; Hong, Yongpyo

    2012-01-01

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

  13. 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

  14. Genetic invalidation of Lp-PLA2 as a therapeutic target: Large-scale study of five functional Lp-PLA2-lowering alleles.

    PubMed

    Gregson, John M; Freitag, Daniel F; Surendran, Praveen; Stitziel, Nathan O; Chowdhury, Rajiv; Burgess, Stephen; Kaptoge, Stephen; Gao, Pei; Staley, James R; Willeit, Peter; Nielsen, Sune F; Caslake, Muriel; Trompet, Stella; Polfus, Linda M; Kuulasmaa, Kari; Kontto, Jukka; Perola, Markus; Blankenberg, Stefan; Veronesi, Giovanni; Gianfagna, Francesco; Männistö, Satu; Kimura, Akinori; Lin, Honghuang; Reilly, Dermot F; Gorski, Mathias; Mijatovic, Vladan; Munroe, Patricia B; Ehret, Georg B; Thompson, Alex; Uria-Nickelsen, Maria; Malarstig, Anders; Dehghan, Abbas; Vogt, Thomas F; Sasaoka, Taishi; Takeuchi, Fumihiko; Kato, Norihiro; Yamada, Yoshiji; Kee, Frank; Müller-Nurasyid, Martina; Ferrières, Jean; Arveiler, Dominique; Amouyel, Philippe; Salomaa, Veikko; Boerwinkle, Eric; Thompson, Simon G; Ford, Ian; Wouter Jukema, J; Sattar, Naveed; Packard, Chris J; Shafi Majumder, Abdulla Al; Alam, Dewan S; Deloukas, Panos; Schunkert, Heribert; Samani, Nilesh J; Kathiresan, Sekar; Nordestgaard, Børge G; Saleheen, Danish; Howson, Joanna Mm; Di Angelantonio, Emanuele; Butterworth, Adam S; Danesh, John

    2017-03-01

    Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA2 enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T > C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA2. We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA2 activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA2-lowering alleles. Results Lp-PLA2 activity was decreased by 64% ( p = 2.4 × 10(-25)) with carriage of any of the four loss-of-function variants, by 45% ( p < 10(-300)) for every allele inherited at Val279Phe, and by 2.7% ( p = 1.9 × 10(-12)) for every allele inherited at Val379Ala. Darapladib 160 mg once-daily reduced Lp-PLA2 activity by 65% ( p < 10(-300)). Causal risk ratios for coronary heart disease per 65% lower Lp-PLA2 activity were: 0.95 (0.88-1.03) with Val279Phe; 0.92 (0.74-1.16) with carriage of any loss-of-function variant; 1.01 (0.68-1.51) with Val379Ala; and 0.95 (0.89-1.02) with darapladib treatment. Conclusions In a large-scale human genetic study, none of a series of Lp-PLA2-lowering alleles was related to coronary heart disease risk, suggesting that Lp-PLA2 is unlikely to be a causal risk factor.

  15. † THE GROUP VIA CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 (iPLA2β)1 PARTICIPATES IN ER STRESS-INDUCED INS-1 INSULINOMA CELL APOPTOSIS BY PROMOTING CERAMIDE GENERATION VIA HYDROLYSIS OF SPHINGOMYELINS BY NEUTRAL SPHINGOMYELINASE

    PubMed Central

    Lei, Xiao-Yong; Zhang, Sheng; Bohrer, Alan; Bao, Shunzhong; Song, Haowei; Ramanadham, Sasanka

    2008-01-01

    β-cell mass is regulated by a balance between β-cell growth and β-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the Group VIA Ca2+-independent phospholipase A2 (iPLA2β), associated with increased ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2β was over expressed (OE INS-1 cells). These findings suggested that iPLA2β and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we addressed this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2β-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and BiP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Ceramide accumulation during ER stress was not associated with changes in mRNA levels of serine palmitoyl-transferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, temporally increased in the INS-1 cells. The increases in NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2β protein and catalytic activity. Pretreatment with BEL inactivated iPLA2β and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2β or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2001-08-31

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

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

    PubMed

    Lizano, Sergio; Domont, Gilberto; Perales, Jonas

    2003-12-15

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

  19. 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.

  20. A novel read-through transcript JMJD7-PLA2G4B regulates head and neck squamous cell carcinoma cell proliferation and survival

    PubMed Central

    Cheng, Yingduan; Wang, Yi; Li, Jiong; Chang, Insoon; Wang, Cun-Yu

    2017-01-01

    Recent findings on the existence of oncogenic fusion genes in a wide array of solid tumors, including head and neck squamous cell carcinoma (HNSCC), suggests that fusion genes have become attractive targets for cancer diagnosis and treatment. In this study, we showed for the first time that a read-through fusion gene JMJD7-PLA2G4B is presented in HNSCC, splicing neighboring jumonji domain containing 7 (JMJD7) and phospholipase A2, group IVB (PLA2G4B) genes together. Ablation of JMJD7-PLA2G4B significantly inhibited proliferation of HNSCC cells by promoting G1 cell cycle arrest and increased starvation-induced cell death compared to JMJD7-only knockdown HNSCC cells. Mechanistically, we found that JMJD7-PLA2G4B modulates phosphorylation of Protein Kinase B (AKT) to promote HNSCC cell survival. Moreover, JMJD7-PLA2G4B also regulated an E3 ligase S-phase kinase-associated protein 2 (SKP2) to control the cell cycle progression from G1 phase to S phase by inhibiting Cyclin-dependent kinase inhibitor 1 (p21) and 1B (p27) expression. Our study provides novel insights into the oncogenic control of JMJD7-PLA2G4B in HNSCC cell proliferation and survival, and suggests that JMJD7-PLA2G4B may serve as an important therapeutic target and prognostic marker for HNSCC development and progression. PMID:28030848

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

    PubMed Central

    Sultan, Md. Tipu; Li, Hong-Mei; Lee, Yong Zu

    2016-01-01

    We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]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 Ca2+ 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 [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption. PMID:26937214

  2. UCN enhances TGF-beta-mediated mitoinhibition of VSMCs via counteracting TGF-beta-induced cPLA2 expression and activation.

    PubMed

    Zhu, Chao; Cao, Changchun; Wang, Xiaofei; Yuan, Jie; Jin, Lai; Li, Shengnan

    2016-11-01

    Urocortins (UCNs) and transforming growth factor-beta (TGF-beta) have been demonstrated to participate in various cardiovascular diseases, many of which involve vascular smooth muscle cells (VSMCs) proliferation. Cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release is an important cause of VSMCs proliferation. The work was to investigate the regulation of VSMCs proliferation by UCN/TGF-beta and whether cPLA2 was a link between their signaling pathways. VSMCs proliferation was measured by colorimetric assay and immunofluorescence microscopy. Using cell flow cytometry, the changes in the cell cycle phases were investigated. Lentiviral Vector Particle was performed to overexpress cPLA2 gene. Both UCN and TGF-beta inhibited VSMCs proliferation and an additive effect was observed when the cells were treated with UCN plus TGF-beta. TGF-beta increased the percentage of cells in G1-phase while UCN increased the cell percentage in G2-phase with a concomitant decrease in S-phase. Furthermore, cPLA2 expression was increased by TGF-beta but decreased by UCN and UCN attenuated TGF-beta-induced cPLA2 expression. In primary VSMCs, TGF-beta induced cPLA2 phosphorylation, and this effect was also attenuated by UCN. Similar to UCN, the cPLA2 inhibitor, pyrrophenone (PYR), also played a role in enhancing TGF-beta-mediated mitoinhibition. Inversely, overexpression of cPLA2 eliminated the effect of UCN on the mitoinhibition. The pretreatment with UCN counteracted TGF-beta-mediated cPLA2 expression and activation, thereby contributing to TGF-beta-mediated mitoinhibition of VSMCs.

  3. The Adipocyte-Inducible Secreted Phospholipases PLA2G5 and PLA2G2E Play Distinct Roles in Obesity

    PubMed Central

    Sato, Hiroyasu; Taketomi, Yoshitaka; Ushida, Ayako; Isogai, Yuki; Kojima, Takumi; Hirabayashi, Tetsuya; Miki, Yoshimi; Yamamoto, Kei; Nishito, Yasumasa; Kobayashi, Tetsuyuki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Ida, Satoshi; Miyamoto, Yuji; Watanabe, Masayuki; Baba, Hideo; Miyata, Keishi; Oike, Yuichi; Gelb, Michael H.; Murakami, Makoto

    2014-01-01

    Summary Metabolic disorders including obesity and insulin resistance have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5−/− and Pla2g2e−/− mice revealed distinct and previously unrecognized roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia and obesiy. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of “metabolic sPLA2s” adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators. PMID:24910243

  4. Evidence for Proteolytic Processing and Stimulated Organelle Redistribution of iPLA2β

    PubMed Central

    Song, Haowei; Bao, Shunzhong; Lei, Xiaoyong; Jin, Chun; Zhang, Sheng; Turk, John; Ramanadham, Sasanka

    2010-01-01

    Over the past decade, important roles for the 84–88 kDa Group VIA Ca2+-independent phospholipase A2 (iPLA2β) in various organs have been described. We demonstrated that iPLA2β participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA2β, and certain stimuli promote perinuclear localization of iPLA2β. To gain a better understanding of its mobilization, iPLA2β was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA2β by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA2β activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA2β activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA2β isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA2β in β-cells and we find here that these stimuli promote differential localization of iPLA2β in subcellular organelles. Further, mass spectrometric analyses identified iPLA2β variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA2β and redistribution of iPLA2β variants in subcellular compartments. It might be proposed that in vivo processing of iPLA2β facilitates its participation in multiple biological processes. PMID:20132906

  5. 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.

  6. 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

  7. 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

  8. Treatment of ovalbumin-induced experimental allergic bronchitis in rats by inhaled inhibitor of secretory phospholipase A2

    PubMed Central

    Shoseyov, D; Bibi, H; Offer, S; Schwob, O; Krimsky, M; Kleiman, M; Yedgar, S

    2005-01-01

    Background: The pathophysiology of asthma involves the action of inflammatory/allergic lipid mediators formed following membrane phospholipid hydrolysis by phospholipase A2 (PLA2). Cysteinyl leukotrienes are considered potent inducers of bronchoconstriction and airway remodelling. Ovalbumin (OVA) induced bronchoconstriction in rats is associated with increased secretory PLA2 (sPLA2) activation and cysteinyl leukotriene production, together with suppression of cytosolic PLA2 and prostaglandin E2. These processes are reversed when the animals are pretreated systemically with an extracellular cell impermeable sPLA2 inhibitor which also suppresses the early allergic reaction to OVA challenge. In this study we examine the capacity of the sPLA2 inhibitor to ameliorate inflammatory and allergic manifestations (early and late bronchoconstriction) of OVA induced allergic bronchitis in rats when the inhibitor was administered by inhalation to confine it to the airways. Methods: Rats sensitised with OVA were treated with the sPLA2 inhibitor hyaluronic acid-linked phosphatidyl ethanolamine (HyPE). The rats were divided into four groups (n = 10 per group): (1) naïve controls (no sensitisation/no treatment); (2) positive controls (sensitisation + challenge with OVA inhalation and subcutaneous injection of 1 ml saline before each challenge; (3) sensitisation + challenge with OVA and HyPE inhalation before every challenge; and (4) sensitisation + challenge with OVA and treatment with subcutaneous dexamethasone (300 µg) before each challenge as a conventional reference. Another group received no treatment with HyPE during the sensitisation process but only before or after challenge of already sensitised rats. Pulmonary function was assessed and changes in the histology of the airways, levels of cysteinyl leukotrienes in BAL fluid, and the production of nitric oxide (No) and tumour necrosis factor α (TNFα) by BAL macrophages were determined. Results: Inhalation of HyPE markedly

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

    PubMed Central

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

    1997-01-01

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

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

  10. Secreted phospholipase A2 inhibitor modulates fatty acid composition and reduces obesity-induced inflammation in Beagle dogs.

    PubMed

    Xu, J; Bourgeois, H; Vandermeulen, E; Vlaeminck, B; Meyer, E; Demeyere, K; Hesta, M

    2015-05-01

    Secreted phospholipase A2 inhibitor (sPLA2i) has been reported to have an anti-inflammatory function by blocking the production of inflammatory mediators. Obesity is characterized by low-grade inflammation and oxidative stress. The aim of this study was to investigate the effects of dietary supplementation of sPLA2i on inflammation, oxidative stress and serum fatty acid profile in dogs. Seven obese and seven lean Beagle dogs were used in a 28-day double blind cross-over design. Dogs were fed a control diet without supplemental sPLA2i or an sPLA2i supplemented diet. The sPLA2i diet decreased plasma fibrinogen levels and increased the protein:fibrinogen ratio in obese dogs to levels similar to those of lean dogs fed the same diet. Obese dogs had a higher plasma concentration of the lipophilic vitamin A with potential antioxidative capacity and a lower ratio of retinol binding protein 4:vitamin A compared to lean dogs, independent of the diets. A higher proportion of myristic acid (C14:0) and a lower proportion of linoleic acid (C18:2n-6) were observed in the dogs fed with the sPLA2i diet compared to dogs fed with the control diet. Furthermore, a higher ratio of n-6 to n-3, a lower proportion of n-3 polyunsaturated fatty acids and lower omega-3 index were observed in obese compared to lean dogs. The results indicate that obese dogs are characterized by a more 'proinflammatory' serum fatty acid profile and that diet inclusion of sPLA2i may reduce inflammation and alter fatty acid profile.

  11. Role of prefrontal cortical calcium-independent phospholipase A2 in antinociceptive effect of the norepinephrine reuptake inhibitor antidepresssant maprotiline.

    PubMed

    Chew, Wee-Siong; Shalini, Suku-Maran; Torta, Federico; Wenk, Markus R; Stohler, Christian; Yeo, Jin-Fei; Herr, Deron R; Ong, Wei-Yi

    2017-01-06

    The prefrontal cortex is essential for executive functions such as decision-making and planning. There is also accumulating evidence that it is important for the modulation of pain. In this study, we investigated a possible role of prefrontal cortical calcium-independent phospholipase A2 (iPLA2) in antinociception induced by the norepinephrine reuptake inhibitor (NRI) and tetracyclic (tricyclic) antidepressant, maprotiline. Intraperitoneal injections of maprotiline increased iPLA2 mRNA and protein expression in the prefrontal cortex. This treatment also reduced grooming responses to von-Frey hair stimulation of the face after facial carrageenan injection, indicating decreased sensitivity to pain. The antinociceptive effect of maprotiline was abrogated by iPLA2 antisense oligonucleotide injection to the prefrontal cortex, indicating a role of this enzyme in antinociception. In contrast, injection of iPLA2 antisense oligonucleotide to the somatosensory cortex did not reduce the antinociceptive effect of maprotiline. Lipidomic analysis of the prefrontal cortex showed decrease in phosphatidylcholine species, but increase in lysophosphatidylcholine species, indicating increased PLA2 activity, and release of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) after maprotiline treatment. Differences in sphingomyelin/ceramide were also detected. These changes were not observed in maprotiline-treated mice that received iPLA2 antisense oligonucleotide to the prefrontal cortex. Metabolites of DHA and EPA may help to strengthen a known supraspinal antinociceptive pathway from the prefrontal cortex to the periaqueductal gray. Together, results indicate a role of prefrontal cortical iPLA2 and its enzymatic products in the antinociceptive effect of maprotiline.

  12. Structure-activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps.

    PubMed

    Sundermann, Tom; Hanekamp, Walburga; Lehr, Matthias

    2016-08-01

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA2α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA2α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA2α and FAAH, respectively.

  13. Structural and evolutionary insights into endogenous alpha-phospholipase A2 inhibitors of Latin American pit vipers.

    PubMed

    Estevão-Costa, Maria Inácia; Fernandes, Carlos Alexandre H; Mudadu, Maurício de Alvarenga; Franco, Glória Regina; Fontes, Marcos Roberto M; Fortes-Dias, Consuelo Latorre

    2016-03-15

    Phospholipases A2 are major components of snake venoms (svPLA2s) and are able to induce multiple local and systemic deleterious effects upon envenomation. Several snake species are provided with svPLA2 inhibitors (sbPLIs) in their circulating blood, which confer a natural resistance against the toxic components of homologous and heterologous venoms. The sbPLIs belong to any of three structural classes named α, β and γ. In the present study, we identified, characterized and performed structural and evolutionary analyses of sbαPLIs transcripts and the encoded proteins, in the most common Latin American pit vipers belonging to Crotalus, Bothrops and Lachesis genera. Mutation data indicated that sbαPLIs from Latin American snakes might have evolved in an accelerated manner, similarly to that reported for sbαPLIs from Asian snakes, and possibly co-evoluted with svPLA2s in response to the diversity of target enzymes. The importance of sbαPLI trimerization for the effective binding and inhibition of acidic svPLA2s is discussed and conserved cationic residues located at the central pore of the inhibitor trimer are suggested to be a significant part of the binding site of sbαPLIs to acidic svPLA2s. Our data contribute to the current body of knowledge on the structural and evolutionary characteristics of sbPLIs, in general, and may assist in the future development of selective inhibitors for secretory PLA2 from several sources.

  14. PLA2-responsive and SPIO-loaded phospholipid micelles

    PubMed Central

    Gao, Qiang; Yan, Lesan; Chiorazzo, Michael; Delikatny, E. James; Tsourkas, Andrew; Cheng, Zhiliang

    2015-01-01

    A PLA2-responsive and superparamagnetic iron oxide (SPIO) nanoparticle-loaded phospholipid micelle was developed. The release of phospholipid-conjugated dye from these micelles was triggered due to phospholipid degradation by phospholipase A2. High relaxivity of the encapsulated SPIO could enable non-invasive magnetic resonance imaging. PMID:26139589

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

    PubMed

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

    2002-01-01

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

  16. Binding to PLA2 may contribute to the anti-inflammatory activity of catechol.

    PubMed

    Dileep, Kalarickal V; Tintu, Ignatius; Mandal, Pradeep K; Karthe, Ponnuraj; Haridas, Madathilkovilakathu; Sadasivan, Chittalakkottu

    2012-01-01

    Inhibiting PLA(2) activity should, in theory, be an effective approach to control the inflammation. Several naturally occurring polyphenolic compounds have been reported as inhibitors of PLA(2) . Among the naturally occurring polyphenols, catechol (1,2-dihydroxybenzene) possesses anti-inflammatory activity. Catechol can inhibit cyclooxygenase and lipo-oxygenase. By means of enzyme kinetic study, it was revealed that catechol can inhibit PLA(2) also. Crystal structure showed that catechol binds to PLA(2) at the opening of the active site cleft. This might stop the entry of substrate into the active site. Hence, catechol can be used as a lead compound for the development of novel anti-inflammatory drugs with PLA(2) as the target.

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

    PubMed Central

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

    2004-01-01

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

  18. Pharmacophore-based discovery of a novel cytosolic phospholipase A2α inhibitor

    PubMed Central

    Noha, Stefan M.; Jazzar, Bianca; Kuehnl, Susanne; Rollinger, Judith M.; Stuppner, Hermann; Schaible, Anja M.; Werz, Oliver; Wolber, Gerhard; Schuster, Daniela

    2012-01-01

    The release of arachidonic acid, a precursor in the production of prostaglandins and leukotrienes, is achieved by activity of the cytosolic phospholipase A2α (cPLA2α). Signaling mediated by this class of bioactive lipids, which are collectively referred to as eicosanoids, has numerous effects in physiological and pathological processes. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the National Cancer Institute (NCI) database, leading to the identification of 4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957) as cPLA2α inhibitor in cell-free and cell-based in vitro assays. PMID:22192589

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

    PubMed

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

    2003-02-27

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

  20. Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction

    PubMed Central

    Castillo-Quan, Jorge Iván; Bartolome, Fernando; Angelova, Plamena R.; Li, Li; Pope, Simon; Cochemé, Helena M.; Khan, Shabana; Asghari, Shabnam; Bhatia, Kailash P.; Hardy, John; Abramov, Andrey Y.; Partridge, Linda

    2015-01-01

    The PLA2G6 gene encodes a group VIA calcium-independent phospholipase A2 beta enzyme that selectively hydrolyses glycerophospholipids to release free fatty acids. Mutations in PLA2G6 have been associated with disorders such as infantile neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type II and Karak syndrome. More recently, PLA2G6 was identified as the causative gene in a subgroup of patients with autosomal recessive early-onset dystonia-parkinsonism. Neuropathological examination revealed widespread Lewy body pathology and the accumulation of hyperphosphorylated tau, supporting a link between PLA2G6 mutations and parkinsonian disorders. Here we show that knockout of the Drosophila homologue of the PLA2G6 gene, iPLA2-VIA, results in reduced survival, locomotor deficits and organismal hypersensitivity to oxidative stress. Furthermore, we demonstrate that loss of iPLA2-VIA function leads to a number of mitochondrial abnormalities, including mitochondrial respiratory chain dysfunction, reduced ATP synthesis and abnormal mitochondrial morphology. Moreover, we show that loss of iPLA2-VIA is strongly associated with increased lipid peroxidation levels. We confirmed our findings using cultured fibroblasts taken from two patients with mutations in the PLA2G6 gene. Similar abnormalities were seen including elevated mitochondrial lipid peroxidation and mitochondrial membrane defects, as well as raised levels of cytoplasmic and mitochondrial reactive oxygen species. Finally, we demonstrated that deuterated polyunsaturated fatty acids, which inhibit lipid peroxidation, were able to partially rescue the locomotor abnormalities seen in aged flies lacking iPLA2-VIA gene function, and restore mitochondrial membrane potential in fibroblasts from patients with PLA2G6 mutations. Taken together, our findings demonstrate that loss of normal PLA2G6 gene activity leads to lipid peroxidation, mitochondrial dysfunction and subsequent mitochondrial membrane

  1. Membranous nephropathy PLA2R+ associated with Chagas disease

    PubMed Central

    Silva, Vanessa dos Santos; Viero, Rosa Marlene

    2015-01-01

    Chagas disease (CD) — a tropical parasitic disease caused by the protozoan Trypanosoma cruzi — is a major health problem in Latin America. The immune response against the parasite is responsible for chronic CD lesions. Currently, there are no reports of an association between CD and membranous nephropathy (MN). The detection of the phospholipase A2 receptor (PLA2R) as a target antigen in idiopathic MN can improve the differential diagnosis of primary and secondary forms of MN. The authors report the case of a male patient with positive serology for CD who presented sudden death and underwent autopsy. Histological sections of the heart showed multifocal inflammatory infiltrate composed mainly of mononuclear cells, leading to myocardiocytes necrosis and interstitial fibrosis. The kidneys showed a MN with positive expression for PLA2R. As far as we know, this is the first report of a case of primary MN in a patient with CD, with severe chronic cardiomyopathy and heart failure. PMID:26558244

  2. P9a(Cdt-PLA2) from Crotalus durissus terrificus as good immunogen to be employed in the production of crotalic anti-PLA2 IgG.

    PubMed

    Fusco, Luciano S; Rodríguez, Juan Pablo; Torres-Huaco, Frank; Huancahuire-Vega, Salomón; Teibler, Pamela; Acosta, Ofelia; Marangoni, Sergio; Ponce-Soto, Luis Alberto; Leiva, Laura C

    2015-10-01

    Four proteins with phospholipase A2 (PLA2) activity, designated P9a(Cdt-PLA2), P9b(Cdt-PLA2), P10a(Cdt-PLA2) and P10b(Cdt-PLA2) were purified from the venom of Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly shows that three of them have a similar abundance. The molecular mass, 14193.8340Da for P9a(Cdt-PLA2), 14134.9102Da for P9b(Cdt-PLA2), 14242.6289Da for P10a(Cdt-PLA2) and 14183.8730Da for P10b(Cdt-PLA2), were initially evaluated by SDS-PAGE and confirmed by ESI-Q-TOF spectrometry, and all of them displayed a monomeric conformation. Also, partial amino acid sequence of each protein was obtained and their alignments with other crotalic PLA2 revealed a high degree of identity among them. Additionally, we studied some pharmacological activities like neurotoxicity, myotoxicity and lethality, which prompted us to pick two of them, P9a(Cdt-PLA2) and P10a(Cdt-PLA2) that resulted to be less toxic that the others, and further characterize them to be used as immunogen. We next injected these last proteins in mice to produce antitoxins against them and ELISA and dot blots reveled that both toxins do not show immunogenic differences, unlike those other pharmacologic activities tested. Furthermore, the antibodies produced cross-reacted with all the isoforms purified demonstrating the feasibility of using only one of them and ensuring the cross-reaction of all. The results obtained show that P9a(Cdt-PLA2) isoform has the lowest toxicity and also a good purification performance; thus this protein may be a promising candidate to be employed in the production of crotalic antitoxins.

  3. The elevation of apoB in hypercholesterolemic patients is primarily attributed to the relative increase of apoB/Lp-PLA2

    PubMed Central

    Tellis, Constantinos C.; Moutzouri, Eliza; Elisaf, Moses; Wolfert, Robert L.; Tselepis, Alexandros D.

    2013-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor of cardiovascular disease. Plasma Lp-PLA2 is mainly associated with apolipoprotein (apo)B-containing lipoproteins, primarily with low density lipoproteins (LDLs). Importantly, only a proportion of circulating lipoproteins contain Lp-PLA2. We determined the plasma levels of Lp-PLA2-bound apoB (apoB/Lp-PLA2) in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed. The plasma apoB/Lp-PLA2 concentration in 50 normolipidemic controls and 53 patients with primary hypercholesterolemia at baseline and at 3 months posttreatment with simvastatin (40 mg/day) was determined by an enzyme-linked immunosorbent assay. The concentration of the apoB-containing lipoproteins that do not bind Lp-PLA2 [apoB/Lp-PLA2(−)] was calculated by subtracting the apoB/Lp-PLA2 from total apoB. The apoB/Lp-PLA2 levels were 3.6-fold higher, while apoB/Lp-PLA2(−) were 1.3-fold higher in patients compared with controls. After 3 months of simvastatin treatment apoB/Lp-PLA2 and apoB/Lp-PLA2(−) levels were reduced by 52% and 33%, respectively. The elevation in apoB-containing lipoproteins in hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic apoB/Lp-PLA2, while simvastatin reduces these particles to a higher extent compared with apoB/Lp-PLA2(−). Considering that Lp-PLA2 is proatherogenic, the predominance of apoB/Lp-PLA2 particles in hypercholesterolemic patients may contribute to their higher atherogenicity and incidence of cardiovascular disease. PMID:24092915

  4. [Anti-NEP and anti-PLA2R antibodies in membranous nephropathy: an update].

    PubMed

    Pozdzik, A A; Debiec, H; I Brochériou; Husson, C; Rorive, S; Broeders, N; Le Moine, A; Ronco, P; Nortier, J

    2015-01-01

    Membranous nephropathy (MN) is the most common cause for nephrotic syndrome in adults and occurs as an idiopathic (primary) or secondary disease. Since the early 2000's, substantial advances have been made in the understanding of the molecular bases of MN. The neutral endopeptidase (NEP) and the receptor for secretory phospholipase A2 (PLA2R) have been identified as target antigens for circulating and deposited antibodies in allo-immune neonatal and adult " idiopathic " MN, respectively. These antibodies recognize specific antigens of podocytes, precipitate as subepithelial immune complexes and activate complement leading to proteinuria. Anti-PLA2R antibodies are of particular clinical importance. Indeed, they are detected in approximately 70% of primary MN in adults, demonstrating that MN actually is an autoimmune condition specific to the kidney. In Europeans, genome-wide studies have shown an association between alleles of PLA2R1 and HLA DQA1 (class II genes of tissue histocompatibility complex) genes and idiopathic MN. Newly developed diagnostic tests detecting circulating anti-PLA2R antibody and PLA2R antigen in glomerular deposits have induced a change in paradigm in the diagnostic approach of idiopathic MN. Measurement of circulating anti-PLA2R antibody is also very useful for the monitoring of MN activity. However, the mechanisms responsible for the formation of anti-PLA2R antibodies as well as those involved in the progression of MN to end-stage renal disease remain to be defined.

  5. Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.

    PubMed Central

    Grass, D S; Felkner, R H; Chiang, M Y; Wallace, R E; Nevalainen, T J; Bennett, C F; Swanson, M E

    1996-01-01

    Group II PLA2 has been implicated in inflammatory processes in both man and other animals and has been shown to be involved in inflammatory conditions, such as arthritis and sepsis. Transgenic mice expressing the human group II PLA2 gene have been generated using a 6.2-kb genomic fragment. These mice express the group II PLA2 gene abundantly in liver, lung, kidney, and skin, and have serum PLA2 activity levels approximately eightfold higher than nontransgenic littermates. The group II PLA2 transgenic mice reported here exhibit epidermal and adnexal hyperplasia, hyperkeratosis, and almost total alopecia. The chronic epidermal hyperplasia and hyperkeratosis seen in these mice is similar to that seen in a variety of dermatopathies, including psoriasis. However, unlike what is seen with these dermatopathies, no significant inflammatory-cell influx was observed in the skin of these animals, or in any other tissue examined. These mice provide an important tool for examining group II PLA2 expression, and for determining the role of group II PLA2 in normal and disease physiology. They serve as an in vivo model for identifying inhibitors of group II PLA2 activity and gene expression. PMID:8636402

  6. Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

    PubMed

    Park, Dae-Won; Kim, Jae-Ryong; Kim, Seong-Yong; Sonn, Jong-Kyung; Bang, Ok-Sun; Kang, Shin-Sung; Kim, Jung-Hye; Baek, Suk-Hwan

    2003-02-15

    The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

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

    PubMed

    Ferri, Nicola; Corsini, Alberto

    2014-12-01

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

  8. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor.

    PubMed Central

    MacPhee, C H; Moores, K E; Boyd, H F; Dhanak, D; Ife, R J; Leach, C A; Leake, D S; Milliner, K J; Patterson, R A; Suckling, K E; Tew, D G; Hickey, D M

    1999-01-01

    A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine. PMID:10024526

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

    PubMed

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

    2014-08-01

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

  10. Structural elements of ligand recognition site in secretory phospho-lipase A2 and structure-based design of specific inhibitors.

    PubMed

    Singh, Nagendra; Somvanshi, Rishi K; Sharma, Sujata; Dey, Sharmistha; Kaur, Punit; Singh, Tej P

    2007-01-01

    Phospholipases A2 (phosphotide 2-acylhydrolases, PLA2s, EC 3.1.1.4) are widely distributed enzymes in the animal world. They catalyze the hydrolysis of the sn-2 acyl ester linkage of phospholipids, producing fatty acids and lysophospholipids. The mammalian type II secreted phospholipase A2 (PLA2-II) is one of the most extensively studied member of low molecular weight (13-18 kDa) PLA2s. PLA2-II contains 120-125 amino acid residues and seven disulphide bridges. The important features of overall structure of PLA2-II contain an N-terminal helix, H1 (residues: 2-12), an external loop (residues: 14-23), a calcium binding loop (Ca2+-loop, residues: 25-35), a second alpha-helix, H2 (residues: 40-55), a short two stranded anti-parallel beta-sheet referred to as beta-wing (residues: 75-84), a third alpha-helix, H3 (residues: 90-108) which is antiparallel to H2 and two single helical turns, SH4 (residues: 114-117) and SH5 (residues: 121-125). The three-dimensional structure of PLA2-II has defined a conserved active site within a hydrophobic channel lined by invariant hydrophobic residues. The active site residues His48, Asp49, Tyr52 and Asp99 are directly connected to the channel. An important water molecule that bridges His48 and Asp49 through hydrogen bonds is a part of catalytic network. Based on the structures of various complexes of group II PLA2, the ligand-recognition site has been divided into six subsites consisting of residues 2-10 (subsite 1), residues 17-23 (subsite 2), residues 28-32 (subsite 3), residues 48-52 (subsite 4), residues 68-70 (subsite 5) and residues 98-106 (subsite 6). It is observed that most of the currently available ligands saturate only part of the ligand-recognition site leaving a wide scope to improve the ligand complementarity. Naturally, the ligands that interact with the largest number of subsites would also correspond to the maximum affinity. Therefore, for the design of potent inhibitors of PLA2, the stereochemical knowledge of the

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

    Franson, R C; Rosenthal, M D

    1997-01-01

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

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

    PubMed

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

    2005-01-01

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

  14. 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.

  15. iPLA2• Knockout Mouse, a Genetic Model for Progressive Human Motor Disorders, Develops Age-Related Neuropathology

    PubMed Central

    Blanchard, Helene; Taha, Ameer Y.; Cheon, Yewon; Kim, Hyung-Wook; Turk, John; Rapoport, Stanley I.

    2015-01-01

    Calcium-independent phospholipase A2 group VIa (iPLA2β) preferentially releases docosahexaenoic acid (DHA) from the sn-2 position of phospholipids. Mutations of its gene, PLA2G6, are found in patients with several progressive motor disorders, including Parkinson disease. At 4 months, PLA2G6 knockout mice (iPLA2β−/−) show minimal neuropathology but altered brain DHA metabolism. By 1 year, they develop motor disturbances, cerebellar neuronal loss, and striatal α-synuclein accumulation. We hypothesized that older iPLA2β−/− mice also would exhibit inflammatory and other neuropathological changes. Real-time polymerase chain reaction and Western blotting were performed on whole brain homogenate from 15 to 20-month old male iPLA2β−/− or wild-type (WT) mice. These older iPLA2β−/− mice compared with WT showed molecular evidence of microglial (CD-11b, iNOS) and astrocytic (glial fibrillary acidic protein) activation, disturbed expression of enzymes involved in arachidonic acid metabolism, loss of neuroprotective brain derived neurotrophic factor, and accumulation of cytokine TNF-α messenger ribonucleic acid, consistent with neuroinflammatory pathology. There was no evidence of synaptic loss, of reduced expression of dopamine active reuptake transporter, or of accumulation of the Parkinson disease markers Parkin or Pink1. iPLA2γ expression was unchanged. iPLA2β deficient mice show evidence of neuroinflammation and associated neuropathology with motor dysfunction in later life. These pathological biomarkers could be used to assess efficacy of dietary intervention, antioxidants or other therapies on disease progression in this mouse model of progressive human motor diseases associated with a PLA2G6 mutation. PMID:24919816

  16. Dual role of the p38 MAPK/cPLA2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

    PubMed Central

    Rukoyatkina, N; Mindukshev, I; Walter, U; Gambaryan, S

    2013-01-01

    p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A2 (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions. PMID:24263105

  17. 1-(5-Carboxyindol-1-yl)propan-2-ones as inhibitors of human cytosolic phospholipase A2alpha: synthesis and properties of bioisosteric benzimidazole, benzotriazole and indazole analogues.

    PubMed

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

    2009-04-15

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

  18. Neutralisation of the pharmacological activities of Bothrops alternatus venom by anti-PLA2 IgGs.

    PubMed

    Garcia Denegri, María E; Maruñak, Silvana; Todaro, Juan S; Ponce-Soto, Luis A; Acosta, Ofelia; Leiva, Laura

    2014-08-01

    Basic phospholipases A2 (PLA2) are toxic and induce a wide spectrum of pharmacological effects, although the acidic enzyme types are not lethal or cause low lethality. Therefore, it is challenging to elucidate the mechanism of action of acidic phospholipases. This study used the acidic non-toxic Ba SpII RP4 PLA2 from Bothrops alternatus as an antigen to develop anti-PLA2 IgG antibodies in rabbits and used in vivo assays to examine the changes in crude venom when pre-incubated with these antibodies. Using Ouchterlony and western blot analyses on B. alternatus venom, we examined the specificity and sensitivity of phospholipase A2 recognition by the specific antibodies (anti-PLA2 IgG). Neutralisation assays using a non-toxic PLA2 antigen revealed unexpected results. The (indirect) haemolytic activity of whole venom was completely inhibited, and all catalytically active phospholipases A2 were blocked. Myotoxicity and lethality were reduced when the crude venom was pre-incubated with anti-PLA2 immunoglobulins. CK levels in the skeletal muscle were significantly reduced at 6 h, and the muscular damage was more significant at this time-point compared to 3 and 12 h. When four times the LD50 was used (224 μg), half the animals treated with the venom-anti PLA2 IgG mixture survived after 48 h. All assays performed with the specific antibodies revealed that Ba SpII RP4 PLA2 had a synergistic effect on whole-venom toxicity. IgG antibodies against the venom of the Argentinean species B. alternatus represent a valuable tool for elucidation of the roles of acidic PLA2 that appear to have purely digestive roles and for further studies on immunotherapy and snake envenoming in affected areas in Argentina and Brazil.

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Park, Youngjin; Kim, Yonggyun; Stanley, David

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

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

    PubMed

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-08-01

    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.

  2. [Effect of calcium-independent phospholipase A(2) inhibitor in reducing hepatocyte lipoapoptosis and improving insulin resistance].

    PubMed

    Shi, H B; Fu, J F; Huang, Y; Liu, L R

    2017-01-20

    Objective: To investigate the effect of calcium-independent phospholipase A(2) (iPLA(2)) inhibitor in reducing hepatocyte lipoapoptosis and improving insulin resistance. Methods: A total of 28 male Sprague-Dawley rats were randomly divided into the following three groups: 12 rats in group I (normal control group) were given normal diet for 18 weeks; 8 rats in group II (non-alcoholic fatty liver disease model group) were given high-fat diet for 18 weeks; 8 rats in group III (iPLA(2) inhibitor group) were given high-fat diet for 18 weeks and intraperitoneal injection of the iPLA(2) inhibitor bromoenol lactone 150 μg/kg once every other day since week 15 (14 times of injection in total). All the rats were sacrificed at the same time, and body weight and liver weight were measured. Blood lipids, serum enzymes, fasting blood glucose, fasting insulin, free fatty acid, and serum iPLA(2) concentration were measured in each group, and liver pathological changes were evaluated. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to measure the level of hepatocyte apoptosis and the apoptotic index was calculated. Quantitative PCR was used to measure the mRNA expression of iPLA(2). The Student-Newman-Keuls test and the chi-square test were used for comparison of parameters between groups I, II, and III. P < 0.05 was considered statistically significant. Results: Compared with group I, group II had significant increases in triglyceride (0.75±0.05 mmol/L vs 1.20±0.13 mmol/L, P < 0.05), cholesterol (1.50±0.12 mmol/L vs 2.94±0.34 mmol/L, P < 0.05), low-density lipoprotein (0.65±0.06 mmol/L vs 1.30±0.16 mmol/L, P < 0.05), free fatty acid (0.58±0.09 mEq/L vs 0.80±0.20 mEq/L, P < 0.05), fasting blood glucose (4.85±0.22 mmol/L vs 6.94±0.65 mmol/L, P < 0.05), and fasting insulin (0.89±0.52 mmol/L vs 1.29±0.52 mmol/L, P < 0.05), and a significant reduction in the insulin sensitivity index (0.52±0.21 vs 0.27±0.11, P < 0.05); group II also had

  3. 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

  4. Discovery of a Novel Series of Imidazo[1,2-a]pyrimidine Derivatives as Potent and Orally Bioavailable Lipoprotein-Associated Phospholipase A2 Inhibitors.

    PubMed

    Chen, Xinde; Xu, Wenwei; Wang, Kai; Mo, Mingguang; Zhang, Wei; Du, Lili; Yuan, Xiaojing; Xu, Yechun; Wang, Yiping; Shen, Jianhua

    2015-11-12

    Inhibition of lipoprotein-associated phospholipase A2 (Lp-PLA2) has been suggested to be a promising therapeutic strategy for several inflammation-associated diseases, including atherosclerosis, Alzheimer's disease, and diabetic macular edema. Herein, we report the discovery of a novel series of Lp-PLA2 inhibitors constructed on an imidazo[1,2-a]pyrimidine scaffold through a conformational restriction strategy. Structure-activity relationship (SAR) analysis resulted in the identification of several compounds with high potency in vitro and good metabolic stability in liver S9 fractions. Compounds 7c and 14b selected for further exploration in vivo demonstrated excellent pharmacokinetic profiles and exhibited significant inhibitory efficacy in SD rats upon oral dosing.

  5. Cross-talk between p(38)MAPK and G iα in regulating cPLA 2 activity by ET-1 in pulmonary smooth muscle cells.

    PubMed

    Chakraborti, Sajal; Chowdhury, Animesh; Chakraborti, Tapati

    2015-02-01

    Endothelin-1 (ET-1) is known as the most potent vasoconstrictor yet described. Infusion of ET-1 into isolated rabbit lung has been shown to cause pulmonary vasoconstriction with the involvement of arachidonic acid metabolites. Given the potency of arachidonic acid metabolites, the activity of phospholipase A2 must be tightly regulated. Herein, we determined the mechanisms by which ET-1 stimulates cPLA2 activity during ET-1 stimulation of bovine pulmonary artery smooth muscle cells. We demonstrated that (i) treatment of bovine pulmonary artery smooth muscle cells with ET-1 stimulates cPLA2 activity in the cell membrane; (ii) ET-1 caused increase in O 2 (·-) production occurs via NADPH oxidase-dependent mechanism; (iii) ET-1-stimulated NADPH oxidase activity is markedly prevented upon pretreatment with PKC-ζ inhibitor, indicating that PKC-ζ plays a prominent role in this scenario; (iv) ET-1-induced NADPH oxidase-derived O 2 (·-) stimulates an aprotinin sensitive protease activity due to prominent increase in [Ca(2+)]i; (v) the aprotinin sensitive protease plays a pivotal role in activating PKC-α, which in turn phosphorylates p(38)MAPK and subsequently Giα leading to the activation of cPLA2. Taken together, we suggest that cross-talk between p(38)MAPK and Giα with the involvement of PKC-ζ, NADPH oxidase-derived O 2 (·-) , [Ca(2+)]i, aprotinin-sensitive protease and PKC-α play a pivotal role for full activation of cPLA2 during ET-1 stimulation of pulmonary artery smooth muscle cells.

  6. Determination of arachidonic acid by on-line solid-phase extraction HPLC with UV detection for screening of cytosolic phospholipase A2α inhibitors.

    PubMed

    Hanekamp, Walburga; Lehr, Matthias

    2012-07-01

    An on-line solid-phase extraction (SPE)-liquid chromatographic method with ultraviolet detection at 200nm for screening of inhibitors of cytosolic phospholipase A(2)α (cPLA(2)α) was developed and validated. cPLA(2)α was isolated from porcine platelets. Enzyme activity was determined by measuring the release of arachidonic acid from a phospholipid substrate using automated on-line sample clean up on a trap column followed by isocratic back-flush elution on a RP18 analytical column. While the use of a conventional RP18 column for trapping the analyte led to peak broadening only after a few runs due to pollution of the column by binding of components present in the enzyme preparation, the application of a turbulent flow column (TurboFlow Cyclone™) resulted in sharp peaks even after a plurality of injections. Interestingly, for sample introduction a turbulent flow of the mobile phase produced by high flow rates was not necessary to maintain good peak shapes. The same result could also be achieved applying low flow rates (0.5 mL/min). Several known cPLA(2inhibitors were used to validate the test system.

  7. 1-(3-biaryloxy-2-oxopropyl)indole-5-carboxylic acids and related compounds as dual inhibitors of human cytosolic phospholipase A2α and fatty acid amide hydrolase.

    PubMed

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

    2011-03-07

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Effects of overweight and the PLA2G7 V279F polymorphism on the association of age with systolic blood pressure

    PubMed Central

    Kim, Minjoo; Kim, Minkyung; Yoo, Hye Jin; Jang, Hye Young; Lee, Sang-Hyun

    2017-01-01

    This prospective study aimed to determine the effects of the persistence of overweight for three years and the PLA2G7 V279F polymorphism, as well as the interaction between these factors, on the association of age with blood pressure (BP). Healthy middle-aged subjects with normotensive BP were divided into the normal-weight and overweight groups. The PLA2G7 V279F genotype, BP, lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, and oxidized low-density lipoprotein (ox-LDL) were determined. Lp-PLA2 activity was lower in the F allele subjects (n = 111) than in those with the VV genotype (n = 389). The overweight individuals with the F allele had lower Lp-PLA2 activity and ox-LDL at both baseline and after three years and lower systolic and diastolic BP and LDL cholesterol after three years compared with those with the VV phenotype. After three years, the overweight subjects with the VV phenotype exhibited greater increases in Lp-PLA2 activity, systolic BP, and ox-LDL than those with the F allele and normal-weight subjects with the VV phenotype. A multivariate analysis revealed that the PLA2G7 V279F genotype, baseline BMI, changes in Lp-PLA2 activity and ox-LDL remained independently and positively associated with changes in systolic BP. The simultaneous presence of the PLA2G7 279VV genotype and persistence of overweight synergistically increases the risk for hypertension, whereas lower Lp-PLA2 activity in PLA2G7 279F allele carriers might offer certain protection against hypertension, even in individuals who have been overweight for over three years. PMID:28334001

  10. Possible involvement of aiPLA2 in the phosphatidylserine-containing liposomes induced production of PGE2 and PGD2 in microglia.

    PubMed

    Takayama, Fumiko; Wu, Zhou; Ma, Hong Mei; Okada, Ryo; Hayashi, Yoshinori; Nakanishi, Hiroshi

    2013-09-15

    Liposomes containing phosphatidylserine (PSL) produce PGE2 after being phagocytosed by microglia, but the precise underlying mechanism behind it still remains unclear. Here, we showed that liposomes consisting of phosphatidylserine and lysophosphatidylcholine, a lipolysis product of phosphatidylcholine by PLA2, were phagocytosed by microglia, but failed to induce secretion of PGE2. Furthermore, PSL-induced PGE2 secretion was significantly inhibited by MJ33, an aiPLA2 inhibitor, but not by AACOCF3, a cPLA2 inhibitor. PSL also produced PGD2 and 15d-PGJ2 in microglia. We thus hypothesize that free arachidonic acid is supplied through aiPLA2-mediated lipolysis of phagocytosed phosphatidylcholine, leading to the production of PGH2 and its downstream metabolites.

  11. Prognostic value of PLA2R autoimmunity detected by measurement of anti-PLA2R antibodies combined with detection of PLA2R antigen in membranous nephropathy: A single-centre study over 14 years

    PubMed Central

    Mihout, Fabrice; Cachanado, Marine; Brocheriou, Isabelle

    2017-01-01

    Introduction Clinical course of membranous nephropathy (MN) is difficult to predict. Measurement of circulating anti-PLA2R autoantibodies (PLA2R-Ab) and detection in immune deposits of PLA2R antigen (PLA2R-Ag) are major advances in disease understanding. We evaluated the clinical significance of these biomarkers. Methods In this 14-year retrospective study, we collected data from 108 MN patients and assessed the relationship between clinical course, PLA2R-Ab and PLA2R-Ag. We also assessed THSD7A status. Results Eighty-five patients suffered from primary MN (PMN) and 23 patients from a secondary form. The median follow-up was 30.4 months [interquartile range, 17.7;56.7]. Among the 77 patients with PMN and available serum and/or biopsy, 69 (89.6%) had PLA2R-related disease as shown by anti-PLA2R-Ab and/or PLA2R-Ag, while 8 patients (8/77, 10.4%) were negative for both. There was no significant difference between these two groups in age at diagnosis and outcome assessed by proteinuria, serum albumin level and eGFR. Two of the 8 negative patients were positive for THSD7A. In patients with PLA2R related PMN, younger age, lower proteinuria, higher eGFR, and lower PLA2R-Ab level at baseline and after 6 months were associated with remission of proteinuria. Initial PLA2R-Ab titer ≤ 97.6 RU/mL and complete depletion of PLA2R-Ab within 6-months were significantly associated with spontaneous remission at the end of follow-up. In rituximab treated patients, lower PLA2R-Ab titer at initiation of treatment, and absence of PLA2R-Ab and higher serum albumin level at 3 months were significantly associated with remission. Noticeably, 81.8% of the patients who achieved remission completely cleared PLA2R-Ab. Depletion of PLA2R-Ab and increase of serum albumin level preceded the decrease of proteinuria. Conclusion Assessment of PLA2R autoimmunity is essential for patient management. Combination of PLA2R-Ab and PLA2R-Ag increases diagnosis sensitivity. PLA2R-Ab titer is a biomarker of

  12. Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells.

    PubMed

    Caporarello, Nunzia; Salmeri, Mario; Scalia, Marina; Motta, Carla; Parrino, Cristina; Frittitta, Lucia; Olivieri, Melania; Cristaldi, Martina; Avola, Roberto; Bramanti, Vincenzo; Toscano, Maria Antonietta; Anfuso, Carmelina Daniela; Lupo, Gabriella

    2016-01-01

    It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

  13. Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells

    PubMed Central

    Scalia, Marina; Motta, Carla; Parrino, Cristina; Frittitta, Lucia; Olivieri, Melania; Cristaldi, Martina; Avola, Roberto; Bramanti, Vincenzo; Toscano, Maria Antonietta; Anfuso, Carmelina Daniela; Lupo, Gabriella

    2016-01-01

    It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion. PMID:27631977

  14. Cytosolic Phospholipase A2 and Lysophospholipids in Tumor Angiogenesis

    PubMed Central

    Linkous, Amanda G.

    2010-01-01

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

  15. A dangerous liaison: Leptin and sPLA2-IIA join forces to induce proliferation and migration of astrocytoma cells

    PubMed Central

    Martín, Rubén; Cordova, Claudia; Gutiérrez, Beatriz; Hernández, Marita; Nieto, María L.

    2017-01-01

    Glioblastoma, the most aggressive type of primary brain tumour, shows worse prognosis linked to diabetes or obesity persistence. These pathologies are chronic inflammatory conditions characterized by altered profiles of inflammatory mediators, including leptin and secreted phospholipase A2-IIA (sPLA2-IIA). Both proteins, in turn, display diverse pro-cancer properties in different cell types, including astrocytes. Herein, to understand the underlying relationship between obesity and brain tumors, we investigated the effect of leptin, alone or in combination with sPLA2-IIA on astrocytoma cell functions. sPLA2-IIA induced up-regulation of leptin receptors in 1321N1 human astrocytoma cells. Leptin, as well as sPLA2-IIA, increased growth and migration in these cells, through activation/phosphorylation of key proteins of survival cascades. Leptin, at concentrations with minimal or no activating effects on astrocytoma cells, enhanced growth and migration promoted by low doses of sPLA2-IIA. sPLA2-IIA alone induced a transient phosphorylation pattern in the Src/ERK/Akt/mTOR/p70S6K/rS6 pathway through EGFR transactivation, and co-addition of leptin resulted in a sustained phosphorylation of these signaling regulators. Mechanistically, EGFR transactivation and tyrosine- and serine/threonine-protein phosphatases revealed a key role in this leptin-sPLA2-IIA cross-talk. This cooperative partnership between both proteins was also found in primary astrocytes. These findings thus indicate that the adipokine leptin, by increasing the susceptibility of cells to inflammatory mediators, could contribute to worsen the prognosis of tumoral and neurodegenerative processes, being a potential mediator of some obesity-related medical complications. PMID:28249041

  16. A dangerous liaison: Leptin and sPLA2-IIA join forces to induce proliferation and migration of astrocytoma cells.

    PubMed

    Martín, Rubén; Cordova, Claudia; Gutiérrez, Beatriz; Hernández, Marita; Nieto, María L

    2017-01-01

    Glioblastoma, the most aggressive type of primary brain tumour, shows worse prognosis linked to diabetes or obesity persistence. These pathologies are chronic inflammatory conditions characterized by altered profiles of inflammatory mediators, including leptin and secreted phospholipase A2-IIA (sPLA2-IIA). Both proteins, in turn, display diverse pro-cancer properties in different cell types, including astrocytes. Herein, to understand the underlying relationship between obesity and brain tumors, we investigated the effect of leptin, alone or in combination with sPLA2-IIA on astrocytoma cell functions. sPLA2-IIA induced up-regulation of leptin receptors in 1321N1 human astrocytoma cells. Leptin, as well as sPLA2-IIA, increased growth and migration in these cells, through activation/phosphorylation of key proteins of survival cascades. Leptin, at concentrations with minimal or no activating effects on astrocytoma cells, enhanced growth and migration promoted by low doses of sPLA2-IIA. sPLA2-IIA alone induced a transient phosphorylation pattern in the Src/ERK/Akt/mTOR/p70S6K/rS6 pathway through EGFR transactivation, and co-addition of leptin resulted in a sustained phosphorylation of these signaling regulators. Mechanistically, EGFR transactivation and tyrosine- and serine/threonine-protein phosphatases revealed a key role in this leptin-sPLA2-IIA cross-talk. This cooperative partnership between both proteins was also found in primary astrocytes. These findings thus indicate that the adipokine leptin, by increasing the susceptibility of cells to inflammatory mediators, could contribute to worsen the prognosis of tumoral and neurodegenerative processes, being a potential mediator of some obesity-related medical complications.

  17. Genome-Wide Association Study of Lp-PLA2 Activity and Mass in the Framingham Heart Study

    PubMed Central

    Suchindran, Sunil; Rivedal, David; Guyton, John R.; Milledge, Tom; Gao, Xiaoyi; Benjamin, Ashlee; Rowell, Jennifer; Ginsburg, Geoffrey S.; McCarthy, Jeanette J.

    2010-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an emerging risk factor and therapeutic target for cardiovascular disease. The activity and mass of this enzyme are heritable traits, but major genetic determinants have not been explored in a systematic, genome-wide fashion. We carried out a genome-wide association study of Lp-PLA2 activity and mass in 6,668 Caucasian subjects from the population-based Framingham Heart Study. Clinical data and genotypes from the Affymetrix 550K SNP array were obtained from the open-access Framingham SHARe project. Each polymorphism that passed quality control was tested for associations with Lp-PLA2 activity and mass using linear mixed models implemented in the R statistical package, accounting for familial correlations, and controlling for age, sex, smoking, lipid-lowering-medication use, and cohort. For Lp-PLA2 activity, polymorphisms at four independent loci reached genome-wide significance, including the APOE/APOC1 region on chromosome 19 (p = 6×10−24); CELSR2/PSRC1 on chromosome 1 (p = 3×10−15); SCARB1 on chromosome 12 (p = 1×10−8) and ZNF259/BUD13 in the APOA5/APOA1 gene region on chromosome 11 (p = 4×10−8). All of these remained significant after accounting for associations with LDL cholesterol, HDL cholesterol, or triglycerides. For Lp-PLA2 mass, 12 SNPs achieved genome-wide significance, all clustering in a region on chromosome 6p12.3 near the PLA2G7 gene. Our analyses demonstrate that genetic polymorphisms may contribute to inter-individual variation in Lp-PLA2 activity and mass. PMID:20442857

  18. PLA2G6 mutations and other rare causes of neurodegeneration with brain iron accumulation.

    PubMed

    McNeill, Alisdair

    2012-08-01

    There is a wide variety of genetic and sporadic causes for neurodegenerative disorders with apparent brain iron accumulation on magnetic resonance imaging. Rare recessive causes include PLA2G6 mutations (infantile neuroaxonal dystrophy), and mutations of ATP13A2 (Kufor Rakeb syndrome) and FA2H. A variety of sporadic neurological disorders can present brain iron accumulation on imaging, including multiple sclerosis and neurological manifestations of HIV infection. The relevant clinical and imaging features will be discussed.

  19. Design, synthesis, and biological evaluation of 3-(1-Aryl-1H-indol-5-yl)propanoic acids as new indole-based cytosolic phospholipase A2α inhibitors.

    PubMed

    Tomoo, Toshiyuki; Nakatsuka, Takashi; Katayama, Toyoko; Hayashi, Yasuhiro; Fujieda, Yusuke; Terakawa, Maki; Nagahira, Kazuhiro

    2014-09-11

    This article describes the design, synthesis, and biological evaluation of new indole-based cytosolic phospholipase A2α (cPLA2α, a group IVA phospholipase A2) inhibitors. A screening-hit compound from our library, (E)-3-{4-[(4-chlorophenyl)thio]-3-nitrophenyl}acrylic acid (5), was used to design a class of 3-(1-aryl-1H-indol-5-yl)propanoic acids as new small molecule inhibitors. The resultant structure-activity relationships studied using the isolated enzyme and by cell-based assays revealed that the 1-(p-O-substituted)phenyl, 3-phenylethyl, and 5-propanoic acid groups on the indole core are essential for good inhibitory activity against cPLA2α. Optimization of the p-substituents on the N1 phenyl group led to the discovery of 56n (ASB14780), which was shown to be a potent inhibitor of cPLA2α via enzyme assay, cell-based assay, and guinea pig and human whole-blood assays. It displayed oral efficacy toward mice tetradecanoyl phorbol acetate-induced ear edema and guinea pig ovalbumin-induced asthma models.

  20. Improved Therapeutic Profiles of PLA2-Free Bee Venom Prepared by Ultrafiltration Method

    PubMed Central

    Lee, Hyunkyoung; Pyo, Min-Jung; Bae, Seong Kyeong; Heo, Yunwi; Kim, Choul Goo; Kang, Changkeun

    2015-01-01

    Bee venom (BV) has long been used in traditional Eastern and Western medicine for chronic inflammation, pain and skin therapy. Human exposure to BV, however, often causes unwanted adverse effects and is even fatal in some cases. Phospholipase A2 (PLA2) of BV is now suspected to play a key role in these adverse effects. We investigated the potential use of PLA2-free bee venom (PBV) as a replacement for BV in cosmetic products. PBV prepared by molecular weight cut-off ultrafiltration exhibits a superior profile in comparison with regular BV, by inhibiting elastase activity and suppressing the induction of nitric oxide (NO) and metalloproteinase-9 (MMP-9), while retaining the effects of cell proliferation and protection against ultraviolet B (UVB)-induced damage in human dermal fibroblast cells. PBV thus appears to be more promising than BV as a cosmetic ingredient with a reduced potential for adverse reactions in the recipient. PMID:25874031

  1. The role of lipoprotein-associated phospholipase A2 in a murine model of experimental autoimmune uveoretinitis.

    PubMed

    Crawford, G L; Boldison, J; Copland, D A; Adamson, P; Gale, D; Brandt, M; Nicholson, L B; Dick, A D

    2015-01-01

    Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp-PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity.

  2. Genetic modulation of islet β-cell iPLA2β expression provides evidence for its impact on β-cell apoptosis and autophagy

    PubMed Central

    Lei, Xiaoyong; Bone, Robert N.; Ali, Tomader; Wohltmann, Mary; Gai, Ying; Goodwin, Karen J.; Bohrer, Alan E.; Turk, John; Ramanadham, Sasanka

    2013-01-01

    β-cell apoptosis is a significant contributor to β-cell dysfunction in diabetes and ER stress is among the factors that contributes to β-cell death. We previously identified that the Ca2+-independent phospholipase A2β (iPLA2β), which in islets is localized in β-cells, participates in ER stress-induced β-cell apoptosis. Here, direct assessment of iPLA2β role was made using β-cell-specific iPLA2β overexpressing (RIP-iPLA2β-Tg) and globally iPLA2β-deficient (iPLA2β-KO) mice. Islets from Tg, but not KO, express higher islet iPLA2β and neutral sphingomyelinase, decrease in sphingomyelins, and increase in ceramides, relative to WT group. ER stress induces iPLA2β, ER stress factors, loss of mitochondrial membrane potential (∆Ψ), caspase-3 activation, and β-cell apoptosis in the WT and these are all amplified in the Tg group. Surprisingly, β-cells apoptosis while reduced in the KO is higher than in the WT group. This, however, was not accompanied by greater caspase-3 activation but with larger loss of ∆Ψ, suggesting that iPLA2β deficiency impacts mitochondrial membrane integrity and causes apoptosis by a caspase-independent manner. Further, autophagy, as reflected by LC3-II accumulation, is increased in Tg and decreased in KO, relative to WT. Our findings suggest that (1) iPLA2β impacts upstream (UPR) and downstream (ceramide generation and mitochondrial) pathways in β-cells and (2) both over- or under-expression of iPLA2β is deleterious to the β-cells. Further, we present for the first time evidence for potential regulation of autophagy by iPLA2β in islet β-cells. These findings support the hypothesis that iPLA2β induction under stress, as in diabetes, is a key component to amplifying β-cell death processes. PMID:23411472

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

    PubMed

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

    2016-07-03

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

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

    SciTech Connect

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

    2016-07-01

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

  5. ASB14780, an Orally Active Inhibitor of Group IVA Phospholipase A2, Is a Pharmacotherapeutic Candidate for Nonalcoholic Fatty Liver Disease.

    PubMed

    Kanai, Shiho; Ishihara, Keiichi; Kawashita, Eri; Tomoo, Toshiyuki; Nagahira, Kazuhiro; Hayashi, Yasuhiro; Akiba, Satoshi

    2016-03-01

    We have previously shown that high-fat cholesterol diet (HFCD)-induced fatty liver and carbon tetrachloride (CCl4)-induced hepatic fibrosis are reduced in mice deficient in group IVA phospholipase A2 (IVA-PLA2), which plays a role in inflammation. We herein demonstrate the beneficial effects of ASB14780 (3-[1-(4-phenoxyphenyl)-3-(2-phenylethyl)-1H-indol-5-yl]propanoic acid 2-amino-2-(hydroxymethyl)propane-1,3-diol salt), an orally active IVA-PLA2 inhibitor, on the development of fatty liver and hepatic fibrosis in mice. The daily coadministration of ASB14780 markedly ameliorated liver injury and hepatic fibrosis following 6 weeks of treatment with CCl4. ASB14780 markedly attenuated the CCl4-induced expression of smooth muscle α-actin (α-SMA) protein and the mRNA expression of collagen 1a2, α-SMA, and transforming growth factor-β1 in the liver, and inhibited the expression of monocyte/macrophage markers, CD11b and monocyte chemotactic protein-1, while preventing the recruitment of monocytes/macrophages to the liver. Importantly, ASB14780 also reduced the development of fibrosis even in matured hepatic fibrosis. Additionally, ASB14780 also reduced HFCD-induced lipid deposition not only in the liver, but also in already established fatty liver. Furthermore, treatment with ASB14780 suppressed the HFCD-induced expression of lipogenic mRNAs. The present findings suggest that an IVA-PLA2 inhibitor, such as ASB14780, could be useful for the treatment of nonalcoholic fatty liver diseases, including fatty liver and hepatic fibrosis.

  6. Lipoprotein-associated phospholipase A2 prognostic role in atherosclerotic complications

    PubMed Central

    Maiolino, Giuseppe; Bisogni, Valeria; Rossitto, Giacomo; Rossi, Gian Paolo

    2015-01-01

    Atherosclerosis manifests itself clinically at advanced stages when plaques undergo hemorrhage and/or rupture with superimposed thrombosis, thus abruptly stopping blood supply. Identification of markers of plaque destabilization at a pre-clinical stage is, therefore, a major goal of cardiovascular research. Promising results along this line were provided by studies investigating the lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of phospholipase A2 proteins family that plays a key role in the metabolism of pro-inflammatory phospholipids, as oxidized low-density lipoproteins, and in the generation of pro-atherogenic metabolites, including lysophosphatidylcholine and oxidized free fatty acids. We herein review the experimental and clinical studies supporting use of Lp-PLA2 activity for predicting cardiovascular events. To his end we considered not only Lp-PLA2 activity and mass, but also Lp-PLA2 gene variations and their association with incident coronary artery disease, stroke, and cardiovascular mortality. Based on these evidences the major scientific societies have included in their guidelines the measurement of Lp-PLA2 activity among the biomarkers that are useful in risk stratification of adult asymptomatic patients at intermediate cardiovascular risk. The results of two recently published major clinical trials with the Lp-PLA2 inhibitor darapladib, which seem to challenge the pathogenic role of Lp-PLA2, will also be discussed. PMID:26516415

  7. Lipoprotein-associated phospholipase A2 prognostic role in atherosclerotic complications.

    PubMed

    Maiolino, Giuseppe; Bisogni, Valeria; Rossitto, Giacomo; Rossi, Gian Paolo

    2015-10-26

    Atherosclerosis manifests itself clinically at advanced stages when plaques undergo hemorrhage and/or rupture with superimposed thrombosis, thus abruptly stopping blood supply. Identification of markers of plaque destabilization at a pre-clinical stage is, therefore, a major goal of cardiovascular research. Promising results along this line were provided by studies investigating the lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of phospholipase A2 proteins family that plays a key role in the metabolism of pro-inflammatory phospholipids, as oxidized low-density lipoproteins, and in the generation of pro-atherogenic metabolites, including lysophosphatidylcholine and oxidized free fatty acids. We herein review the experimental and clinical studies supporting use of Lp-PLA2 activity for predicting cardiovascular events. To his end we considered not only Lp-PLA2 activity and mass, but also Lp-PLA2 gene variations and their association with incident coronary artery disease, stroke, and cardiovascular mortality. Based on these evidences the major scientific societies have included in their guidelines the measurement of Lp-PLA2 activity among the biomarkers that are useful in risk stratification of adult asymptomatic patients at intermediate cardiovascular risk. The results of two recently published major clinical trials with the Lp-PLA2 inhibitor darapladib, which seem to challenge the pathogenic role of Lp-PLA2, will also be discussed.

  8. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-02

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

  11. Non-steroidal anti-inflammatory drugs as potent inhibitors of phospholipase A2: structure of the complex of phospholipase A2 with niflumic acid at 2.5 Angstroms resolution.

    PubMed

    Jabeen, Talat; Singh, Nagendra; Singh, Rajendra K; Sharma, Sujata; Somvanshi, Rishi K; Dey, Sharmistha; Singh, Tej P

    2005-12-01

    Phospholipase A(2) (PLA(2); EC 3.1.3.4) catalyzes the first step of the production of proinflammatory compounds collectively known as eicosanoids. The binding of phospholipid substrates to PLA(2) occurs through a well formed hydrophobic channel. Surface plasmon resonance studies have shown that niflumic acid binds to Naja naja sagittifera PLA(2) with an affinity that corresponds to a dissociation constant (K(d)) of 4.3 x 10(-5) M. Binding studies of PLA(2) with niflumic acid were also carried out using a standard PLA(2) kit that gave an approximate binding constant, K(i), of 1.26 +/- 0.05 x 10(-6) M. Therefore, in order to establish the viability of PLA(2) as a potential target molecule for drug design against inflammation, arthritis and rheumatism, the three-dimensional structure of the complex of PLA(2) with the known anti-inflammatory agent niflumic acid [2-[3-(trifluoromethyl)anilino]nicotinic acid] has been determined at 2.5 Angstroms resolution. The structure of the complex has been refined to an R factor of 0.187. The structure determination reveals the presence of one niflumic acid molecule at the substrate-binding site of PLA(2). It shows that niflumic acid interacts with the important active-site residues His48 and Asp49 through two water molecules. It is observed that the niflumic acid molecule is completely buried in the substrate-binding hydrophobic channel. The conformations of the binding site in PLA(2) as well as that of niflumic acid are not altered upon binding. However, the orientation of the side chain of Trp19, which is located at the entry of the substrate-binding site, has changed from that found in the native PLA(2), indicating its familiar role.

  12. Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases

    PubMed Central

    Layne, Joseph D.; Shridas, Preetha; Webb, Nancy R.

    2015-01-01

    Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells. PMID:25623068

  13. The 763C>G Polymorphism of The Secretory PLA2IIa Gene Is Associated with Endometriosis in Iranian Women

    PubMed Central

    Sahmani, Mehdi; Darabi, Masoud; Darabi, Maryam; Dabaghi, Talaat; Alizadeh, Safar Ali; Najafipour, Reza

    2015-01-01

    Background Endometriosis is a chronic gynecological disease resulting from complex interactions between genetic, hormonal, environmental and oxidative stress and intrinsic inflammatory components. The aim of this study was to investigate the potential association of the 763C>G polymorphism in the secretory phospholipase A2 group IIa gene (PLA2G2A) with the risk of endometriosis in Iranian women. Materials and Methods Ninety seven patients with endometriosis along with 107 women who were negative for endometriosis after laparoscopy and laparatomy, and served as the control group, were enrolled for this cross-sectional study. Samples were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. Results Multivariate analysis was used to examine the association between the risk of endometriosis and the 763C>G polymorphism of PLA2G2A. Genotype distributions of PLA2G2A were significantly different between patients and the controls (p<0.001, OR=0.22, 95% CI=0.21-0.39). Correlation analysis showed that there was a significant association between the normal homozygous genotype and susceptibility to endometriosis (p<0.001). Conclusion The present study suggests that the 763C>G polymorphism of PLA2G2A plays an important role as an independent factor in the risk of endometriosis in Iranian women. PMID:25780526

  14. HLA-DRB1*15:01 and HLA-DRB3*02:02 in PLA2R-Related Membranous Nephropathy.

    PubMed

    Le, Wei-Bo; Shi, Jing-Song; Zhang, Tao; Liu, Lei; Qin, Hua-Zhang; Liang, ShaoShan; Zhang, Yuan-Wei; Zheng, Cun-Xia; Jiang, Song; Qin, Wei-Song; Zhang, Hai-Tao; Liu, Zhi-Hong

    2016-12-27

    Idiopathic membranous nephropathy (MN) is associated with HLA; however, the HLA allele involved remains unknown. To identify the HLA risk alleles associated with phospholipase A2 receptor (PLA2R)-related MN in the Chinese population, we sequenced the entire MHC region in DNA samples from 99 patients with PLA2R-related MN, 50 patients with PLA2R-unrelated MN, and 100 healthy subjects. Two HLA risk alleles, HLA-DRB1*15:01 and HLA-DRB3*02:02, independently and strongly associated with an increased risk of PLA2R-related MN. After adjusting for HLA-DRB1*15:01 and HLA-DRB3*02:02, no other alleles showed significant association with PLA2R-related MN. A replication study in an independent cohort of 293 participants with PLA2R-related MN and 285 healthy controls validated these findings. In a joint analysis, a multivariate logistic regression model confirmed that HLA-DRB1*15:01 (odds ratio [OR], 24.9; 95% confidence interval [95% CI], 15.3 to 42.6; P=2.3×10(-35)) and HLA-DRB3*02:02 (OR, 17.7; 95% CI, 11.0 to 30.3; P=8.0×10(-29)) independently and strongly associated with PLA2R-related MN. As many as 98.7% of patients with PLA2R-related MN, compared with 43.9% of control subjects, carried at least one HLA risk allele. Subjects with either risk allele had higher odds of developing PLA2R-related MN than those without a risk allele (OR, 98.9; 95% CI, 44.4 to 281.7; P=2.5×10(-23)). These HLA risk alleles also associated with the age at disease onset in patients with PLA2R-related MN. In conclusion, our findings provide clear evidence that the HLA-DRB1*15:01 and HLA-DRB3*02:02 alleles independently and strongly associate with PLA2R-related MN in the Chinese population.

  15. PLA2G16 promotes osteosarcoma metastasis and drug resistance via the MAPK pathway.

    PubMed

    Li, Lin; Liang, Shoulei; Wasylishen, Amanda R; Zhang, Yanqin; Yang, Xueli; Zhou, Bingzheng; Shan, Luling; Han, Xiuxin; Mu, Tianyang; Wang, Guowen; Xiong, Shunbin

    2016-04-05

    The prognosis of metastatic osteosarcoma is dismal and a better understanding of the mechanisms underlying disease progression is essential to improve treatment options and patient outcomes. We previously demonstrated Pla2g16 overexpression in mouse osteosarcoma contributes to metastasis phenotypes and increased expression of PLA2G16 is associated with metastasis and poor prognosis in human tumors. To further examine the mechanisms through which PLA2G16 contributes to human osteosarcoma metastasis and explore the potential of PLA2G16 as a therapeutic target in osteosarcoma, we generated a panel of human osteosarcoma cell lines expressing different levels of PLA2G16. The functional analyses of these cell lines demonstrated high levels of PLA2G16 expression increased osteosarcoma cell migration, invasion, clonogenic survival, and anchorage-independent colony formation. Importantly, this activity was dependent on the phospholipase activity of PLA2G16. Additionally, PLA2G16 overexpression decreased the sensitivity of cells to a panel of chemotherapeutic agents. Analysis of downstream pathways revealed the pro-metastasis functions of PLA2G16 were mediated through the MAPK pathway, as knockdown of PLA2G16 decreased ERK1/2 phosphorylation and pharmacological inhibition of MEK significantly repressed PLA2G16 mediated cell migration and clonogenic survival. Furthermore, PLA2G16 overexpression promoted xenograft tumor growth in vivo, and these tumors exhibit increased ERK1/2 phosphorylation. Lastly, the expression of PLA2G16 is strongly correlated with the increased ERK1/2 phosphorylation in human osteosarcoma samples, and the combined lesions are associated with reduced overall and metastasis-free survival. Collectively, these results demonstrate increased PLA2G16 expression activates the MAPK pathway to enhance osteosarcoma metastasis and may be a novel therapeutic target for these cancers.

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

    PubMed

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

    2010-12-01

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

  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. 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

  19. 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.

  20. Ectopically expressed pro-group X secretory phospholipase A2 is proteolytically activated in mouse adrenal cells by furin-like proprotein convertases: implications for the regulation of adrenal steroidogenesis.

    PubMed

    Layne, Joseph D; Shridas, Preetha; Webb, Nancy R

    2015-03-20

    Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.

  1. Novel transglutaminase inhibitors reverse the inflammation of allergic conjunctivitis

    PubMed Central

    Sohn, Joonhong; Kim, Tae-Im; Yoon, Young-Hee; Kim, Joo-Yong; Kim, Soo-Youl

    2003-01-01

    Steroidal anti-inflammatory drugs induce proteins that inhibit phospholipase A2 (PLA2), including uteroglobin and lipocortin-1 (annexin I). Uteroglobin and lipocortin-1 retain several conserved sequences. Based on these sequences, several nonapeptides (antiflammins) were synthesized. These nonapeptides were shown to have anti-inflammatory effects in vitro and in vivo, possibly by inhibiting PLA2. Subsequent research showed that PLA2 is activated by transglutaminase 2 (TGase 2). We hypothesize here that TGase 2 inhibitors may increase the anti-inflammatory efficacy of inhibiting PLA2 activity. To test this theory, we constructed recombinant peptides containing sequences from pro-elafin (for inhibition of TGase 2), and from lipocortin-1, lipocortin-5, and uteroglobin (for inhibition of PLA2). The recombinant peptides, which had dual inhibitory effects on purified TGase 2 and PLA2, reversed the inflammation of allergic conjunctivitis to ragweed in a guinea pig model. The present work suggests that novel recombinant peptides may be safe and effective agents for the treatment of various inflammatory diseases. PMID:12511595

  2. A Role for Phospholipase A2 Activity in Membrane Tubule Formation and TGN Trafficking

    PubMed Central

    Schmidt, John A.; Kalkofen, Danielle N.; Donovan, Kirk W.; Brown, William J.

    2015-01-01

    We have investigated the role of phospholipase A2 (PLA2) enzymes in generating membrane tubules at the trans-Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over-expressing kinase dead protein kinase D were inhibited by the PLA2 inhibitors ONO-RS-082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes. Finally, use of the reversible antagonist ONO and time-lapse imaging revealed for the first time that PLA2 antagonists inhibit the initiation of membrane tubule formation at the TGN. Thus, PLA2 enzymes appear to have an important role in the earliest steps of membrane tubule formation at the TGN, which are utilized for membrane trafficking. PMID:20874826

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

    PubMed

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

    2007-04-03

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

  4. Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

    PubMed Central

    Lin, Wan-W; Chen, Bin C

    1998-01-01

    The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively.PMA at 1 μM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%.Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII,

  5. Insights on the structure of native CNF, an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake.

    PubMed

    Fortes-Dias, Consuelo Latorre; Ortolani, Paula Ladeira; Fernandes, Carlos Alexandre H; Lobo, Kelli Roberta; Amaral de Melo, Lutiana; Borges, Márcia Helena; Pazin, Wallance Moreira; Neto, Mário de Oliveira; Fernandez, Roberto Morato; Fontes, Marcos Roberto M

    2014-09-01

    Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

  6. sPLA2 -IIA Overexpression in Mice Epidermis Depletes Hair Follicle Stem Cells and Induces Differentiation Mediated Through Enhanced JNK/c-Jun Activation.

    PubMed

    Sarate, Rahul M; Chovatiya, Gopal L; Ravi, Vagisha; Khade, Bharat; Gupta, Sanjay; Waghmare, Sanjeev K

    2016-09-01

    Secretory phospholipase A2 Group-IIA (sPLA2 -IIA) catalyzes the hydrolysis of the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. sPLA2 -IIA is deregulated in various cancers; however, its role in hair follicle stem cell (HFSC) regulation is obscure. Here we report a transgenic mice overexpressing sPLA2 -IIA (K14-sPLA2 -IIA) showed depletion of HFSC pool. This was accompanied with increased differentiation, loss of ortho-parakeratotic organization and enlargement of sebaceous gland, infundibulum and junctional zone. The colony forming efficiency of keratinocytes was significantly reduced. Microarray profiling of HFSCs revealed enhanced level of epithelial mitogens and transcription factors, c-Jun and FosB that may be involved in proliferation and differentiation. Moreover, K14-sPLA2 -IIA keratinocytes showed enhanced activation of EGFR and JNK1/2 that led to c-Jun activation, which co-related with enhanced differentiation. Further, depletion of stem cells in bulge is associated with high levels of chromatin silencing mark, H3K27me3 and low levels of an activator mark, H3K9ac suggestive of alteration in gene expression contributing toward stem cells differentiation. Our results, first time uncovered that overexpression of sPLA2 -IIA lead to depletion of HFSCs and differentiation associated with altered histone modification. Thus involvement of sPLA2 -IIA in stem cells regulation and disease pathogenesis suggest its prospective clinical implications. Stem Cells 2016;34:2407-2417.

  7. 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.

  8. Nanomolar vitamin E alpha-tocotrienol inhibits glutamate-induced activation of phospholipase A2 and causes neuroprotection.

    PubMed

    Khanna, Savita; Parinandi, Narasimham L; Kotha, Sainath R; Roy, Sashwati; Rink, Cameron; Bibus, Douglas; Sen, Chandan K

    2010-03-01

    Our previous works have elucidated that the 12-lipoxygenase pathway is directly implicated in glutamate-induced neural cell death, and that such that toxicity is prevented by nM concentrations of the natural vitamin E alpha-tocotrienol (TCT). In the current study we tested the hypothesis that phospholipase A(2) (PLA(2)) activity is sensitive to glutamate and mobilizes arachidonic acid (AA), a substrate for 12-lipoxygenase. Furthermore, we examined whether TCT regulates glutamate-inducible PLA(2) activity in neural cells. Glutamate challenge induced the release of [(3)H]AA from HT4 neural cells. Such response was attenuated by calcium chelators (EGTA and BAPTA), cytosolic PLA(2) (cPLA(2))-specific inhibitor (AACOCF(3)) as well as TCT at 250 nM. Glutamate also caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of phospholipid-esterified AA in neural cells. Furthermore, glutamate induced a time-dependent translocation and enhanced serine phosphorylation of cPLA(2) in the cells. These effects of glutamate on fatty acid levels and on cPLA(2) were significantly attenuated by nM TCT. The observations that AACOCF(3), transient knock-down of cPLA(2) as well as TCT significantly protected against the glutamate-induced death of neural cells implicate cPLA(2) as a TCT-sensitive mediator of glutamate induced neural cell death. This work presents first evidence recognizing glutamate-induced changes in cPLA(2) as a novel mechanism responsible for neuroprotection observed in response to nanomolar concentrations of TCT.

  9. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A2 Pathways

    PubMed Central

    Seo, Jihui; Maeng, Jeehye; Kim, Hwa-Jung

    2016-01-01

    The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [3H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i), the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2) in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions. PMID:27783042

  10. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities[S

    PubMed Central

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

    2016-01-01

    A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase®, guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement. PMID:27194811

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

    PubMed Central

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

    2009-01-01

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

  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. 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

  14. 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

  15. TNF-α-Induced cPLA2 Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells

    PubMed Central

    Lin, Chih-Chung; Lin, Wei-Ning; Cho, Rou-Ling; Wang, Chen-yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-01-01

    Tumor necrosis factor-α (TNF-α) triggers activation of cytosolic phospholipase A2 (cPLA2) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-α induced cPLA2 expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-α-stimulated increases in cPLA2 mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE2 secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE2 ELISA kit. These TNF-α-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS [N-acetyl cysteine, (NAC)], NF-κB (Bay11-7082) and transfection with siRNA of ASK1, p47phox, TRAF2, NIK, IKKα, IKKβ, or p65. TNF-α markedly stimulated NADPH oxidase activation and ROS including superoxide and hydrogen peroxide production which were inhibited by pretreatment with a TNFR1 neutralizing antibody, APO, DPI or transfection with siRNA of TRAF2, ASK1, or p47phox. In addition, TNF-α also stimulated p47phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-α induced TNFR1, TRAF2, ASK1, and p47phox complex formation in HPAEpiCs, which were attenuated by a TNF-α neutralizing antibody. We found that pretreatment with NAC, DPI, or APO also attenuated the TNF-α-stimulated IKKα/β and NF-κB p65 phosphorylation, NF-κB (p65) translocation, and NF-κB promoter activity in HPAEpiCs. Finally, we observed that TNF-α-stimulated NADPH oxidase activation and ROS generation activates NF-κB through the NIK/IKKα/β pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA2 by TNF-α is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-κB pathway. PMID:27932980

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2011-09-01

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

  20. Phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in MCII mast cells mediated by high-affinity Fc receptor for IgE.

    PubMed Central

    Currie, S; Roberts, E F; Spaethe, S M; Roehm, N W; Kramer, R M

    1994-01-01

    In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the

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

    PubMed

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

    2010-10-01

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

  2. 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.

  3. High-Density Lipoprotein (HDL) Counter-Regulates Serum Amyloid A (SAA)-Induced sPLA2-IIE and sPLA2-V Expression in Macrophages

    PubMed Central

    Wang, Angelina; Wong, Sarabeth; Bao, Guoqiang; Li, Jianhua; Yang, Huan; Tracey, Kevin J.; D’Angelo, John

    2016-01-01

    Human serum amyloid A (SAA) has been demonstrated as a chemoattractant and proinflammatory mediator of lethal systemic inflammatory diseases. In the circulation, it can be sequestered by a high-density lipoprotein, HDL, which carries cholesterol, triglycerides, phospholipids and apolipoproteins (Apo-AI). The capture of SAA by HDL results in the displacement of Apo-AI, and the consequent inhibition of SAA’s chemoattractant activities. It was previously unknown whether HDL similarly inhibits SAA-induced sPLA2 expression, as well as the resultant HMGB1 release, nitric oxide (NO) production and autophagy activation. Here we provided compelling evidence that human SAA effectively upregulated the expression and secretion of both sPLA2-IIE and sPLA2-V in murine macrophages, which were attenuated by HDL in a dose-dependent fashion. Similarly, HDL dose-dependently suppressed SAA-induced HMGB1 release, NO production, and autophagy activation. In both RAW 264.7 cells and primary macrophages, HDL inhibited SAA-induced secretion of several cytokines (e.g., IL-6) and chemokines (e.g., MCP-1 and RANTES) that were likely dependent on functional TLR4 signaling. Collectively, these findings suggest that HDL counter-regulates SAA-induced upregulation and secretion of sPLA2-IIE/V in addition to other TLR4-dependent cytokines and chemokines in macrophage cultures. PMID:27898742

  4. 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.

  5. Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands*

    PubMed Central

    Shridas, Preetha; Bailey, William M.; Boyanovsky, Boris B.; Oslund, Rob C.; Gelb, Michael H.; Webb, Nancy R.

    2010-01-01

    We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A2 (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A2 (sPLA2), but not a catalytically inactive mutant form of GX sPLA2, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA2 inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA2-overexpressing Y1 cells, ruling out a role for this sPLA2 receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA2. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA2 antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA2 is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression. PMID:20421306

  6. Group X secretory phospholipase A2 regulates the expression of steroidogenic acute regulatory protein (StAR) in mouse adrenal glands.

    PubMed

    Shridas, Preetha; Bailey, William M; Boyanovsky, Boris B; Oslund, Rob C; Gelb, Michael H; Webb, Nancy R

    2010-06-25

    We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A(2) (GX KO). These mice have approximately 80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A(2) (sPLA(2)), but not a catalytically inactive mutant form of GX sPLA(2), significantly reduced steroid production 30-40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA(2) inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA(2)-overexpressing Y1 cells, ruling out a role for this sPLA(2) receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was approximately 2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA(2). Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA(2) antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA(2) is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.

  7. Role of group V phospholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption*

    PubMed Central

    Satake, Yoshiyuki; Diaz, Bruno L.; Balestrieri, Barbara; Lam, Bing K.; Kanaoka, Yoshihide; Grusby, Michael J.; Arm, Jonathan P.

    2005-01-01

    SUMMARY Conclusions regarding the contribution of low molecular weight secretory phospholipase A2 (sPLA2) enzymes in eicosanoid generation have relied on data obtained from transfected cells or the use of inhibitors that fail to discriminate between individual members of the large family of mammalian sPLA2 enzymes. To elucidate the role of group V sPLA2, we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C4 and prostaglandin E2 was attenuated ~50% in peritoneal macrophages from group V sPLA2-null mice compared to macrophages from wild-type littermates. Furthermore, the early phase of plasma exudation in response to intraperitoneal injection of zymosan and the accompanying in vivo generation of cysteinyl leukotrienes were markedly attenuated in group V sPLA2-null mice compared to wild-type controls. These data provide clear evidence of a role for group V sPLA2 in regulating eicosanoid generation in response to an acute innate stimulus of the immune response both in vitro and in vivo, suggesting a role for this enzyme in innate immunity. PMID:14761945

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

    PubMed

    Romero, Paco; Gandía, Mónica; Alférez, Fernando

    2013-09-01

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

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

    PubMed

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

    2006-11-16

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

  10. Role of secretory phospholipase A(2) in rhythmic contraction of pulmonary arteries of rats with monocrotaline-induced pulmonary arterial hypertension.

    PubMed

    Tanabe, Yoshiyuki; Saito-Tanji, Maki; Morikawa, Yuki; Kamataki, Akihisa; Sawai, Takashi; Nakayama, Koichi

    2012-01-01

    Excessive stretching of the vascular wall in accordance with pulmonary arterial hypertension (PAH) induces a variety of pathogenic cellular events in the pulmonary arteries. We previously reported that indoxam, a selective inhibitor for secretory phospholipase A(2) (sPLA(2)), blocked the stretch-induced contraction of rabbit pulmonary arteries by inhibition of untransformed prostaglandin H(2) (PGH(2)) production. The present study was undertaken to investigate involvement of sPLA(2) and untransformed PGH(2) in the enhanced contractility of pulmonary arteries of experimental PAH in rats. Among all the known isoforms of sPLA(2), sPLA(2)-X transcript was most significantly augmented in the pulmonary arteries of rats with monocrotaline-induced pulmonary hypertension (MCT-PHR). The pulmonary arteries of MCT-PHR frequently showed two types of spontaneous contraction in response to stretch; 27% showed rhythmic contraction, which was sensitive to indoxam and SC-560 (selective COX-1 inhibitor), but less sensitive to NS-398 (selective COX-2 inhibitor); and 47% showed sustained incremental tension (tonic contraction), which was insensitive to indoxam and SC-560, but sensitive to NS-398 and was attenuated to 45% of the control. Only the rhythmically contracting pulmonary arteries of MCT-PHR produced a substantial amount of untransformed PGH(2), which was abolished by indoxam. These results suggest that sPLA(2)-mediated PGH(2) synthesis plays an important role in the rhythmic contraction of pulmonary arteries of MCT-PHR.

  11. Leukotriene receptor antagonists, LY293111 and ONO-1078, protect neurons from the sPLA2-IB-induced neuronal cell death independently of blocking their receptors.

    PubMed

    Yagami, Tatsurou; Yamamoto, Yasuhiro; Kohma, Hiromi

    2013-09-01

    In the ischemic brain, leukotrienes (LTs) are increased and their receptor antagonists protect neurons. However, it has not yet been sufficiently clarified how antagonists for LT receptors exhibit neuroprotective effects. In the present study, we evaluated protective effects of receptor antagonists for LTB4 (LY293111) and cysteinyl LTs (ONO-1078) in the primary culture of rat cortical neurons. The group IB secretory phospholipase A2 (sPLA2-IB)-induced neuronal cell death had been established as the in vitro model for cerebral ischemia. sPLA2-IB triggered the influx of Ca(2+) into neurons via L-type voltage-dependent calcium channel (L-VDCC). Subsequently, the enzyme produced eicosanoids including LTB4 before neuronal cell death. Neither administration of LTB4 nor cysteinyl LTs such as LTC4, LTD4 and LTE4 killed neurons. However, both LY293111 and ONO-1078 significantly prevented neurons from the neurotoxicity of sPLA2-IB, suggesting that the two LT receptor blockers protected neurons through alternative pathways beside LT receptors. An L-VDCC blocker does not only inhibit the influx of Ca(2+) into neurons but also rescues neurons from the sPLA2-IB-induced neuronal cell death. The two LT receptor antagonists also blocked the sPLA2-IB-induced Ca(2+) influx significantly. Thus, LTs exhibited no neurotoxicity, but their receptor antagonists protected neurons directly in the in vitro ischemic model. Furthermore, the suppression of L-VDCC appeared to be involved in the neuroprotective effects of LY293111 and ONO-1078 independent of blocking their receptors.

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

    PubMed

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

    2010-02-01

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

  13. 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

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

    PubMed

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

    2013-10-15

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

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

    PubMed Central

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

    2015-01-01

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

  16. 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.

  17. Associations of MDR1, TBXA2R, PLA2G7, and PEAR1 genetic polymorphisms with the platelet activity in Chinese ischemic stroke patients receiving aspirin therapy

    PubMed Central

    Peng, Ling-ling; Zhao, Yuan-qi; Zhou, Zi-yi; Jin, Jing; Zhao, Min; Chen, Xin-meng; Chen, Ling-yan; Cai, Ye-feng; Li, Jia-li; Huang, Min

    2016-01-01

    Aim: Aspirin resistance has an incidence of 5%–65% in patients with ischemic stroke, who receive the standard dose of aspirin, but the platelet function is inadequately inhibited, thereby leading to thrombotic events. Numerous evidence shows that thromboxane A2 receptor (TXA2 receptor, encoded by TBXA2R), lipoprotein-associated phospholipase A2 (Lp-PLA2, encoded by PLA2G7) and platelet endothelial aggregation receptor-1 (PEAR1, encoded by PEAR1) are crucial in regulating platelet activation, and P-glycoprotein (P-gp, encoded by MDR1) influences the absorption of aspirin in the intestine. In this study we examined the correlation between MDR1, TBXA2R, PLA2G7, PEAR1 genetic polymorphisms and platelet activity in Chinese ischemic stroke patients receiving aspirin therapy. Methods: A total of 283 ischemic stroke patients receiving 100 mg aspirin for 7 d were genotyped for polymorphisms in MDR1 C3435T, TBXA2R (rs1131882), PLA2G7 (rs1051931, rs7756935), and PEAR1 (rs12566888, rs12041331). The platelet aggregation response was measured using an automatic platelet aggregation analyzer and a commercially available TXB2 ELISA kit. Results: Thirty-three patients (11.66%) were insensitive to aspirin treatment. MDR1 3435TT genotype carriers, whose arachidonic acid (AA) or adenosine diphosphate (ADP)-induced platelet aggregation was lower than that of CC+CT genotype carriers, were less likely to suffer from aspirin resistance (odds ratio=0.421, 95% CI: 0.233–0.759). The TBXA2R rs1131882 CC genotype, which was found more frequently in the aspirin-insensitive group (81.8% vs 62.4%) than in the sensitive group, was identified as a risk factor for aspirin resistance (odds ratio=2.712, 95% CI: 1.080–6.810) with a higher level of AA-induced platelet aggregation. Due to the combined effects of PLA2G7 rs1051931 and rs7756935, carriers of the AA-CC haplotype had a higher level of ADP-induced platelet aggregation, and were at considerably higher risk of aspirin resistance than

  18. 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.

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

    PubMed

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

    2015-12-07

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

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

    PubMed Central

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

    2005-01-01

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

  1. Intensive Lifestyle Modification Reduces Lp-PLA2 in Dyslipidemic HIV/HAART Patients

    PubMed Central

    Wooten, Joshua S.; Nambi, Preethi; Gillard, Baiba K.; Pownall, Henry J.; Coraza, Ivonne; Scott, Lynne W.; Nambi, Vijay; Ballantyne, Christie M.; Balasubramanyam, Ashok

    2013-01-01

    Patients with dyslipidemia associated with HIV-1 infection and highly active antiretroviral therapy (HAART) have elevated levels of Lp-PLA2 and CCL5/RANTES, which may increase risk of cardiovascular disease. Purpose This study aimed to determine whether an intensive diet and exercise (D/E) program, independently or combined with fenofibrate or niacin, could reduce Lp-PLA2 or RANTES. Methods Hypertriglyceridemic HIV patients on stable HAART (n=107) were randomized to one of five interventions: 1) Usual Care (UC); 2) D/E with placebos; 3) D/E with fenofibrate and placebo; 4) D/E with niacin and placebo; or 5) D/E with fenofibrate and niacin for 24 weeks. Lp-PLA2 and RANTES concentrations were measured in fasting plasma samples at baseline and post-intervention. General linear models were used to compare Lp-PLA2 and RANTES levels between the five groups post-intervention, controlling for baseline levels, age, BMI, CD4+ T-cell count, viral load, duration of infection, and HAART. Results At baseline, fasting plasma Lp-PLA2 (388.5 ± 127.5 ng/mL) and RANTES (43.8 ± 25.5 ng/mL) levels were elevated when compared to healthy controls. Post-treatment Lp-PLA2 mass was lower in patients who received D/E only (323.0 ± 27.2 ng/mL), D/E plus fenofibrate (327.2 ± 25.9 ng/mL) and D/E plus niacin (311.1 ± 27.8 ng/mL) when compared to patients receiving UC (402.2 ± 25.3 ng/mL). RANTES concentrations were not significantly affected by any intervention. Conclusions Elevated plasma Lp-PLA2 mass can be reduced by an intensive diet and exercise program in patients with HIV/HAART-associated dyslipidemia. RANTES is elevated but is not reduced by lifestyle modification, fenofibrate or niacin. PMID:23299761

  2. PLA2G6-associated neurodegeneration (PLAN): further expansion of the clinical, radiological and mutation spectrum associated with infantile and atypical childhood-onset disease.

    PubMed

    Illingworth, M A; Meyer, E; Chong, W K; Manzur, A Y; Carr, L J; Younis, R; Hardy, C; McDonald, F; Childs, A M; Stewart, B; Warren, D; Kneen, R; King, M D; Hayflick, S J; Kurian, M A

    2014-06-01

    Phospholipase A2 associated neurodegeneration (PLAN) is a major phenotype of autosomal recessive Neurodegeneration with Brain Iron Accumulation (NBIA). We describe the clinical phenotypes, neuroimaging features and PLA2G6 mutations in 5 children, of whom 4 presented with infantile neuroaxonal dystrophy (INAD). One other patient was diagnosed with the onset of PLAN in childhood, and our report highlights the diagnostic challenges associated with this atypical PLAN subtype. In this series, the neuroradiological relevance of classical PLAN features as well as apparent claval hypertrophy' is explored. Novel PLA2G6 mutations were identified in all patients. PLAN should be considered not only in patients presenting with a classic INAD phenotype but also in older patients presenting later in childhood with non-specific progressive neurological features including social communication difficulties, gait disturbance, dyspraxia, neuropsychiatric symptoms and extrapyramidal motor features.

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

    PubMed

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

    2014-05-01

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

  4. 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.

  5. Structural Basis for the Inhibition of a Phospholipase A2-Like Toxin by Caffeic and Aristolochic Acids

    PubMed Central

    Fernandes, Carlos A. H.; Cardoso, Fábio Florença; Cavalcante, Walter G. L.; Soares, Andreimar M.; Dal-Pai, Maeli; Gallacci, Marcia; Fontes, Marcos R. M.

    2015-01-01

    One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from Bothropspirajai venom, complexed with two compounds present in several plants used in folk medicine against snakebites. These ligands partially neutralized the myotoxic activity of PrTX-I towards binding on the two independent sites of interaction between Lys49-PLA2 and muscle membrane. Our results corroborate the previously proposed mechanism of action of PLA2s-like and provide insights for the design of structure-based inhibitors that could prevent the permanent injuries caused by these proteins in snakebite victims. PMID:26192963

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

    PubMed Central

    Leslie, Christina C.

    2015-01-01

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

  7. 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

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

    PubMed

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

    2002-10-01

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

  9. 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

  10. Inhibition of secreted phospholipase A2. 4-glycerol derivatives of 4,5-dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one with broad activities.

    PubMed

    Touaibia, Mohamed; Djimdé, Atimé; Cao, Fei; Boilard, Eric; Bezzine, Sofiane; Lambeau, Gérard; Redeuilh, Catherine; Lamouri, Aazdine; Massicot, France; Chau, François; Dong, Chang-Zhi; Heymans, Françoise

    2007-04-05

    Secreted phospholipases A2 (sPLA2s) have been reported to play an important role in various inflammatory conditions and thus represent an attractive therapeutic target. Previous SAR studies from our laboratory have revealed certain important features of our recently discovered specific hGIIA sPLA2 inhibitors, and we report here the synthesis and biological activities of glycerol-containing derivatives of our lead compound III (Figure 1). Efficient and selective synthesis methods have been developed to make glycerol trisubstituted by different groups on desired positions. In terms of biological activities, the best compounds (A3, A6, and A15) are more active than III (Figure 1), as potent as Me-Indoxam, an sPLA2s inhibitor of reference, against hGIIA, hGV, and hGX sPLA2s and at least 10 times less active toward the GIB enzymes in two in vitro assay systems. By synthesis of enantiopure (S)-A6, we demonstrated that no important improvement of the inhibitory potency could be achieved by this approach. Furthermore, the results show that the global lipophilicity is likely responsible for the anti-PLA2 activity and two oxadiazolone moieties seem too big to be accommodated by the active site of the hGIIA enzyme.

  11. Down-regulation by prostaglandins of type-II phospholipase A2 expression in guinea-pig alveolar macrophages: a possible involvement of cAMP.

    PubMed Central

    Vial, D; Arbibe, L; Havet, N; Dumarey, C; Vargaftig, B; Touqui, L

    1998-01-01

    We have demonstrated previously that isolated guinea-pig alveolar macrophages (AM) synthesize type-II phospholipase A2 (PLA2-II) through a tumour necrosis factor-alpha (TNF-alpha)-dependent process. This synthesis is enhanced by lipopolysaccharide (LPS) and accompanied by a release of prostaglandin E2 (PGE2) into the medium. Because agents elevating intracellular cAMP, such as PGE2, have been shown to stimulate PLA2-II expression in various cell types, we investigated the modulation of PLA2-II synthesis by cAMP in AM. Surprisingly, incubation of AM with PGE2, dibutyryl-cAMP, cholera toxin or rolipram (an inhibitor of specific cAMP-phosphodiesterase) inhibited both basal and LPS-stimulated PLA2-II expression. The inhibitory effect of PGE2 was observed at concentrations similar to those released by AM. Moreover, treatment of AM with either aspirin or neutralizing PGE2 monoclonal antibody stimulated PLA2-II synthesis. These effects were closely correlated with the ability of these agents to modulate TNF-alpha release, which was decreased by dibutyryl-cAMP and exogenous PGE2, whereas neutralizing PGE2 antibody markedly increased this release. Hence, in contrast to other cell systems, we report that: (i) agents elevating intracellular cAMP levels down-regulate both basal and LPS-induced PLA2-II synthesis, (ii) prostaglandins exert a negative feedback effect on this synthesis, probably through an elevation of intracellular cAMP levels, and (iii) inhibition of TNF-alpha release may account, at least in part, for the down-regulation of PLA2-II expression by endogenously produced prostaglandins and cAMP-elevating agents. PMID:9461495

  12. Prehypertension-Associated Elevation in Circulating Lysophosphatidlycholines, Lp-PLA2 Activity, and Oxidative Stress

    PubMed Central

    Kim, Minjoo; Jung, Saem; Kim, Su Yeon; Lee, Sang-Hyun; Lee, Jong Ho

    2014-01-01

    Prehypertension is a risk factor for atherosclerosis. We investigated alterations in plasma metabolites that are associated with prehypertension. A group of 53 individuals was identified who remained within the range of prehypertension during repeated measurements in a 3-year period. This group was compared with the control group of 53 normotensive subjects who were matched for age and gender. Metabolomic profiles were analyzed with UPLC-LTQ-Orbitrap mass spectrometry. The prehypertensive group showed higher levels of lysophosphatidylcholines (lysoPCs) containing C14:0, C16:1, C16:0, C18:2, C18:1, C18:0, C20:5, C20:4, C20:3, and C22:6, higher circulating Lp-PLA2 activity, oxidized LDL (ox-LDL), interleukin 6 (IL-6), urinary 8-epi-PGF2α, and higher brachial-ankle pulse wave velocity (ba-PWV), before and after adjusting for BMI, WHR, smoking, alcohol consumption, serum lipid profiles, glucose, and insulin. LysoPC (16:0) was the most important plasma metabolite for evaluating the difference between control and prehypertensive groups, with a variable important in the projection (VIP) value of 17.173, and it showed a positive and independent association with DBP and SBP. In the prehypertensive group, the levels of lysoPC (16:0) positively and significantly correlated with ox-LDL, Lp-PLA2 activity, 8-epi-PGF2α, ba-PWV, and IL-6 before and after adjusting for confounding variables. Prehypertension-associated elevations in lysoPCs, Lp-PLA2 activity, ox-LDL, urinary 8-epi-PGF2α, IL-6, and ba-PWV could indicate increased oxidative stress from Lp-PLA2-catalyzed PC hydrolysis during increased LDL oxidation, thereby enhancing proinflammation and arterial stiffness. PMID:24800806

  13. Orai, STIM1 and iPLA2β: a view from a different perspective

    PubMed Central

    Bolotina, Victoria M

    2008-01-01

    The mechanism of store-operated Ca2+ entry (SOCE) remains one of the intriguing mysteries in the field of Ca2+ signalling. Recent discoveries have resulted in the molecular identification of STIM1 as a Ca2+ sensor in endoplasmic reticulum, Orai1 (CRACM1) as a plasma membrane channel that is activated by the store-operated pathway, and iPLA2β as an essential component of signal transduction from the stores to the plasma membrane channels. Numerous studies have confirmed that molecular knock-down of any one of these three molecules impair SOCE in a wide variety of cell types, but their mutual relations are far from being understood. This report will focus on the functional roles of Orai1, STIM1 and iPLA2β, and will address some specific questions about Orai1 and TRPC1, and their relation to SOC channels in excitable and non-excitable cells. Also, it will analyse the novel role of STIM1 as a trigger for CIF production, and the complex relationship between STIM1 and Orai1 expression, puncta formation and SOCE activation. It will highlight some of the most recent findings that may challenge simple conformational coupling models of SOCE, and will offer some new perspectives on the complex relationships between Orai1, STIM1 and iPLA2β in the SOCE pathway. PMID:18499724

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2003-10-15

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

  16. Secreted phospholipase A2 and mast cells.

    PubMed

    Murakami, Makoto; Taketomi, Yoshitaka

    2015-01-01

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

  17. The expression of ERα, OTR, cPLA(2), COX-2, and PPARγ in the cervix of the ewe during the estrous cycle.

    PubMed

    Falchi, L; Scaramuzzi, R J

    2013-01-01

    The ovine cervix relaxes at estrus allowing easier entry of spermatozoa into the uterus. The mechanism responsible for this relaxation is not fully elucidated and we hypothesized that cervical relaxation at estrus is induced by ovarian and pituitary hormones stimulating the local production of prostaglandin E(2) via a biosynthetic pathway involving a number of mediators including oxytocin, phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor gamma (PPARγ). The aim of this study was to investigate the cervical expression of estradiol receptor alpha (ERα), oxytocin receptor (OTR), cPLA(2), COX-2, and PPARγ at three stages of the estrous cycle (the luteal phase and two times during the follicular phase, just before and just after the LH surge). An experiment was conducted during the breeding season, in 25 ewes to test this hypothesis. Samples of cervical tissue were collected from groups of ewes at three stages of the estrous cycle: the luteal (N = 8), "pre-LH surge" (N = 8), and "post-LH surge" (N = 9) stages. Cervical tissue from uterine, mid, and vaginal regions of the cervix were analyzed by Western immunoblot analysis for ERα, OTR, cPLA(2,) COX-2, and PPARγ. The results showed that the levels of all five proteins were lowest during the luteal phase of the estrous cycle in all regions of the cervix. The levels of all except cPLA(2), increased significantly during the "pre-LH surge" stage. The levels of cPLA(2) and ERα increased in the "post-LH surge" stage and those for OTR and PPARγ were unchanged and those for COX-2 were lower. These data show that the cervical levels of all five of the intermediates in the synthesis of prostaglandin E(2) that were examined in this study were higher in the "pre-" and "post-LH surge" stages compared with the luteal phase of the estrous cycle and these findings are consistent with our hypothesis.

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

    PubMed

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

    2014-12-01

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

  19. Role of cytosolic and calcium independent phospholipases A(2) in insulin secretion impairment of INS-1E cells infected by S. aureus.

    PubMed

    Caporarello, N; Salmeri, M; Scalia, M; Motta, C; Parrino, C; Frittitta, L; Olivieri, M; Toscano, M A; Anfuso, C D; Lupo, G

    2015-12-21

    Cytosolic PLA2 (cPLA2) and Ca(2+)-independent PLA2 (iPLA2) play a significant role in insulin β-cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS-1 cells alters glucose-induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho-cPLA2, phospho-PKCα and phospho-ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX-2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX-2 inhibitor.

  20. Clinacanthus nutans Extracts Modulate Epigenetic Link to Cytosolic Phospholipase A2 Expression in SH-SY5Y Cells and Primary Cortical Neurons.

    PubMed

    Tan, Charlene Siew-Hon; Ho, Christabel Fung-Yih; Heng, Swan-Ser; Wu, Jui-Sheng; Tan, Benny Kwong-Huat; Ng, Yee-Kong; Sun, Grace Y; Lin, Teng-Nan; Ong, Wei-Yi

    2016-09-01

    Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.

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

    PubMed Central

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

    1992-01-01

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

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

    PubMed

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

    2001-01-01

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

  3. Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom

    PubMed Central

    Sucasaca-Monzón, Georgina; Randazzo-Moura, Priscila; Rocha, Thalita; Vilca-Quispe, Augusto; Ponce-Soto, Luis Alberto; Marangoni, Sérgio; da Cruz-Höfling, Maria Alice; Rodrigues-Simioni, Léa

    2015-01-01

    A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°–45°C. Full Bp-13 PLA2 activity required Ca2+; its PLA2 activity was inhibited by Mg2+, Mn2+, Sr2+, and Cd2+ in the presence and absence of 1 mM Ca2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca2+ by Sr2+ and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom. PMID:25789175

  4. Circulating (CD3(-)CD19(+)CD20(-)IgD(-)CD27(high)CD38(high)) Plasmablasts: A Promising Cellular Biomarker for Immune Activity for Anti-PLA2R1 Related Membranous Nephropathy?

    PubMed

    Pozdzik, Agnieszka; Beukinga, Ingrid; Gu-Trantien, Chunyan; Willard-Gallo, Karen; Nortier, Joëlle; Pradier, Olivier

    2016-01-01

    Membranous nephropathy (MN) is a kidney specific autoimmune disease mainly mediated by anti-phospholipase A2 receptor 1 autoantibody (PLA2R1 Ab). The adequate assessment of chimeric anti-CD20 monoclonal antibody, rituximab (RTX), efficacy is still needed to improve clinical outcome of patient with MN. We evaluated the modification of plasmablasts (CD3(-)CD19(+)CD20(-)IgD(-)CD27(high)CD38(high)), a useful biomarker of RTX response in other autoimmune diseases, and memory (CD3(-)CD19(+)CD20(+)IgD(-)CD27(+)CD38(-)) and naive (CD3(-)CD19(+)CD20(+)IgD(+)CD27(-)CD38(low)) B cells by fluorescence-activated cell sorter analysis in PLA2R1 related MN in one patient during the 4 years of follow-up after RTX. RTX induced complete disappearance of CD19(+) B cells, plasmablasts, and memory B cells as soon as day 15. Despite severe CD19(+) lymphopenia, plasmablasts and memory B cells reemerged early before naive B cells (days 45, 90, and 120, resp.). During the follow-up, plasmablasts decreased more rapidly than memory B cells but still remained elevated as compared to day 0 of RTX. Concomitantly, anti-PLA2R1 Ab increased progressively. Our single case report suggests that, besides monitoring of serum anti-PLA2R1 Ab level, enumeration of circulating plasmablasts and memory B cells represents an attractive and complementary tool to assess immunological activity and efficacy of RTX induced B cells depletion in anti-PLA2R1 Ab related MN.

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

    PubMed

    Bae, Jong-Sup

    2015-08-01

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

  6. Cytosolic phospholipase A2 regulates alcohol-mediated astrocyte inflammatory responses in HIV-associated neurocognitive disorders

    PubMed Central

    Pandey, R; Ghorpade, A

    2015-01-01

    Alcohol (EtOH) abuse and HIV-1 infection remain leading public health problems not only in the United States but also across the world. Alcohol abusers have a significantly greater risk of HIV-1 infection than non-drinkers globally. In the United States, prevalence of EtOH abuse is over two-fold higher in HIV-1-positive individuals than that of the general population. Although alcohol abusers show neurodegeneration, exacerbated neuroinflammation and oxidative damage, the mechanism(s) by which EtOH regulates astrocyte inflammatory responses in HIV-associated neurocognitive disorders is unknown. Thus, we explored signaling pathway(s) involved in EtOH-mediated activation of human astrocytes with HIV-1 and subsequent alterations in their inflammatory functions. Alcohol exposure altered the morphology of astrocytes, proinflammatory responses and induced cytotoxicity in a dose-dependent manner. Time-dependent changes were also evaluated. EtOH and HIV-1 cotreatment decreased cell viability and proliferation, while increasing apoptosis and mitochondrial depolarization. EtOH and HIV-1 together increased the levels of proinflammatory molecules, interleukin-1β, tumor necrosis factor-α, CXCL8, tissue inhibitor of metalloproteinases-1 and more importantly, arachidonic acid, a known downstream target of cytosolic phospholipase A2 (cPLA2). Consistent with this observation, phospho-cPLA2 levels were augmented in HIV-1 and EtOH cotreatment as compared with HIV-1 or EtOH alone. Cyclooxygenase 2 was upregulated as measured by real-time PCR and western blot, whereas cotreatment of HIV-1 and EtOH decreased cytochrome P450-2E1 levels as compared with EtOH alone. Furthermore, we confirmed that blocking cPLA2 with arachidonyl tri floro methyl ketone, a cPLA2-specific inhibitor, effectively prevented cPLA2 phosphorylation and downstream outcomes. Thus, the present findings suggest that cPLA2 has a critical role in alcohol and HIV-induced astrocyte inflammation. In the future, cPLA2

  7. [Prognostic implications of GP3a glucoprotein gene PLA1/PLA2 allele in prostatic cancer: pilot results of the study].

    PubMed

    Loran, O B; Itkes, A V; Seregin, A A; Miandina, G I

    2005-01-01

    We studied the role of integrins, primarily, the role of allele distribution of GP3a gene in development of prostatic cancer (PC) and assessment of its prognostic significance. From November 2003 to May 2004 we examined 32 patients with PC: 11 patients with local PC T1-2N0M0; 14 patients with locally advanced cancer T3N0M0 and 7 patients with invasive and/or metastatic cancer T3-4N10-1 or T3-4N0-1M1. The blood from all the patients we studied with PCR for alleles of GP3a gene, PSA. Seventeen patients were found to have alleles PLA1A1, 14(44%)--alleles PLA1A2, 1(3%)--alleles PLA2A2. Alleles PLA1A2 occurred significantly more often than in the population (p < 0.005). The group analysis has found that 8 patients with local PC had alleles PLA1A1, 3 patients--alleles PLA1A2 (27%). We discovered alleles PLA2A2, PLA1A1 and PLA1A2 in 1(7%), 5(36%) and 8(57%) patients with locally advanced PC, respectively. Among patients with metastatic and/or invasive prostatic cancer, there were 4 (57%) and 3 (43%) cases of alleles PLA1A1 and PLA1A2, respectively. Our study demonstrated influence of carriage of PLA2 allele on occurrence of PC risk (5-fold higher) and its invasive forms (10-fold higher and more). Probability to develop local invasion among patients with prostatic cancer--carriers allele PLA1A2 is 6 times higher than among carriers of alleles PLA1A1. A PC course in carriers of alleles PLA1A2 may be characterized by faster development of local invasion and metastasizing vs carriers of alleles PLA1A1. These findings can be used in design of nomograms for prognostication of invasion of clinically small tumors in verification of significance on greater number of the patients.

  8. Fetal akinesia deformation sequence and neuroaxonal dystrophy without PLA2G6 mutation.

    PubMed

    Rakheja, Dinesh; Uddin, Naseem; Mitui, Midori; Cope-Yokoyama, Sandy; Hogan, Robert N; Burns, Dennis K

    2010-01-01

    We present autopsy findings of a stillborn female infant at 20 to 21 weeks' gestation with neuroaxonal dystrophy. External examination showed features of fetal akinesia deformation sequence. Internal examination showed hypoplasia of the cerebellum, corpus callosum, and optic nerves, as well as nuclear cataracts. Light and electron microscopic examinations showed widespread axonal spheroids in the central and peripheral nervous systems. Gene sequencing failed to reveal PLA2G6 mutations, indicating that fetal neuroaxonal dystrophy presenting as fetal akinesia deformation sequence is genetically distinct from infantile neuroaxonal dystrophy and related disorders. In addition, placental examination showed α-fetoprotein-positive, eosinophilic, globular inclusions in the cytoplasm of a few villous macrophages. The significance of this novel histologic finding is unclear.

  9. PLA2G6-associated Dystonia–Parkinsonism: Case Report and Literature Review

    PubMed Central

    Karkheiran, Siamak; Shahidi, Gholam Ali; Walker, Ruth H.; Paisán-Ruiz, Coro

    2015-01-01

    Background Phospholipase-associated neurodegeneration (PLAN) caused by PLA2G6 mutations is a recessively inherited disorder with three known phenotypes: the typical infantile onset neuroaxonal dystrophy (INAD); an atypical later onset form (atypical NAD); and the more recently recognized young-onset dystonia–parkinsonism (PLAN-DP). Case Report We report the clinical, radiological, and genetic findings of a young Pakistani male with PLAN-DP. We review 11 previously published case reports cited in PubMed, and summarize the demographic, clinical, genetic, and radiological data of the 23 patients described in those articles. Discussion PLAN-DP presents with diverse motor, autonomic, and neuropsychiatric features and should be considered in the differential diagnosis of patients with young-onset neurodegenerative disorders. PMID:26196026

  10. PLA2G6 Mutations Related to Distinct Phenotypes: A New Case with Early-onset Parkinsonism

    PubMed Central

    Giri, Anamika; Guven, Gamze; Hanagasi, Hasmet; Hauser, Ann-Kathrin; Erginul-Unaltuna, Nihan; Bilgic, Basar; Gurvit, Hakan; Heutink, Peter; Gasser, Thomas; Lohmann, Ebba; Simón-Sánchez, Javier

    2016-01-01

    Background PLA2G6-associated neurodegeneration (PLAN) is a recessive neurodegenerative disorder characterized by three distinct phenotypes: infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy (atypical NAD), and PLA2G6-related dystonia–parkinsonism. Methods A consanguineous index case from Turkey was diagnosed with early-onset Parkinsonism at the Istanbul Faculty of Medicine. She and her unaffected brother were subjected to whole-genome sequencing. Results In this report, we describe a 33-year-old index case with parental consanguinity and early-onset Parkinsonism. Whole-genome sequencing of this individual revealed that a homozygous p.R747W mutation in PLA2G6 segregates with the disease in this family Discussion This result supports the importance of prioritizing this gene in mutational analysis of autosomal recessive Parkinsonism, and confirms the clinical heterogeneity of PLAN. PMID:27127721

  11. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen.

    PubMed

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka

    2015-03-24

    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  12. Monocyte-derived factors including PLA2G7 induced by macrophage-nasopharyngeal carcinoma cell interaction promote tumor cell invasiveness

    PubMed Central

    Low, Heng Boon; Png, Chin Wen; Li, Chunwei; Wang, De Yun; Wong, Soon Boon Justin; Zhang, Yongliang

    2016-01-01

    The non-keratinizing undifferentiated subtype of nasopharyngeal carcinoma (NPC) is a malignancy characterized by an intimate relationship between neoplastic cells and a non-neoplastic lymphoid component. Tumor-associated macrophages (TAMs) foster tumor progression through production of soluble mediators that support proliferation, angiogenesis, survival and invasion of malignant cells. However, the role of macrophages in the progression of NPC remains poorly understood. This study aims to investigate the functional and phenotypic changes that occur to macrophages in macrophage-NPC cell co-culture systems, and how these changes influence tumor cells. We found that monocytes, including THP-1 cells and primary human monocytes, co-cultured with C666-1 NPC cells upregulate expression of pro-inflammatory cytokines at the early stages, followed by the induction of metastasis-related genes and interferon-stimulated genes at the later stage of coculture, indicating that TAMs are “educated” by NPC cells for cancer progression. Importantly, the induction of these factors from the TAMs was also found to enhance the migratory capabilities of the NPC cells. We have also identified one of these macrophage-derived factor, phospholipase A2 Group 7 (PLA2G7), to be important in regulating tumor cell migration and a novel tumor-promoting factor in NPC. Further studies to characterize the role of PLA2G7 in tumor metastasis may help determine its potential as a therapeutic target in NPC. PMID:27487154

  13. Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining.

    PubMed

    Daniel, Bron; DeCoster, Mark A

    2004-08-01

    The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) stain is in wide use for measuring apoptosis in neurons, as well as in other cell types. TUNEL may give false positive results due to variations in labeling technique as well as staining of cells that have undergone non-apoptotic DNA strand breaks. Therefore, in isolation, TUNEL is not a certain indicator of apoptosis. Recently, we have demonstrated the potent apoptotic effect of secreted phospholipase A2 from group III (sPLA2-III) on primary cortical neurons from rat. Here we describe a computer-assisted method for quantifying TUNEL-positive neurons after sPLA2-III induced apoptosis. Extent of TUNEL is normalized to total nuclear content using 4',6-diamidino-2-phenylindole (DAPI) staining. Furthermore, DAPI counterstaining allows for determination of a nuclear morphology indicator, based on nuclear size and roundness, which we call the nuclear area factor. We found that the nuclear area factor is an early indicator of cell death (significant after 4 h post treatment), while TUNEL staining is significant at later times (26 h). Thus, the independent staining techniques using TUNEL and DAPI complement each other, and with commercially available image analysis software, may be used to indicate early as well as delayed cell injury processes.

  14. Molecular cloning of phospholipases A(2) from venom glands of Echis carpet vipers.

    PubMed

    Bharati, K; Hasson, S S; Oliver, J; Laing, G D; Theakston, R D G; Harrison, R A

    2003-06-01

    Venom toxin-specific antibodies offer a more rational treatment of snake envenoming than conventional antivenom. Here, we describe novel cDNAs encoding phospholipase A(2) (PLA(2)) isoforms from venom gland RNA of Echis pyramidum leakeyi (Epl), Echis sochureki (Es) and Echis ocellatus (Eo). The deduced amino acid sequences of these cDNAs encoded proteins with high overall sequence identity to the viper group II PLA(2) protein family, including the 14 cysteine residues capable of forming seven disulphide bonds that characterize this group of PLA(2) enzymes. Comparison of the PLA(2) sequences from Echis with those from related vipers failed to make significant geographic, taxonomic or PLA(2)-function distinctions between these Echis PLA(2) isoforms. However, their deduced hydrophilicity profiles revealed a conserved tertiary structure that we will exploit, by epidermal DNA immunization, to generate PLA(2)-neutralizing antibodies with polyspecific potential.

  15. Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway.

    PubMed

    Levi, Roberto; Seyedi, Nahid; Schaefer, Ulrich; Estephan, Rima; Mackins, Christina J; Tyler, Eleanor; Silver, Randi B

    2007-04-15

    We hypothesized that the histamine H(3)-receptor (H(3)R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Galpha(i)-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gbetagamma(i)-mediated activation of the MAPK-PLA(2) cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE(2)). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H(3)R-mediated attenuation of K(+)-induced NE exocytosis was prevented by MAPK and PLA(2) inhibitors, and by cyclooxygenase and EP(3)-receptor (EP(3)R) antagonists. Moreover, H(3)R activation resulted in MAPK phosphorylation in H(3)R-transfected SH-SY5Y neuroblastoma cells, and in PLA(2) activation and PGE(2) production in cardiac synaptosomes; H(3)R-induced MAPK phosphorylation was prevented by an anti-betagamma peptide. Synergism between H(3)R and EP(3)R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE(2) may be a downstream effector of the anti-exocytotic effect of H(3)R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca(2+)-channel antagonist omega-conotoxin GVIA, and prevented by an anti-Gbetagamma peptide. Our findings imply that an EP(3)R Gbetagamma(i)-induced decrease in Ca(2+) influx through N-type Ca(2+)-channels is involved in the PGE(2)/EP(3)R-mediated attenuation of NE exocytosis elicited by H(3)R activation. Conceivably, activation of the Gbetagamma(i) subunit of H(3)R and EP(3)R may also inhibit Ca(2+) entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H(3)R activation is cardioprotective. Accordingly, this novel H(3)R signaling pathway may ultimately bear therapeutic significance in hyper

  16. ROLE OF INTRACELLULAR CALCIUM AND PHOSPHOLIPASE A2 IN ARACHIDONIC ACID-INDUCED TOXICITY IN LIVER CELLS OVEREXPRESSING CYP2E1*

    PubMed Central

    Caro, Andres A.; Cederbaum, Arthur I.

    2007-01-01

    Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (α -tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by α -tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or α -tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; iii) does not depend on increased influx of extracellular calcium, and iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1. PMID:17118330

  17. Primary structures and partial toxicological characterization of two phospholipases A2 from Micrurus mipartitus and Micrurus dumerilii coral snake venoms.

    PubMed

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

    2017-03-14

    Snake venom phospholipases A2 (PLA2) share high sequence identities and a conserved structural scaffold, but show important functional differences. Only a few PLA2s have been purified and characterized from coral snake (Micrurus spp.) venoms, and their role in envenomation remains largely unknown. In this report, we describe the isolation, sequencing and partial functional characterization of two Micrurus PLA2s: MmipPLA2 from Micrurus mipartitus and MdumPLA2 from Micrurus dumerilii, two species of clinical importance in Colombia. MmipPLA2 consisted of 119 amino acid residues with a predicted pI of 8.4, whereas MdumPLA2 consisted of 117 residues with a pI of 5.6. Both PLA2s showed the conserved 'group I' cysteine pattern and were enzymatically active, although MdumPLA2 had higher activity. The two enzymes differed notably in their toxicity, with MmipPLA2 being highly lethal to mice and mildly myotoxic, whereas MdumPLA2 was not lethal (up to 3 μg/g body weight) but strongly myotoxic. MdumPLA2 displayed higher anticoagulant activity than MmipPLA2in vitro and caused more sustained edema in the mouse footpad assay. Neither of these enzymes was cytolytic to cultured skeletal muscle C2C12 myotubes. Based on their structural differences, the two enzymes were placed in separate lineages in a partial phylogeny of Micrurus venom PLA2s and this classification agreed with their divergent biological activities. Overall, these findings highlight the structural and functional diversity of Micrurus venom PLA2.

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

    PubMed Central

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

    2015-01-01

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

  19. Biochemical, Pharmacological, and Structural Characterization of New Basic PLA2 Bbil-TX from Bothriopsis bilineata Snake Venom

    PubMed Central

    Corasolla Carregari, Victor; Stuani Floriano, Rafael; Rodrigues-Simioni, Lea; Winck, Flavia V.; Baldasso, Paulo Aparecido; Ponce-Soto, Luis Alberto; Marangoni, Sergio

    2013-01-01

    Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25–37°C. Maximum PLA2 activity required Ca2+ and in the presence of Cd2+, Zn2+, Mn2+, and Mg2+ it was reduced in the presence or absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena. PMID:23509754

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

    PubMed Central

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

    1997-01-01

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

  1. Crystal structure of a phospholipase A2 from Bothrops asper venom: Insights into a new putative "myotoxic cluster".

    PubMed

    Salvador, Guilherme H M; Dos Santos, Juliana I; Lomonte, Bruno; Fontes, Marcos R M

    2017-02-01

    Snake venoms from the Viperidae and Elapidae families often have several phospholipases A2 (PLA2s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA2s from these venoms are generally divided into two classes: (i) catalytic PLA2s (or Asp49-PLA2s) and (ii) non-catalytic PLA2-like toxins (or Lys49-PLA2s). In many Viperidae venoms, a subset of the basic Asp49-PLA2s displays some functional and structural characteristics of PLA2-like proteins and group within the same phylogenetic clade, but their myotoxic mechanism is still largely unknown. In the present study, we have crystallized and solved the structure of myotoxin I (MT-I), a basic myotoxic Asp49-PLA2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA2s and Lys49-PLA2s and has been confirmed by other biophysical and bioinformatics techniques. This arrangement suggests a possible cooperative action between both monomers to exert myotoxicity via two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS). This mechanism would resemble that proposed for Lys49-PLA2s, but the sites involved appear to be situated in a different region. Thus, as both sites are close to one another, they form a "myotoxic cluster", which is also found in two other basic myotoxic Asp49-PLA2s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA2s found in viperid snake venoms.

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

    PubMed

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

    2015-05-01

    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.

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

    PubMed

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

    1997-02-01

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

  4. Identification of Novel Compound Mutations in PLA2G6-Associated Neurodegeneration Patient with Characteristic MRI Imaging.

    PubMed

    Guo, Sen; Yang, Liu; Liu, Huijie; Chen, Wei; Li, Jinchen; Yu, Ping; Sun, Zhong Sheng; Chen, Xiang; Du, Jie; Cai, Tao

    2016-07-09

    Neurodegeneration with brain iron accumulation comprises a heterogeneous group of disorders characterized clinically by progressive motor dysfunction. Accurate identification of de novo and rare inherited mutations is important for determining causative genes of undiagnosed neurological diseases. In the present study, we report a unique case with cerebellar ataxia symptoms and social communication difficulties in an intermarriage family. MRI showed a marked cerebellar atrophy and the "eye-of-the-tiger"-like sign in the medial globus pallidus. Potential genetic defects were screened by whole-exome sequencing (WES) for the patient and four additional family members. A previously undescribed de novo missense mutation (c.1634A>G, p.K545R) in the exon 12 of the PLA2G6 gene was identified. A second rare variant c.1077G>A at the end of exon 7 was also identified, which was inherited from the mother, and resulted in a frame-shift mutation (c.1074_1077del.GTCG) due to an alternative splicing. In conclusion, the identification of the "eye-of-the-tiger"-like sign in the globus pallidus of the patient expands the phenotypic spectrum of PLA2G6-associated disorders and reveals its value in differential diagnosis of PLA2G6-associated disorders.

  5. BmPLA2 containing conserved domain WD40 affects the metabolic functions of fat body tissue in silkworm, Bombyx mori.

    PubMed

    Orville Singh, Chabungbam; Xin, Hu-Hu; Chen, Rui-Ting; Wang, Mei-Xian; Liang, Shuang; Lu, Yan; Cai, Zi-Zheng; Miao, Yun-Gen

    2016-02-01

    PLA2 enzyme hydrolyzes arachidonic acid, and other polyunsaturated fatty acids, from the sn-2 position to release free arachidonic acid and a lysophospholipid. Previous studies reported that the PLA2 in invertebrate organisms participates in lipid signaling molecules like arachidonic acid release in immune-associated tissues like hemocytes and fat bodies. In the present study, we cloned the BmPLA2 gene from fat body tissue of silkworm Bombyx mori, which has a total sequence of 1.031 kb with a 31.90 kDa protein. In silico results of BmPLA2 indicated that the protein has a putative WD40 conserved domain and its phylogeny tree clustered with Danaus plexippus species. We investigated the transcriptional expression in development stages and tissues. The highest expression of BmPLA2 was screened in fat body among the studied tissues of third day fifth instar larva, with a high expression on third day fifth instar larva followed by a depression of expression in the wandering stage of the fifth instar larva. The expression of BmPLA2 in female pupa was higher than that of male pupa. Our RNAi-mediated gene silencing results showed highest reduction of BmPLA2 expression in post-24 h followed by post-48 and post-72 h. The BmPLA2-RNAi larvae and pupa could be characterized by pharate adult lethality and underdevelopment. The phenotypic characters of fat body cells in RNAi-induced larva implied that BmPLA2 affects the metabolic functions of fat body tissue in silkworm Bombyx mori.

  6. iPLA2β deficiency attenuates obesity and hepatic steatosis in ob/ob mice through hepatic fatty-acyl phospholipid remodeling.

    PubMed

    Deng, Xiuling; Wang, Jiliang; Jiao, Li; Utaipan, Tanyarath; Tuma-Kellner, Sabine; Schmitz, Gerd; Liebisch, Gerhard; Stremmel, Wolfgang; Chamulitrat, Walee

    2016-05-01

    PLA2G6 or GVIA calcium-independent PLA2 (iPLA2β) is identified as one of the NAFLD modifier genes in humans, and thought to be a target for NAFLD therapy. iPLA2β is known to play a house-keeping role in phospholipid metabolism and remodeling. However, its role in NAFLD pathogenesis has not been supported by results obtained from high-fat feeding of iPLA2β-null (PKO) mice. Unlike livers of human NAFLD and genetically obese rodents, fatty liver induced by high-fat diet is not associated with depletion of hepatic phospholipids. We therefore tested whether iPLA2β could regulate obesity and hepatic steatosis in leptin-deficient mice by cross-breeding PKO with ob/ob mice to generate ob/ob-PKO mice. Here we observed an improvement in ob/ob-PKO mice with significant reduction in serum enzymes, lipids, glucose, insulin as well as improved glucose tolerance, and reduction in islet hyperplasia. The improvement in hepatic steatosis measured by liver triglycerides, fatty acids and cholesterol esters was associated with decreased expression of PPARγ and de novo lipogenesis genes, and the reversal of β-oxidation gene expression. Notably, ob/ob livers contained depleted levels of lysophospholipids and phospholipids, and iPLA2β deficiency in ob/ob-PKO livers lowers the former, but replenished the latter particularly phosphatidylethanolamine (PE) and phosphatidylcholine (PC) that contained arachidonic (AA) and docosahexaenoic (DHA) acids. Compared with WT livers, PKO livers also contained increased PE and PC containing AA and DHA. Thus, iPLA2β deficiency protected against obesity and ob/ob fatty liver which was associated with hepatic fatty-acyl phospholipid remodeling. Our results support the deleterious role of iPLA2β in severe obesity associated NAFLD.

  7. Novel Natural Inhibitors of CYP1A2 Identified by in Silico and in Vitro Screening

    PubMed Central

    Zhu, Ruixin; Hu, Liwei; Li, Haiyun; Su, Juan; Cao, Zhiwei; Zhang, Weidong

    2011-01-01

    Inhibition of cytochrome P450 (CYP) is a major cause of herb–drug interactions. The CYP1A2 enzyme plays a major role in the metabolism of drugs in humans. Its broad substrate specificity, as well as its inhibition by a vast array of structurally diverse herbal active ingredients, has indicated the possibility of metabolic herb–drug interactions. Therefore nowadays searching inhibitors for CYP1A2 from herbal medicines are drawing much more attention by biological, chemical and pharmological scientists. In our work, a pharmacophore model as well as the docking technology is proposed to screen inhibitors from herbal ingredients data. Firstly different pharmaphore models were constructed and then validated and modified by 202 herbal ingredients. Secondly the best pharmaphore model was chosen to virtually screen the herbal data (a curated database of 989 herbal compounds). Then the hits (147 herbal compounds) were continued to be filtered by a docking process, and were tested in vitro successively. Finally, five of eighteen candidate compounds (272, 284, 300, 616 and 817) were found to have inhibition of CYP1A2 activity. The model developed in our study is efficient for in silico screening of large herbal databases in the identification of CYP1A2 inhibitors. It will play an important role to prevent the risk of herb–drug interactions at an early stage of the drug development process. PMID:21686183

  8. Conjugated linoleic acid-enriched butter improved memory and up-regulated phospholipase A2 encoding-genes in rat brain tissue.

    PubMed

    Gama, Marco A S; Raposo, Nádia R B; Mury, Fábio B; Lopes, Fernando C F; Dias-Neto, Emmanuel; Talib, Leda L; Gattaz, Wagner F

    2015-10-01

    Reduced phospholipase A2 (PLA2) activity has been reported in blood cells and in postmortem brains of patients with Alzheimer disease (AD), and there is evidence that conjugated linoleic acid (CLA) modulates the activity of PLA2 groups in non-brain tissues. As CLA isomers were shown to be actively incorporated and metabolized in the brains of rats, we hypothesized that feeding a diet naturally enriched in CLA would affect the activity and expression of Pla 2 -encoding genes in rat brain tissue, with possible implications for memory. To test this hypothesis, Wistar rats were trained for the inhibitory avoidance task and fed a commercial diet (control) or experimental diets containing either low CLA- or CLA-enriched butter for 4 weeks. After this period, the rats were tested for memory retrieval and killed for tissue collection. Hippocampal expression of 19 Pla 2 genes was evaluated by qPCR, and activities of PLA2 groups (cPLA2, iPLA2, and sPLA2) were determined by radioenzymatic assay. Rats fed the high CLA diet had increased hippocampal mRNA levels for specific PLA2 isoforms (iPla 2 g6γ; cPla 2 g4a, sPla 2 g3, sPla 2 g1b, and sPla 2 g12a) and higher enzymatic activity of all PLA2 groups as compared to those fed the control and the low CLA diet. The increment in PLA2 activities correlated significantly with memory enhancement, as assessed by increased latency in the step-down inhibitory avoidance task after 4 weeks of treatment (rs = 0.69 for iPLA2, P < 0.001; rs = 0.81 for cPLA2, P < 0.001; and rs = 0.69 for sPLA2, P < 0.001). In face of the previous reports showing reduced PLA2 activity in AD brains, the present findings suggest that dairy products enriched in cis-9, trans-11 CLA may be useful in the treatment of this disease.

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

    PubMed Central

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

    2016-01-01

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

  10. Involvement of Ca2+-independent phospholipase A2 isoforms in oxidant-induced neural cell death.

    PubMed

    Peterson, Brianna; Knotts, Taylor; Cummings, Brian S

    2007-01-01

    This study determined the roles of Ca2+-independent PLA2 (iPLA2) in phospholipid chemistry and oxidant-induced cell death in human astrocytes. A172 cells expressed both cytosolic Group VIA (iPLA2beta) and microsomal Group VIB (iPLA2gamma) PLA2 as determined by activity assays and immunoblot analysis. Inhibition of total iPLA2 activity using racemic bromoenol lactone (BEL, 2.5 microM) decreased the expression of 14:0-16:0 phosphatidylcholine (PtdCho) 15% and increased 18:0-18:1-PtdCho expression 15%. Treatment of cells with the iPLA2gamma specific inhibitor R-BEL decreased 14:0-16:0-PtdCho 35%, 16:0-16:0-PtdCho 15% and induced a 35% increase in 18:0-18:1-PtdCho. In contrast, treatment of cells with the iPLA2beta inhibitor S-BEL did not alter any phospholipid studied. To determine the roles of iPLA2 in oxidant-induced cell death, A172 cells were exposed to hydrogen peroxide (H2O2) or tert-butylhydroperoxide (TBHP); both induced time- and concentration-dependent increases in cell death as assessed by annexin V and propidium iodide staining. Treatment of cells with racemic-BEL alone did not induce cell death. However, pretreatment with BEL prior to H2O2 (500 microM) or TBHP (200 microM) significantly increased necrosis as determined by increases in propidium iodide staining. Treatment with BEL prior to exposure to oxidants accelerated the loss of ATP levels, but not the formation of reactive oxygen species. These data support the hypothesis that iPLA2 mediates oxidant-induced neural cell death and demonstrates differential roles of iPLA2 isoforms in physiological and pathological events.

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

    PubMed

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

    2012-10-19

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

  12. Chloroquine and Hydroxychloroquine Are Novel Inhibitors of Human Organic Anion Transporting Polypeptide 1A2.

    PubMed

    Xu, Chenghao; Zhu, Ling; Chan, Ting; Lu, Xiaoxi; Shen, Weiyong; Madigan, Michele C; Gillies, Mark C; Zhou, Fanfan

    2016-02-01

    Chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used to treat malaria and inflammatory diseases, long-term usage of which often causes severe side effects, especially retinopathy. Solute carrier transporters (SLCs) are important proteins responsible for the cellular uptake of endogenous and exogenous substances. Inhibitors competing with transporter substrates for SLCs often results in unfavorable toxicities and unsatisfactory therapeutic outcomes. We investigated the inhibitory effect of CQ and HCQ on substrate uptake mediated through a range of important SLC transporters in overexpressing human embryonic kidney (HEK293) cells. Our data revealed that both CQ and HCQ potently inhibit the uptake activity of organic anion transporting polypeptide 1A2 (OATP1A2). We recently reported OATP1A2 to be expressed in human retinal pigment epithelium (RPE), where it mediates cellular uptake of all-trans-retinol (atROL), a key step in the classical visual cycle. In this study, we demonstrate that CQ and HCQ could markedly impair atROL uptake in OATP1A2-expressing HEK293 cells and more importantly, in primary human RPE cells. Our study shows that CQ and HCQ are novel inhibitors of OATP1A2 and significantly impair OATP1A2-mediated substrate uptake, particularly transport of atROL into the RPE. This effect may compromise the function of the classic visual cycle leading to vision impairment and contribute to the retinopathy observed clinically in patients using CQ or HCQ.

  13. Annexin A2 is a natural extrahepatic inhibitor of the PCSK9-induced LDL receptor degradation.

    PubMed

    Seidah, Nabil G; Poirier, Steve; Denis, Maxime; Parker, Rex; Miao, Bowman; Mapelli, Claudio; Prat, Annik; Wassef, Hanny; Davignon, Jean; Hajjar, Katherine A; Mayer, Gaétan

    2012-01-01

    Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2(-/-) mice revealed: i) a ∼1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ∼2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2(-/-) tissues revealed that the LDLR was decreased by ∼50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9≡LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-25

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2001-11-01

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

  18. 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.

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

    PubMed Central

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

    2010-01-01

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

  20. Expression of group IIA phospholipase A2 is an independent predictor of favorable outcome for patients with gastric cancer.

    PubMed

    Wang, Xi; Huang, Chun-Jin; Yu, Guan-Zhen; Wang, Jie-Jun; Wang, Rui; Li, Yu-Mei; Wu, Qiong

    2013-10-01

    Growing evidence suggests that phospholipase A2 (PLA2) plays a pivotal role in tumorigenesis in human gastrointestinal cancer. One of the well-studied isoforms of PLA2, group IIA PLA2 (PLA2G2A), appears to exert its protumorigenic or antitumorigenic effects in a tissue-specific manner. The present study was designed to determine the expression profile and prognostic value of PLA2G2A in gastric cancer in a large Chinese cohort. By using real-time polymerase chain reaction, the amount of PLA2G2A messenger RNA in 60 pairs of fresh gastric tumors and adjacent noncancerous mucosa was measured. The immunostaining of PLA2G2A in 866 gastric cancers with paired noncancerous tissues was assayed. No expression of PLA2G2A was found in normal gastric mucosa, and focal expression of PLA2G2A was noticed in intestinal metaplasia, whereas significantly increased expression of PLA2G2A was observed in the cytoplasm of gastric cancer cells. Furthermore, the extent of PLA2G2A expression was associated with tumor size (P < .001), tumor differentiation (P = .001), T class (P < .001), N class (P < .001), and TNM stage (P < .001) of gastric cancer. Multivariate analysis showed that PLA2G2A expression was an independent predictor of survival for patients with gastric cancer (P = .024). Expression of PLA2G2A seems to be protective for patients with gastric cancer (hazard ratio, 1.423; 95% confidence interval, 1.047-1.935), and it may be a target for achieving better treatment outcomes.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

    Cavigiolio, Giorgio; Jayaraman, Shobini

    2014-01-01

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

  3. The Rickettsia prowazekii ExoU Homologue Possesses Phospholipase A1 (PLA1), PLA2, and Lyso-PLA2 Activities and Can Function in the Absence of Any Eukaryotic Cofactors In Vitro ▿

    PubMed Central

    Housley, Nicole A.; Winkler, Herbert H.; Audia, Jonathon P.

    2011-01-01

    Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA2 and lyso-PLA2 activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA1 activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs. PMID:21764940

  4. Cytosolic phospholipaseA2 inhibition with PLA-695 radiosensitizes tumors in lung cancer animal models.

    PubMed

    Thotala, Dinesh; Craft, Jeffrey M; Ferraro, Daniel J; Kotipatruni, Rama P; Bhave, Sandeep R; Jaboin, Jerry J; Hallahan, Dennis E

    2013-01-01

    Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted.

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

    PubMed

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

    2002-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  7. Characterization of phospholipase A(2) activity in serum of the American alligator (Alligator mississippiensis).

    PubMed

    Merchant, Mark; Heard, Raphiel; Monroe, Caleb

    2009-11-01

    PLA(2) is a diverse class of enzymes with a broad spectrum of physiological functions. Secretory PLA(2) isoforms have been reported to exhibit important innate immune function in higher vertebrates. This study was conducted to characterize PLA(2) activity in the serum of the American alligator (Alligator mississippiensis). We used a glycerophospholipid with a fatty acid in the sn-2 position labeled with a fluorescent probe (BODIPY) to detect and quantify alligator serum PLA(2) activity. Incubation of BODIPY-labeled bacteria with different concentrations of alligator serum resulted in a concentration-dependent detection of PLA(2) activity. Kinetic studies showed that product formation was rapid, with substantial activity within 5 min, and maximal activity at approximately 20 min. The alligator PLA(2) activity was temperature-dependent, with activity at lower temperatures (5-10 degrees C) approximately half of that observed at temperatures of 30-40 degrees C. In addition, the generation of fluorescent product was reduced by a specific inhibitor (p-bromophenacyl bromide) of secretory PLA(2) in a concentration-dependent manner, enforcing the idea that the observed activities are due to a secretory PLA(2) enzyme in alligator serum.

  8. 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

  9. Effect of Chlorogenic Acid (5-Caffeoylquinic Acid) Isolated from Baccharis oxyodonta on the Structure and Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus terrificus

    PubMed Central

    Toyama, Daniela O.; Ferreira, Marcelo J. P.; Romoff, Paulete; Fávero, Oriana A.; Gaeta, Henrique H.; Toyama, Marcos H.

    2014-01-01

    The aim of this paper was to investigate the effect of chlorogenic acid (5-caffeoylquinic acid, 5CQA), isolated from Baccharis oxyodonta, on the structure and pharmacological effect of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus. All in vitro and in vivo experiments were conducted using a purified sPLA2 compared under the same experimental conditions with sPLA2 : 5CQA. 5CQA induced several discrete modifications in the secondary structure and the hydrophobic characteristics of native sPLA2 that induced slight changes in the α-helical content, increase in the random coil structure, and decrease of fluorescence of native sPLA2. Moreover, 5CQA significantly decreased the enzymatic activity and the oedema and myonecrosis induced by native sPLA2. As the catalytic activity of sPLA2 plays an important role in several of its biological and pharmacological properties, antibacterial activity was used to confirm the decrease in its enzymatic activity by 5CQA, which induced massive bacterial cell destruction. We found that 5CQA specifically abolished the enzymatic activity of sPLA2 and induced discrete protein unfolding that mainly involved the pharmacological site of sPLA2. These results showed the potential application of 5CQA in the snake poisoning treatment and modulation of the pathological effect of inflammation induced by secretory PLA2. PMID:25258715

  10. Phospholipase A2 inhibits cisplatin-induced acute kidney injury by modulating regulatory T cells by the CD206 mannose receptor.

    PubMed

    Kim, Hyunseong; Lee, Hyojung; Lee, Gihyun; Jang, Hyunil; Kim, Sung-Su; Yoon, Heera; Kang, Geun-Hyung; Hwang, Deok-Sang; Kim, Sun Kwang; Chung, Hwan-Suck; Bae, Hyunsu

    2015-09-01

    Previously, we found that Foxp3-expressing CD4(+) regulatory T (Treg) cells attenuate cisplatin-induced acute kidney injury in mice and that bee venom and its constituent phospholipase A2 (PLA2) are capable of modulating Treg cells. Here we tested whether PLA2 could inhibit cisplatin-induced acute kidney injury. As a result of treatment with PLA2, the population of Treg cells was significantly increased, both in vivo and in vitro. PLA2-injected mice showed reduced levels of serum creatinine, blood urea nitrogen, renal tissue damage, and pro-inflammatory cytokine production upon cisplatin administration. These renoprotective effects were abolished by depletion of Treg cells. Furthermore, PLA2 bound to CD206 mannose receptors on dendritic cells, essential for the PLA2-mediated protective effects on renal dysfunction. Interestingly, PLA2 treatment increased the secretion of IL-10 in the kidney from normal mice. Foxp3(+)IL-10(+) cells and CD11c(+)IL-10(+) cells were increased by PLA2 treatment. The anticancer effects of repeated administrations of a low dose of cisplatin were not affected by PLA2 treatment in a tumor-bearing model. Thus, PLA2 may prevent inflammatory responses in cisplatin-induced acute kidney injury by modulating Treg cells and IL-10 through the CD206 mannose receptor.

  11. Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2.

    PubMed

    Fuentes, Lucía; Pérez, Rebeca; Nieto, María L; Balsinde, Jesús; Balboa, María A

    2003-11-07

    Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.

  12. 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.

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

    PubMed Central

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

    2014-01-01

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

  14. Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2

    PubMed Central

    Rebres, Robert A.; Moon, Christina; DeCamp, Dianne; Lin, Keng-Mean; Fraser, Iain D.; Milne, Stephen B.; Roach, Tamara I. A.; Brown, H. Alex; Seaman, William E.

    2010-01-01

    Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca2+ responses to Gαi-linked receptors, including the C5aR, but reduced responses to Gαq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca2+ responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCβ isoform-deficient BMDM, we found that ToxB inhibited Ca2+ signaling through PLCβ4 but enhanced signaling through PLCβ3. Effects of ToxB on GPCR Ca2+ responses correlated with GPCR use of PLCβ3 versus PLCβ4. ToxB inhibited UDP Ca2+ signaling without reducing InsP3 production or the sensitivity of cellular Ca2+ stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca2+coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca2+ signaling by C5a was prevented by inhibition of PLA2 or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca2+ signaling by different GPCR-linked PLCβ isoforms in macrophages. PMID:20200401

  15. Extracellular phospholipase A2 secretion is a common effector pathway of interleukin-1 and tumour necrosis factor action.

    PubMed

    Vadas, P; Pruzanski, W; Stefanski, E; Ellies, L G; Aubin, J E; Sos, A; Melcher, A

    1991-06-01

    Inflammatory processes are characterized by increased levels of extracellular phospholipase A2 (PLA2) and cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF). IL-1, TNF and PLA2 share a number of proinflammatory, arthritogenic effects. The sequential induction, first of the cytokines followed by PLA2, suggests that these cytokines may regulate synthesis and secretion of PLA2. To test this postulate, foetal rat calvarial bone-forming cells (FRCC) were treated with recombinant human IL-1 and TNF and extracellular PLA2 release was quantitated. Both IL-1 and TNF induced the de novo synthesis of PLA2 in a concentration-dependent manner. Continuous exposure of FRCC in primary culture to IL-1 (50 units/ml) over 15 days resulted in as much as 100-fold increase in PLA2 secretion. IL-1 (50 units/ml) added to post-confluent cultures for a 48-h pulse increased PLA2 activity 9.4-fold. The combination of IL-1 (50 units/ml) and TNF (500 units/ml) was synergistic with an observed increase in extracellular PLA2 secretion of 146-fold following a 48-h pulse. Interleukin-6, alone or in combination with IL-1 or TNF, did not further enhance PLA2 synthesis of secretion. Cytokine-induced synthesis of PLA2 was inhibited 80% by 10 microM cycloheximide but not by dexamethasone over the range of 10(-6) to 10(-8) M. FRCC-derived PLA2 was neutral-active with a pH optimum of 6-7.5 and was calcium-dependent with optimal activity in the presence of 2-7 mM calcium. It had absolute 2-acyl specificity using micellar phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin.

    PubMed

    Chiou, Yi-Ling; Lin, Shinne-Ren; Hu, Wan-Ping; Chang, Long-Sen

    2014-12-15

    Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    2010-01-01

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

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

    PubMed

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

    2010-05-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2017-03-07

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

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

    PubMed

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

    2017-03-20

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

  3. Effect of NGF on the subcellular localization of group IIA secretory phospholipase A(2) (GIIA) in PC12 cells: role in neuritogenesis.

    PubMed

    Ferrini, M; Nardicchi, V; Mannucci, R; Arcuri, C; Nicoletti, I; Donato, R; Goracci, G

    2010-12-01

    Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.

  4. 1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid.

    PubMed

    Schwartz, Z; Shaked, D; Hardin, R R; Gruwell, S; Dean, D D; Sylvia, V L; Boyan, B D

    2003-05-01

    1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2012-10-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

    We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A. PMID:26836161

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

    PubMed

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

    2016-01-01

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

  11. Antiphospholipase A2 Receptor Autoantibodies: A Step Forward in the Management of Primary Membranous Nephropathy

    PubMed Central

    Obrisca, Bogdan; Ismail, Gener; Jurubita, Roxana; Baston, Catalin; Andronesi, Andreea; Mircescu, Gabriel

    2015-01-01

    Since the identification of PLA2R (M-type phospholipase A2 receptor) as the first human antigenic target in primary membranous nephropathy (MN), perpetual progress has been made in understanding the pathogenesis of this disease. Accumulating clinical data support a pathogenic role for the anti-PLA2R antibodies (PLA2R ABs), but confirmation in an animal model is still lacking. However, PLA2R ABs were related to disease activity and outcome, as well as to response therapy. Accordingly, PLA2R ABs assay seems to be promising tool not only to diagnose MN but also to predict the course of the disease and could open the way to personalize therapy. Nevertheless, validation of a universal assay with high precision and definition of cut-off levels, followed by larger studies with a prolonged follow-up period, are needed to confirm these prospects. PMID:26576418

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

    PubMed

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

    1998-04-01

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

  13. Towards novel radiosensitizing agents: the role of cytosolic PLA2α in combined modality cancer therapy

    PubMed Central

    Craft, Jeffrey M; Hallahan, Dennis

    2012-01-01

    The radioresistant nature of some tumors serves as an obstacle to curative therapy for several poor-prognosis malignancies. The radiosensitivity of a cancer is dependent not only on the intrinsic ability of tumor cells to recover from radiation-induced damage, but also the ability of stromal elements (e.g., vasculature) in the tumor microenvironment to survive and continue proliferating in the face of ionizing radiation. In this regard, it is important to understand the initial events activating radiation-induced signal transduction pathways. Among these events is the activation of cytosolic phospholipase A2α and the subsequent production of the lipid second messengers. These events occur within minutes following exposure to ionizing radiation, and have been shown to enhance cell viability through a number of prosurvival signaling pathways. Furthermore, inhibition of cytosolic phospholipase A2α has now been shown to reduce the viability of endothelial cells in culture after exposure to ionizing radiation, as well as slowing the growth of tumors in animal models of cancer. PMID:21644828

  14. Effects of mexiletine, a CYP1A2 inhibitor, on tizanidine pharmacokinetics and pharmacodynamics.

    PubMed

    Momo, Kenji; Homma, Masato; Osaka, Yoshiko; Inomata, Shin-ichi; Tanaka, Makoto; Kohda, Yukinao

    2010-03-01

    The aim of this study was to determine whether mexiletine, a CYP1A2 inhibitor, altered the pharmacokinetics and pharmacodynamics of tizanidine. The pharmacokinetics of tizanidine were examined in an open-label study in 12 healthy participants after a single dose of tizanidine (2 mg) with and without mexiletine coadministration (50 mg, 3 times as a pretreatment for a day and 2 times on the study day). Compared with tizanidine alone, mexiletine coadministration increased the peak plasma concentration (1.8 +/- 0.8 vs 5.3 +/- 1.8 ng/mL), area under the curve (4.5 +/- 2.2 vs 15.4 +/- 6.5 ng x h/mL), and the half-life (1.3 +/- 0.2 vs 1.8 +/- 0.7 h) of tizanidine, respectively (P < .05). Reduction in systolic blood pressure (-10 +/- 8 vs -24 +/- 7 mm Hg) and diastolic blood pressure (-10 +/- 7 vs -18 +/- 8 mm Hg) after tizanidine administration was also significantly enhanced by coadministration of mexiletine (P < .01). Of the 15 patients treated with tizanidine and mexiletine, 4 suffered tizanidine-induced adverse effects such as drowsiness and dry mouth in the retrospective survey. Present results suggested that coadministration of mexiletine increased blood tizanidine concentrations and enhanced tizanidine pharmacodynamics in terms of reduction in blood pressure and adverse symptoms.

  15. A 2-gene classifier for predicting response to the farnesyltransferase inhibitor tipifarnib in acute myeloid leukemia.

    PubMed

    Raponi, Mitch; Lancet, Jeffrey E; Fan, Hongtao; Dossey, Lesley; Lee, Grace; Gojo, Ivana; Feldman, Eric J; Gotlib, Jason; Morris, Lawrence E; Greenberg, Peter L; Wright, John J; Harousseau, Jean-Luc; Löwenberg, Bob; Stone, Richard M; De Porre, Peter; Wang, Yixin; Karp, Judith E

    2008-03-01

    At present, there is no method available to predict response to farnesyltransferase inhibitors (FTIs). We analyzed gene expression profiles from the bone marrow of patients from a phase 2 study of the FTI tipifarnib in older adults with previously untreated acute myeloid leukemia (AML). The RASGRP1/APTX gene expression ratio was found to predict response to tipifarnib with the greatest accuracy using a "leave one out" cross validation (LOOCV; 96%). RASGRP1 is a guanine nucleotide exchange factor that activates RAS, while APTX (aprataxin) is involved in DNA excision repair. The utility of this classifier for predicting response to tipifarnib was validated in an independent set of 58 samples from relapsed or refractory AML, with a negative predictive value (NPV) and positive predictive value (PPV) of 92% and 28%, respectively (odds ratio of 4.4). The classifier also predicted for improved overall survival (154 vs 56 days; P < .001), which was independent of other covariates, including a previously described prognostic gene expression classifier. Therefore, these data indicate that a 2-gene expression assay may have utility in categorizing a population of patients with AML who are more likely to respond to tipifarnib.

  16. Cloning and Sequence Analysis of cDNAs Encoding Two Acidic PLA(2) from venom of Ophiophagus hannah(King Cobra), Guangxi Species.

    PubMed

    Wang, Qiu-Yan; Shu, Yu-Yan; Zhuang, Mao-Xing; Lin, Zheng-Jiong

    2001-01-01

    Total RNA was extracted from venom glands of Ophiophagus hannah, Guangxi species. The cDNAs encoding PLA(2) were amplified by RT-PCR and cloned into the PUCm-T vector. The positive clones encoding two acidic PLA(2) (APLA(2)-1 and APLA(2)-2) were selected and bidirectionally sequenced. Their complete amino acid sequences were deduced and found to be identical to the known amino acid sequences. Their isoelectric points calculated by computer agreed with the values determined with their protein. Homology analysis indicated that the mature peptide of APLA(2)-1 had high homology with PLA(2) from venoms of Ophiophagus hannah, Fujian and Taiwan species, but APLA(2)-2 had lower homology. The most striking difference between APLA(2)-2 and other PLA(2) from Ophiophagus hannah venoms is the missing of a extra "pancreatic loop" at residues 62--66 in APLA(2)-2, and it may be related to their species evolution and biological activity.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-01-01

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

  19. Long non-coding RNA SNHG14 promotes microglia activation by regulating miR-145-5p/PLA2G4A in cerebral infarction.

    PubMed

    Qi, Xu; Shao, Ming; Sun, Hongjing; Shen, Yue; Meng, Delong; Huo, Wei

    2017-04-21

    Activated microglia cells (MCs) are able to release a large amount of inflammatory cytokines after ischemic stroke, which exacerbates neuron damage. In this study, we explored the functional involvement of long non-coding RNA (lncRNA) SNHG14 and its potential regulatory mechanism in the activation of MCs. The mouse model of middle cerebral artery occlusion (MCAO) and microglia cell model of oxygen/glucose deprivation (OGD) were made. The expression of SNHG14, miR-145-5p and PLA2G4A protein expression was determined by quantitative real time PCR and western blot, respectively. Dual-luciferase assay was used to verify the direct binding of miR-145-5p and PLA2G4A. Flow cytometry was applied to measure neurons' apoptosis. SNHG14 highly expressed in ischemic cerebral tissues and BV-2 cells after OGD treatment. SNHG14 knockdown could remarkably inhibit BV-2 cells activation induced by OGD; while SNHG14 overexpression significantly promoted BV-2 cells activation, showing an increase of TNF-α and NO production and neurons' apoptosis rate. Additionally, SNHG14 knockdown promoted the expression of miR-145-5p and reduced PLA2G4A. Contrarily, SNHG14 overexpression inhibited miR-145-5p expression and increased PLA2G4A. Moreover, miR-145-5p overexpression also reversed the effect of OGD on BV-2 cells activation. Bioinformatics analysis and dual-luciferase assay supported that SNHG14 could bind directly to miR-145-5p and miR-145-5p-binding site was existed on 3'-UTR of PLA2G4A. MiR-145-5p mimic reversed the increase of PLA2G4A and reduced the high levels of TNF-α and NO in BV-2 cells induced by SNHG14 overexpression. SNHG14 increased the expression of PLA2G4A by inhibition of miR-145-5p, which resulted in the activation of MCs in cerebral infarction.

  20. Identification of a secretory phospholipase A2 from Papaver somniferum L. that transforms membrane phospholipids.

    PubMed

    Jablonická, Veronika; Mansfeld, Johanna; Heilmann, Ingo; Obložinský, Marek; Heilmann, Mareike

    2016-09-01

    The full-length sequence of a new secretory phospholipase A2 was identified in opium poppy seedlings (Papaver somniferum L.). The cDNA of poppy phospholipase A2, denoted as pspla2, encodes a protein of 159 amino acids with a 31 amino acid long signal peptide at the N-terminus. PsPLA2 contains a PLA2 signature domain (PA2c), including the Ca(2+)-binding loop (YGKYCGxxxxGC) and the catalytic site motif (DACCxxHDxC) with the conserved catalytic histidine and the calcium-coordinating aspartate residues. The aspartate of the His/Asp dyad playing an important role in animal sPLA2 catalysis is substituted by a serine residue. Furthermore, the PsPLA2 sequence contains 12 conserved cysteine residues to form 6 structural disulfide bonds. The calculated molecular weight of the mature PsPLA2 is 14.0 kDa. Based on the primary structure PsPLA2 belongs to the XIB group of PLA2s. Untagged recombinant PsPLA2 obtained by expression in Escherichia coli, renaturation from inclusion bodies and purification by cation-exchange chromatography was characterized in vitro. The pH optimum for activity of PsPLA2 was found to be pH 7, when using mixed micelles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Triton X-100. PsPLA2 specifically cleaves fatty acids from the sn-2 position of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and shows a pronounced preference for PC over phosphatidyl ethanolamine, -glycerol and -inositol. The active recombinant enzyme was tested in vitro against natural phospholipids isolated from poppy plants and preferably released the unsaturated fatty acids, linoleic acid and linolenic acid, from the naturally occurring mixture of substrate lipids.

  1. Platelet Aggregation Unchanged by Lipoprotein-Associated Phospholipase A2 Inhibition: Results from an In Vitro Study and Two Randomized Phase I Trials

    PubMed Central

    Shaddinger, Bonnie C.; Xu, Yanmei; Roger, James H.; Macphee, Colin H.; Handel, Malcolm; Baidoo, Charlotte A.; Magee, Mindy; Lepore, John J.; Sprecher, Dennis L.

    2014-01-01

    Background We explored the theorized upregulation of platelet-activating factor (PAF)– mediated biologic responses following lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibition using human platelet aggregation studies in an in vitro experiment and in 2 clinical trials. Methods and Results Full platelet aggregation concentration response curves were generated in vitro to several platelet agonists in human plasma samples pretreated with rilapladib (selective Lp-PLA2 inhibitor) or vehicle. This was followed by a randomized, double-blind crossover study in healthy adult men (n = 26) employing a single-agonist dose assay of platelet aggregation, after treatment of subjects with 250 mg oral rilapladib or placebo once daily for 14 days. This study was followed by a second randomized, double-blind parallel-group trial in healthy adult men (n = 58) also treated with 250 mg oral rilapladib or placebo once daily for 14 days using a full range of 10 collagen concentrations (0–10 µg/ml) for characterizing EC50 values for platelet aggregation for each subject. Both clinical studies were conducted at the GlaxoSmithKline Medicines Research Unit in the Prince of Wales Hospital, Sydney, Australia. EC50 values derived from multiple agonist concentrations were compared and no pro-aggregant signals were observed during exposure to rilapladib in any of these platelet studies, despite Lp-PLA2 inhibition exceeding 90%. An increase in collagen-mediated aggregation was observed 3 weeks post drug termination in the crossover study (15.4% vs baseline; 95% confidence interval [CI], 3.9–27.0), which was not observed during the treatment phase and was not observed in the parallel-group study employing a more robust EC50 examination. Conclusions Lp-PLA2 inhibition does not enhance platelet aggregation. Trial Registration 1) Study 1: ClinicalTrials.gov NCT01745458 2) Study 2: ClinicalTrials.gov NCT00387257 PMID:24475026

  2. Spectroscopic investigation of phenolic groups ionization in the vipoxin neurotoxic phospholipase A 2: comparison with the X-ray structure in the region of the tyrosyl residues

    NASA Astrophysics Data System (ADS)

    Georgieva, Dessislava Nikolova; Genov, Nicolay; Rajashankar, Kanagalaghatta R.; Aleksiev, Boris; Betzel, Christian

    1998-12-01

    The neurotoxin vipoxin is the major lethal component of the venom of Vipera ammodites meridionalis, the most toxic snake in Europe. It is a complex between a toxic phospholipase A 2 (PLA 2) and a non-toxic protein inhibitor (Inh). Tyrosyl residues are involved in the catalytic site (Tyr 52 and 73) and in the substrate binding (Tyr 22). Spectroscopic studies demonstrated differences in the ionization behavior of the various phenolic hydroxyl groups in the toxic PLA 2. The tyrosyl side chains of the enzyme can be classified into three groups: (a) three phenolic hydroxyls are accessible to the solvent and titrate normally, with a p Keff=10.45; (b) three residues are partially 'buried' and participate in hydrogen bonds with neighboring functional groups. They titrate anomalously with a p Keff=12.17; (c) two tyrosines with a p Keff=13.23 are deeply 'buried' in the hydrophobic interior of PLA 2. They became accessible to the titrating agent only after alkaline denaturation of the protein molecule. The spectroscopic data are related to the X-ray structure of the vipoxin PLA 2. The refined model was investigated in the region of the tyrosyl side chains. The accessible surface area of each tyrosyl residue and each phenolic hydroxyl group was calculated. A good correlation between the spectrophotometric and the crystallographic data was observed. The ionization behavior of the phenolic groups is explained by peculiarities of the protein three-dimensional structure and the participation of tyrosines in the catalytic site hydrogen bond network. Attempts are made to assign the calculated p Keff values to individual residues. The high degree of 'exposure' on the protein surface of Tyr 22 and 75 is probably important for their function as parts of the substrate binding and pharmacological sites.

  3. 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

  4. Effects of Statins and Xuezhikang on the Expression of Secretory Phospholipase A2, Group IIA in Rat Vascular Smooth Muscle Cells.

    PubMed

    Xie, Qiang; Zhang, Dan

    2017-02-07

    Atherosclerosis is a multifactorial vascular disease characterized by formation of inflammatory lesions. Secretory phospholipase A2, group IIA (sPLA2-IIA) is involved in this process and plays a critical role. However, the exact role of sPLA2-IIA in cardiovascular inflammation is more complicated and remains unclear. Furthermore, both statins and Xuezhikang (XZK) are widely used in the prevention and treatment of cardiovascular disease risk because of their pleiotropic effects on the cardiovascular system. However, their effects on sPLA2-IIA are still controversial. We investigated the regulation of sPLA2-IIA by rat thoracic aorta smooth muscle cells (VSMCs) in culture. Cells were first incubated with IL-1β alone to induce expression of sPLA2-IIA and then treated with several concentrations of statins or XZK for different times in the absence or presence of IL-1β. We tested the expression of sPLA2-IIA, including sPLA2-IIA mRNA, protein, as well as activity. We found that statins or IL-1β increase the expression of sPLA2-IIA in VSMCs and the effect is based on a synergetic relationship between them. However, for the first time, we observed that XZK effectively reduces sPLA2-IIA expression in IL-1β-treated VSMCs. Our findings may shine a new light on the clinical use of XZK and statins in the prevention and treatment of atherosclerosis-related thrombosis.

  5. 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.

  6. 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.

  7. Lipoprotein-associated phospholipase A2 and risk of incident peripheral arterial disease in a multi-ethnic cohort: The Multi-Ethnic Study of Atherosclerosis.

    PubMed

    Garg, Parveen K; Jorgensen, Neal W; McClelland, Robyn L; Jenny, Nancy S; Criqui, Michael H; Allison, Matthew A; Greenland, Philip; Rosenson, Robert S; Siscovick, David S; Cushman, Mary

    2017-02-01

    Prospective studies supporting a relationship between elevated lipoprotein-associated phospholipase A2 (Lp-PLA2) and incident peripheral arterial disease (PAD) are limited. We evaluated the association of Lp-PLA2 with incident PAD in a multi-ethnic cohort without clinical cardiovascular disease. A total of 4622 participants with measurement of Lp-PLA2 mass and Lp-PLA2 activity and an ankle-brachial index (ABI) between 0.9 and 1.4 were followed for the development of PAD (median follow-up = 9.3 years), defined as an ABI ⩽0.9 and decline from baseline ⩾0.15. There were 158 incident PAD events during follow-up. In adjusted logistic regression models, each higher standard deviation of both Lp-PLA2 activity and mass did not confer an increased risk of developing PAD [odds ratios, (95% confidence intervals)]: 0.92 (0.66-1.27) for Lp-PLA2 activity and 1.06 (0.85-1.34) for mass. Additionally, no significant interaction was found according to ethnicity: p=0.43 for Lp-PLA2 activity and p=0.55 for Lp-PLA2 mass. We found no evidence of an association between Lp-PLA2 and incident PAD.

  8. 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

  9. Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

    PubMed Central

    Beck, Goichi; Shinzawa, Koei; Hayakawa, Hideki; Baba, Kousuke; Yasuda, Toru; Sumi-Akamaru, Hisae; Tsujimoto, Yoshihide; Mochizuki, Hideki

    2015-01-01

    Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA2β), which is encoded by the PLA2G6 gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA2β-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA2β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA2β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA2β-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA2β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction. PMID:26506412

  10. 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

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

    PubMed

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

    2014-03-01

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

  12. Unusual venom phospholipases A2 of two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneensis.

    PubMed

    Wang, Ying-Ming; Peng, Hao-Fan; Tsai, Inn-Ho

    2005-06-01

    To explore the venom diversity of Asian pit vipers, we investigated the structure and function of venom phospholipase A2 (PLA2) derived from two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneensis. We purified six novel PLA2s from T. puniceus venom and another three from T. borneensis venom. All cDNAs encoding these PLA2s except one were cloned, and the molecular masses and N-terminal sequences of the purified enzymes closely matched those predicted from the cDNA. Three contain K49 and lack a disulfide bond at C61-C91, in contrast with the D49-containing PLA2s in both venom species. They are less thermally stable than other K49-PLA2s which contain seven disulfide bonds, as indicated by a decrease of 8.8 degrees C in the melting temperature measured by CD spectroscopy. The M110D mutation in one of the K49-PLA2s apparently reduced its edematous potency. A phylogenetic tree based on the amino-acid sequences of 17 K49-PLA2s from Asian pit viper venoms illustrates close relationships among the Trimeresurus species and intergeneric segregations. Basic D49-PLA2s with a unique Gly6 substitution were also purified from both venoms. They showed edema-inducing and anticoagulating activities. It is notable that acidic PLA2s from both venoms inhibited blood coagulation rather than platelet aggregation, and this inhibition was only partially dependent on enzyme activity. These results contribute to our understanding of the evolution of Trimeresurus pit vipers and the structure-function relationships between various subtypes of crotalid venom PLA2.

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  15. 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.

  16. 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

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

    PubMed

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

    2014-03-15

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

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed

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

    2003-06-01

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

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

    PubMed

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

    2005-09-01

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

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

    PubMed

    Bacha, Abir Ben

    2014-10-01

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

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

    PubMed

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

    2016-02-26

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

  3. Pantothenate kinase-associated neurodegeneration (PKAN) and PLA2G6-associated neurodegeneration (PLAN): review of two major neurodegeneration with brain iron accumulation (NBIA) phenotypes.

    PubMed

    Kurian, Manju A; Hayflick, Susan J

    2013-01-01

    Neurodegeneration with brain iron accumulation (NBIA) comprises a heterogeneous group of disorders characterized by the presence of radiologically discernible high brain iron, particularly within the basal ganglia. A number of childhood NBIA syndromes are described, of which two of the major subtypes are pantothenate kinase-associated neurodegeneration (PKAN) and PLA2G6-associated neurodegeneration (PLAN). PKAN and PLAN are autosomal recessive NBIA disorders due to mutations in PANK2 and PLA2G6, respectively. Presentation is usually in childhood, with features of neurological regression and motor dysfunction. In both PKAN and PLAN, a number of classical and atypical phenotypes are reported. In this chapter, we describe the clinical, radiological, and genetic features of these two disorders and also discuss the pathophysiological mechanisms postulated to play a role in disease pathogenesis.

  4. Roles of PKA, PI3K, and cPLA2 in the NO-mediated negative inotropic effect of beta2-adrenoceptor agonists in guinea pig right papillary muscles.

    PubMed

    Faucher, Fabien A; Gannier, François E; Lignon, Jacques M; Cosnay, Pierre; Malécot, Claire O

    2008-01-01

    Although beta(2)-adrenoceptors represent 15-25% of beta-adrenoceptors in the guinea pig heart, their functionality is controversial. We assessed the inotropic effects of beta(2)-adrenoceptor partial agonists in right papillary muscles. Salbutamol induced a small but significant concentration-dependent negative inotropic effect (NIE, -5% at 60 nM) followed by a moderate positive inotropic effect (+36% at 6 microM) due to activation of beta(1)-adrenoceptors. In the presence of 4 microM atenolol, the concentration-dependent NIE (-12% at 6 microM) was biphasic, best described by a double logistic equation with respective EC(50) values of 3 and approximately 420 nM, and was insensitive to SR59230A. In muscles from pertussis toxin-treated guinea pigs, the salbutamol-induced positive inotropic effect was sensitive to low concentrations of ICI-118551 in an unusual manner. Experiments in reserpinized animals revealed the importance of the phosphorylation-dephosphorylation processes. PKA inhibition reduced and suppressed the effects obtained at low and high concentrations, respectively, indicating that its activation was a prerequisite to the NIE. The effect occurring at nanomolar concentrations depended upon PKA/phosphatidylinositol 3-kinase/cytosolic phospholipase A(2) (cPLA(2)) activations leading to nitric oxide (NO) release via the arachidonic acid/cyclooxygenase pathway. NO release via PKA-dependent phosphorylation of the receptor was responsible for the inotropic effect observed at submicromolar concentrations, which is negatively controlled by cPLA(2). The possibility that these effects are due to an equilibrium between different affinity states of the receptor (G(s)/G(i) coupled and G(i) independent with different signaling pathways) that can be displaced by ICI-118551 is discussed. We conclude that beta(2)-adrenoceptors are functional in guinea pig heart and can modulate the inotropic state.

  5. Novel PLA2G6 mutations associated with an exonic deletion due to non-allelic homologous recombination in a patient with infantile neuroaxonal dystrophy

    PubMed Central

    Yamamoto, Toshiyuki; Shimojima, Keiko; Shibata, Takashi; Akiyama, Mari; Oka, Makio; Akiyama, Tomoyuki; Yoshinaga, Harumi; Kobayashi, Katsuhiro

    2015-01-01

    Novel PLA2G6 mutations associated with p.Asp283Asn and a unique intragenic deletion of exons 4 and 5 due to non-allelic homologous recombination were identified in a Japanese female patient with typical infantile neuroaxonal dystrophy. The patient showed progressive tetraplegia beginning at 9 months. An electroencephalogram showed a diffuse increase in fast waves, and brain magnetic resonance imaging showed progressive brain atrophy and T2 hypointensity in the globus pallidus. PMID:27081553

  6. 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

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

    PubMed

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

    2010-05-01

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

  8. Screening of the Pan-African natural product library identifies ixoratannin A-2 and boldine as novel HIV-1 inhibitors.

    PubMed

    Tietjen, Ian; Ntie-Kang, Fidele; Mwimanzi, Philip; Onguéné, Pascal Amoa; Scull, Margaret A; Idowu, Thomas Oyebode; Ogundaini, Abiodun Oguntuga; Meva'a, Luc Mbaze; Abegaz, Berhanu M; Rice, Charles M; Andrae-Marobela, Kerstin; Brockman, Mark A; Brumme, Zabrina L; Fedida, David

    2015-01-01

    The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world.

  9. Screening of the Pan-African Natural Product Library Identifies Ixoratannin A-2 and Boldine as Novel HIV-1 Inhibitors

    PubMed Central

    Tietjen, Ian; Ntie-Kang, Fidele; Mwimanzi, Philip; Onguéné, Pascal Amoa; Scull, Margaret A.; Idowu, Thomas Oyebode; Ogundaini, Abiodun Oguntuga; Meva’a, Luc Mbaze; Abegaz, Berhanu M.; Rice, Charles M.; Andrae-Marobela, Kerstin; Brockman, Mark A.; Brumme, Zabrina L.; Fedida, David

    2015-01-01

    The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world. PMID:25830320

  10. Interfacial Catalysis: The Mechanism of Phospholipase A2

    PubMed Central

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

    2012-01-01

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

  11. Atomic resolution structure of prokaryotic phospholipase A2: analysis of internal motion and implication for a catalytic mechanism.

    PubMed

    Matoba, Yasuyuki; Sugiyama, Masanori

    2003-05-15

    We have found a secreted phospholipase A(2) (PLA(2), EC 3.1.1.4) from Streptomyces violaceoruber A-2688, which is the first PLA(2) identified in prokaryote, and determined its tertiary structure by NMR and X-ray analyses. In this study, we collected the X-ray diffraction data of the bacterial PLA(2) at room temperature (297 K) using conventional MoK(alpha) radiation and refined the structure at a 1.05 A resolution. The atomic resolution analysis led us to introduce disordered conformations and hydrogen atoms into a full anisotropic model. The molecular motion, which is expressed as the sum of rigid-body motion and internal motion of protein, is roughly estimated as the thermal motion when the X-ray diffraction data are collected at room temperature. In this study, we applied a TLS (rigid-body motion in terms of translation, libration, and screw motions) model to analyze the rigid-body motion of the bacterial PLA(2) and calculated the internal motion by subtracting the estimate of the rigid-body motion from the observed anisotropic temperature factor. We also subjected the TLS model to estimate the internal motion of the bovine pancreatic PLA(2) using the anisotropic temperature factor deposited in the Protein Data Bank. Both results indicate that the localization of regions exhibiting larger internal motion in the bacterial PLA(2) is almost the same as that in the bovine pancreatic PLA(2), suggesting that although the tertiary structure of the bacterial PLA(2) is strikingly different from that of the bovine pancreatic PLA(2), the internal motion, which is associated with the calcium(II) ion-binding, phospholipid-binding, and allosteric interfacial activation, is commonly observed in both PLA(2)s.

  12. Alleviation of high-fat diet-induced fatty liver damage in group IVA phospholipase A2-knockout mice.

    PubMed

    Ii, Hiromi; Yokoyama, Naoki; Yoshida, Shintaro; Tsutsumi, Kae; Hatakeyama, Shinji; Sato, Takashi; Ishihara, Keiichi; Akiba, Satoshi

    2009-12-01

    Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A(2) (IVA-PLA(2)), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA(2)-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA(2)-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA(2)-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA(2)-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA(2)-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E(2), which has a fat storage effect, was lower in IVA-PLA(2)-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA(2) alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA(2) metabolites, such as prostaglandin E(2). IVA-PLA(2) could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.

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

    PubMed Central

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

    2016-01-01

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

  14. 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

  15. Comparative proteomics and subtyping of venom phospholipases A2 and disintegrins of Protobothrops pit vipers.

    PubMed

    Tsai, Inn-Ho; Chen, Yi-Hsuan; Wang, Ying-Ming

    2004-10-01

    To explore the venom diversity and systematics of pit vipers under the genus Protobothrops, the venom phospholipases A2 (PLA2s) of P. mangshanensis, P. elegans and P. tokarensis were purified and characterized for the first time. The results were compared with the corresponding venom data of other co-generic species including P. mucrosquamatus, P. flavoviridis and P. jerdonii. Based on sequence features at the N-terminal regions, we identified five PLA2 subtypes, i.e., the Asp49-PLA2s with N6, E6 or R6 substitution and the Lys49-PLA2. However, not all subtypes were expressed in each of the species. Venom N6-PLA2s from P. mangshanensis and P. tokarensis venom were weakly neurotoxic toward chick biventer cervicis tissue preparations. The venoms of P. tokarensis and P. flavoviridis contained identical PLA2 isoforms. In most Protobothrop disintegrins, sequences flanking the RGD-motif are conserved. Phylogenetic analyses based on amino acid sequences of both families of the acidic PLA2s and the disintegrins clarify that these species could belong to a monophyletic group.

  16. Predictive three-dimensional quantitative structure-activity relationship of cytochrome P450 1A2 inhibitors.

    PubMed

    Korhonen, Laura E; Rahnasto, Minna; Mähönen, Niina J; Wittekindt, Carsten; Poso, Antti; Juvonen, Risto O; Raunio, Hannu

    2005-06-02

    The purpose of this study was to determine the cytochrome P450 1A2 (CYP1A2) inhibition potencies of structurally diverse compounds to create a comprehensive three-dimensional quantitative structure-activity relationship (3D-QSAR) model of CYP1A2 inhibitors and to use this model to predict the inhibition potencies of an external set of compounds. Fifty-two compounds including naphthalene, lactone and quinoline derivatives were assayed in a 96-well plate format for CYP1A2 inhibition activity using 7-ethoxyresorufin O-dealkylation as the probe reaction. The IC50 values of the tested compounds varied from 2.3 microM to over 40,000 microM. On the basis of this data set, a comparative molecular field analysis (CoMFA) and GRID/GOLPE models were created that yielded novel structural information about the interaction between inhibitory molecules and the CYP1A2 active site. The created CoMFA model was able to accurately predict inhibitory potencies of several structurally unrelated compounds, including selective inhibitors of other cytochrome P450 forms.

  17. Nitro-oleic acid downregulates lipoprotein-associated phospholipase A2 expression via the p42/p44 MAPK and NFκB pathways

    PubMed Central

    Wang, Gangqi; Ji, Yuan; Li, Zhuang; Han, Xiaolei; Guo, Nannan; Song, Qi; Quan, Longquan; Wang, Tiedong; Han, Wenyu; Pang, Daxin; Ouyang, Hongsheng; Tang, Xiaochun

    2014-01-01

    Nitro-oleic acid (OA-NO2), acting as anti-inflammatory signaling mediators, are involved in multiple signaling pathways. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is well known as a cardiovascular risk biomarker. Our results showed that OA-NO2 downregulated the expression of Lp-PLA2 in a time- and dose-dependent manner, whereas native OA had no such effect. Furthermore, OA-NO2 could repress Lp-PLA2 expression in the peripheral blood mononuclear cells of apo CIII-transgenic (apo CIII TG) pigs, which exhibited higher Lp-PLA2 expression and activity than did wild-type (WT) pigs. OA-NO2 inhibited Lp-PLA2 expression in macrophages, independent of nitric oxide formation and PPARγ-activation. However, OA-NO2 downregulates Lp-PLA2 by inhibiting the p42/p44 mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NFκB) pathways. When used to mediate anti-inflammatory signaling, the regulation of inflammatory cytokines and SOD by OA-NO2 might be associated with the reduction of Lp-PLA2. These results suggested that OA-NO2 might exert a vascular-protective effect partially via Lp-PLA2 inhibition. PMID:24809325

  18. Nitro-oleic acid downregulates lipoprotein-associated phospholipase A2 expression via the p42/p44 MAPK and NFκB pathways.

    PubMed

    Wang, Gangqi; Ji, Yuan; Li, Zhuang; Han, Xiaolei; Guo, Nannan; Song, Qi; Quan, Longquan; Wang, Tiedong; Han, Wenyu; Pang, Daxin; Ouyang, Hongsheng; Tang, Xiaochun

    2014-05-09

    Nitro-oleic acid (OA-NO2), acting as anti-inflammatory signaling mediators, are involved in multiple signaling pathways. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is well known as a cardiovascular risk biomarker. Our results showed that OA-NO2 downregulated the expression of Lp-PLA2 in a time- and dose-dependent manner, whereas native OA had no such effect. Furthermore, OA-NO2 could repress Lp-PLA2 expression in the peripheral blood mononuclear cells of apo CIII-transgenic (apo CIII TG) pigs, which exhibited higher Lp-PLA2 expression and activity than did wild-type (WT) pigs. OA-NO2 inhibited Lp-PLA2 expression in macrophages, independent of nitric oxide formation and PPARγ-activation. However, OA-NO2 downregulates Lp-PLA2 by inhibiting the p42/p44 mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NFκB) pathways. When used to mediate anti-inflammatory signaling, the regulation of inflammatory cytokines and SOD by OA-NO2 might be associated with the reduction of Lp-PLA2. These results suggested that OA-NO2 might exert a vascular-protective effect partially via Lp-PLA2 inhibition.

  19. Cloning and characterization of cDNAs encoding three isoforms of phospholipase A2 in Malayan spitting cobra (Naja naja sputatrix) venom.

    PubMed

    Armugam, A; Earnest, L; Chung, M C; Gopalakrishnakone, P; Tan, C H; Tan, N H; Jeyaseelan, K

    1997-01-01

    cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

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

    PubMed Central

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

    2014-01-01

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

  1. Genetic ablation of calcium-independent phospholipase A2gamma leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype.

    PubMed

    Mancuso, David J; Sims, Harold F; Han, Xianlin; Jenkins, Christopher M; Guan, Shao Ping; Yang, Kui; Moon, Sung Ho; Pietka, Terri; Abumrad, Nada A; Schlesinger, Paul H; Gross, Richard W

    2007-11-30

    Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

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

    PubMed Central

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

    2009-01-01

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

  3. The first convergent total synthesis of penarolide sulfate A2, a novel α-glucosidase inhibitor.

    PubMed

    Gao, Yangguang; Shan, Qiuli; Liu, Jun; Wang, Linlin; Du, Yuguo

    2014-04-07

    Penarolide sulfate A2, a 31-membered macrolide encompassing a proline residue and three sulfate groups, was firstly synthesized in 16 linear steps with 4.8% overall yield. Three consecutive stereogenic centers in penarolide sulfate A2 were efficiently derived from natural chiral template l-arabinose. The crucial assembly reactions included Brown asymmetric allylation, olefin cross-metathesis, alkyne-epoxide coupling, and macrolactamization. The anti-yeast α-glucosidase activities of penarolide sulfate A2 and its fully desulfated derivative were examined showing IC50 values of 4.87 and 10.74 μg mL(-1), respectively.

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    1999-03-01

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

  8. Genetic Analysis of PLA2G6 in 22 Indian Families with Infantile Neuroaxonal Dystrophy, Atypical Late-Onset Neuroaxonal Dystrophy and Dystonia Parkinsonism Complex

    PubMed Central

    Rather, Mohammad Iqbal; Bhat, Vishwanath; Gopinath, Sindhura; Bindu, Parayil Sankaran; Taly, Arun B.; Sinha, Sanjib; Nagappa, Madhu; Bharath, Rose Dawn; Mahadevan, Anita; Narayanappa, Gayathri; Chickabasaviah, Yasha T.; Kumar, Arun

    2016-01-01

    Mutations in PLA2G6 were identified in patients with a spectrum of neurodegenerative conditions, such as infantile neuroaxonal dystrophy (INAD), atypical late-onset neuroaxonal dystrophy (ANAD) and dystonia parkinsonism complex (DPC). However, there is no report on the genetic analysis of families with members affected with INAD, ANAD and DPC from India. Therefore, the main aim of this study was to perform genetic analysis of 22 Indian families with INAD, ANAD and DPC. DNA sequence analysis of the entire coding region of PLA2G6 identified 13 different mutations, including five novel ones (p.Leu224Pro, p.Asp283Asn, p.Arg329Cys, p.Leu491Phe, and p.Arg649His), in 12/22 (54.55%) families with INAD and ANAD. Interestingly, one patient with INAD was homozygous for two different mutations, p.Leu491Phe and p.Ala516Val, and thus harboured four mutant alleles. With these mutations, the total number of mutations in this gene reaches 129. The absence of mutations in 10/22 (45.45%) families suggests that the mutations could be in deep intronic or promoter regions of this gene or these families could have mutations in a yet to be identified gene. The present study increases the mutation landscape of PLA2G6. The present finding will be useful for genetic diagnosis, carrier detection and genetic counselling to families included in this study and other families with similar disease condition. PMID:27196560

  9. Restoration of on-time embryo implantation corrects the timing of parturition in cytosolic phospholipase A2 group IVA deficient mice.

    PubMed

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

    2009-12-01

    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.

  10. High-density Association Mapping and Interaction Analysis of PLA2R1 and HLA Regions with Idiopathic Membranous Nephropathy in Japanese

    PubMed Central

    Thiri, Myo; Honda, Kenjiro; Kashiwase, Koichi; Mabuchi, Akihiko; Suzuki, Hodaka; Watanabe, Kimio; Nakayama, Masaaki; Watanabe, Tsuyoshi; Doi, Kent; Tokunaga, Katsushi; Noiri, Eisei

    2016-01-01

    Although recent studies showed anti-PLA2R antibody plays a crucial role in idiopathic membranous nephropathy (IMN), detailed HLA mapping and interaction between the HLA genes and PLA2R1 have not been investigated in IMN. We genotyped across the PLA2R1 gene and the HLA region, using 183 IMN patients and 811 healthy controls. Five SNPs around the PLA2R1 gene were significantly associated with IMN. In addition to the two SNPs previously reported to be strongly associated with IMN, rs3749119 and rs35771982 (OR 3.02 and 2.93, P = 3.24E-14 and 4.64E-14, respectively), two novel intronic SNPs (rs2715928 and rs16844715) were also identified as IMN-associated SNPs (OR = 2.30 and 2.51, P = 3.15E-10 and 5.66E-13, respectively). In the HLA gene analysis, DRB1*1501 and DQB1*0602 were strongly associated with IMN (P = 1.14E-11 and 1.25E-11, respectively). The interaction was strongest between HLA-DRB1*15:01 - HLA-DQB1*06:02 and the intronic SNP rs2715928 (OR = 17.53, P = 4.26E-26). Furthermore, positive interaction was also observed between HLA-DRB1*15:01 - HLA-DQB1*06:02 and the missense SNP rs35771982 (OR = 15.91, P = 2.76E-29), which is in strong linkage disequilibrium with 5′UTR SNP rs3749119, and intronic SNP rs16844715 (OR = 15.91, P = 2.30E-26) for IMN. Neither HLA-DRB1*15:01 nor HLA-DQB1*06:02 was associated with steroid responsiveness, overall survival and renal survival during the observation period of mean 11 years though limited number of analysis. PMID:27934873

  11. 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

  12. Cytoplasmic Phospholipase A2 Antagonists Inhibit Multiple Endocytic Membrane Trafficking Pathways

    PubMed Central

    Doody, Anne M.; Antosh, Amy L.; Brown, William J.

    2009-01-01

    Previous studies have suggested a role for cytosolic Ca2+-independent phospholipase A2 (PLA2) activity in the formation of endosome membrane tubules that participate in the export of transferrin (Tf) and transferrin receptors (TfR) from sorting endosomes (SEs) and the endocytic recycling compartment (ERC). Here we show that the PLA2 requirement is a general feature of endocytic trafficking. The reversible cytoplasmic PLA2-antagonist ONO-RS-082 (ONO) produced a concentration-dependent, differential block in the endocytic recycling of both low-density lipoprotein receptor (LDLR) and TfRs, and in the degradative pathways of LDL and epidermal growth factor (EGF). These results are consistent with the model that a cytoplasmic PLA2 plays a general role in the export of cargo from multiple endocytic compartments by mediating the formation of membrane tubules. PMID:19695219

  13. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2015-04-01

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

  17. 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.

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

    PubMed Central

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

    2016-01-01

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

  19. Monitoring Phospholipase A2 Activity with Gd-encapsulated Phospholipid Liposomes

    PubMed Central

    Cheng, Zhiliang; Tsourkas, Andrew

    2014-01-01

    To date, numerous analytical methods have been developed to monitor phospholipase A2 (PLA2) activity. However, many of these methods require the use of unnatural PLA2 substrates that may alter enzyme kinetics, and probes that cannot be extended to applications in more complex environments. It would be desirable to develop a versatile assay that monitors PLA2 activity based on interactions with natural phospholipids in complex biological samples. Here, we developed an activatable T1 magnetic resonance (MR) imaging contrast agent to monitor PLA2 activity. Specifically, the clinically approved gadolinium (Gd)-based MR contrast agent, gadoteridol, was encapsulated within nanometer-sized phospholipid liposomes. The encapsulated Gd exhibited a low T1-weighted signal, due to low membrane permeability. However, when the phospholipids within the liposomal membrane were hydrolyzed by PLA2, encapsulated Gd was released into bulk solution, resulting in a measureable change in the T1-relaxation time. These activatable MR contrast agents can potentially be used as nanosensors for monitoring of PLA2 activity in biological samples with minimal sample preparation. PMID:25376186

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1996-10-01

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

  2. Synthesis, characterization and bioactivity studies of novel 1,3,4-oxadiazole small molecule that targets basic phospholipase A2 from Vipera russelli.

    PubMed

    Kameshwar, Vivek Hamse; R, Kumar J; Priya, Babu S; Swamy, S Nanjunda

    2017-02-01

    Secretory phospholipase A2 (sPLA2) is a key enzyme participating in the inflammatory cascade followed by the action of cyclooxygenase-2 and lipoxygenases. Therefore, inhibitors of sPLA2 could be used as potent anti-inflammatory agents to treat the early phase of inflammation. In this study, we have prepared the fenoprofen and ibuprofen analogs containing 1,3,4-oxadiazole nucleus and tested against Vipera russelli venom's basic sPLA2 (VRV-PL-VIIIa). Among the tested ligands 5(a-t),2-(2-chlorophenyl)-5-(1-(4-phenoxyphenyl) ethyl)-1,3,4-oxadiazole (5m) inhibited the catalytic activity of VRV-PL-VIIIa with an IC50 value of 11.52 µM. Biophysical studies revealed that the 5m quenches the intrinsic fluorescence of VRV-PL-VIIIa, in a concentration dependent manner. Also, the compound 5m affected VRV-PL-VIIIa conformation, which was observed by circular dichroism spectra that recorded the prominent shift in the α-helix peak and the random coil formation of VRV-PL-VIIIa. Further, molecular docking analysis revealed that the compound 5m possess strong hydrophobic interactions at catalytic triad region of the VRV-PL-VIIIa. Evident to in vitro and in silico studies, 5m strongly inhibited the hemolysis of red blood cells. Our in vivo pharmacological studies revealed that the compound 5m inhibited the edematogenic activity of VRV-PL-VIIIa in mouse foot pad. Additionally, the 5m inhibited VRV-PL-VIIIa-induced myotoxicity and lung hemorrhage in mice. Overall, our ADMET results depicted that 5m possess better druggable property. Thus, this study explored the new fenoprofen and ibuprofen analog 5m as the lead-structure that serves as an anti-inflammatory agent.

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

    PubMed

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

    2015-10-19

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

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

    PubMed Central

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

    2015-01-01

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

  5. 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.

  6. 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

  7. Inflammatory oedema induced by phospholipases A2 isolated from Crotalus durissus sp. in the rat dorsal skin: a role for mast cells and sensory C-fibers.

    PubMed

    Câmara, Paula R S; Esquisatto, Laura C M; Camargo, Enilton A; Ribela, Maria Teresa C P; Toyama, Marcos H; Marangoni, Sergio; De Nucci, Gilberto; Antunes, Edson

    2003-06-01

    The ability of the phospholipases A(2) (PLA(2)s) from Crotalus durissus cascavella, Crotalus durissus collilineatus and Crotalus durissus terrificus venoms and crotapotin to increase the vascular permeability in the rat skin as well as the contribution of both mast cells and sensory C-fibers have been investigated in this study. Vascular permeability was measured as the plasma extravascular accumulation at skin sites of intravenously injected 125I-human serum albumin. Intradermal injection of crotalic PLA(2)s (0.05-0.5 microg/site) in the rat skin resulted in dose-dependent increase in plasma extravascular whereas crotapotin (1 microg/site) failed to affect this response. Co-injection of crotapotin (1 microg/site) did not modify the increased vascular permeability induced by the PLA(2)s (0.05-0.5 microg/site). Previous treatment (30 min) of the animals with cyproheptadine (2 mg/kg, i.p.) markedly reduced PLA(2) (0.5 microg/site)-induced oedema. In rats treated neonatally with capsaicin to deplete neuropeptides, the plasma extravasation induced by all PLA(2)s (0.5 microg/site) was also significantly reduced. Similarly, the tachykinin NK(1) receptor antagonist SR140333 (1nmol/site) significantly reduced the PLA(2)-induced oedema. In addition, the combination of SR140333 with cyproheptadine further reduced the increased plasma extravasation by PLA(2) from C. d. cascavella venom, but not by PLA(2) from C. d. terrificus and C. d. collilineatus venoms. Our results suggest that increase in skin vascular permeability by crotalic PLA(2)s is mediated by activation of sensory C-fibers culminating in the release of substance P, as well as by activation of mast cells which in turn release amines such as histamine and serotonin.

  8. Mice with Genetic Deletion of Group VIA Phospholipase A2β Exhibit Impaired Macrophage Function and Increased Parasite Load in Trypanosoma cruzi-Induced Myocarditis

    PubMed Central

    Sharma, Janhavi; Blase, Jennifer R.; Hoft, Daniel F.; Marentette, John O.; Turk, John

    2016-01-01

    Trypanosoma cruzi infection, which is the etiological agent of Chagas disease, is associated with intense inflammation during the acute and chronic phases. The pathological progression of Chagas disease is influenced by the infiltration and transmigration of inflammatory cells across the endothelium to infected tissues, which are carefully regulated processes involving several molecular mediators, including adhesion molecules and platelet-activating factor (PAF). We have shown that PAF production is dependent upon calcium-independent group VIA phospholipase A2β (iPLA2β) following infection of human coronary artery endothelial cells (HCAECs) with T. cruzi, suggesting that the absence of iPLA2β may decrease the recruitment of inflammatory cells to the heart to manage parasite accumulation. Cardiac endothelial cells isolated from iPLA2β-knockout (iPLA2β-KO) mice infected with T. cruzi demonstrated decreased PAF production compared to that by cells isolated from wild-type (WT) mice but demonstrated increases in adhesion molecule expression similar to those seen in WT mice. Myocardial inflammation in iPLA2β-KO mice infected with T. cruzi was similar in severity to that in WT mice, but the iPLA2β-KO mouse myocardium contained more parasite pseudocysts. Upon activation, macrophages from iPLA2β-KO mice produced significantly less nitric oxide (NO) and caused less T. cruzi inhibition than macrophages from wild-type mice. Thus, the absence of iPLA2β activity does not influence myocardial inflammation, but iPLA2β is essential for T. cruzi clearance. PMID:26857573

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

    PubMed

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

    1996-03-15

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

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

    PubMed

    Xia, Wencui; Hu, Zhongyang; Song, Zhi

    2017-02-28

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

  11. 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

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

    PubMed

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

    2011-01-01

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

  13. Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.

    PubMed

    Heinze, Michael; Steighardt, Jörg; Gesell, Andreas; Schwartze, Wieland; Roos, Werner

    2007-12-01

    Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-04-01

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

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

    PubMed

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

    2009-05-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    2011-01-01

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

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

    PubMed

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

    2015-04-01

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

  3. [Adult-onset case of idiopathic neurodegeneration with brain iron accumulation without mutations in the PANK2 and PLA2G6 genes].

    PubMed

    Saiki, Shinji; Sekine, Takeshi; Ueno, Yuji; Yoshino, Hiroyo; Takahashi, Junko; Tani, Yoshihiko; Kambe, Yasunori; Motoi, Yumiko; Hattori, Nobutaka

    2009-08-01

    A 47-year-old man with a 15-year history of bipolar disorder treated with anti-depressants, lithium carbonate or neuroleptics was admitted because of marked difficulty in gait and speech. At the age 45, he was unable to walk without bilateral assists and became a wheel-chair state. There was no family history and his mother, father and younger sister were neurologically free. General physical examinations revealed no abnormalities. Neurologically, he was moderately demented (mini mental state examination: 18/30) and showed bilateral horizontal gaze nystagmus, parkinsonism, cerebellar ataxia, dysarthria and moderate spastic paraparesis. No involuntary movements were noted. Wet blood smear showed acanthocytes, while blood chemistries revealed no abnormalities including levels of serum creatine kinase, hepatic enzymes and blood beta-lipoprotein. Kell antigen expressions of the red blood cells were within normal limit. Western blot analysis with anti-chorein antibody detected normal chorein expression levels of the red blood cells. Cranial MRI showed severe symmetric atrophy of the frontotemporal lobes, caudate nuclei, putamen, and brainstem. Also, MRI-gradient echo showed symmetric iron accumulation in the medial portion of the globus pallidus without surrounding high intensity areas, so called "eye-of-the-tiger sign". Genetic analyses revealed no mutations in the PANK2 and PLA2G6 genes. Therefore, he was diagnosed as idiopathic neurodegeneration with brain iron accumulation (NBIA). These findings suggest that NBIA is heterogeneous and other additional genes remain to be found.

  4. 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

  5. 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.

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

    PubMed Central

    Balsinde, Jesús

    2002-01-01

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

  7. Preconditioning stress prevents cold restraint stress-induced gastric lesions in rats: roles of COX-1, COX-2, and PLA2.

    PubMed

    Tanaka, Akiko; Hatazawa, Ryo; Takahira, Yuka; Izumi, Nahoko; Filaretova, Ludmila; Takeuchi, Koji

    2007-02-01

    We investigated the protective effect of mild stress on gastric lesions induced by cold-restraint stress, especially concerning prostaglandins (PGs)/cyclo-oxygenase (COX) isozymes. Rats were exposed to severe stress (cold-restraint stress at 10 degrees C for 6 hr) or mild stress (cold-restraint stress at 10 degrees C for 30 min and kept at room temperature for 60 min) followed by severe stress. Severe stress induced gastric lesions, with a concomitant decrease in body temperature (BT). The ulcerogenic response was inhibited by atropine but worsened by indomethacin and SC-560 but not rofecoxib, although none of these agents had any effect on the change in BT. Mild stress suppressed the gastric ulceration and the decrease in BT induced by severe stress, and these effects were reversed by both COX-1 and COX-2 inhibitors. The expression of COX-2 in the stomach was up-regulated from 4 hr after severe stress and this response was slightly expedited by mild stress. COX-2 was also expressed in the hypothalamus under normal and stressed conditions. Quinacrine (phospholipase A(2) inhibitor) attenuated the protective effect of mild stress on the ulceration and decrease in BT caused by severe stress. TA-0910 (TRH analogue) at a low dose also prevented the gastric ulceration and the decrease in BT induced by severe stress. These results suggest that mild stress protects against cold-restraint stress-induced gastric ulceration, and the effect is peripherally and centrally mediated by PGs derived from both COX-1 and COX-2 through the activation of phospholipase A(2). TRH may also be involved in the protective effect of mild stress, probably through regulation of the thermogenic system.

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

    PubMed Central

    Evans, R. J.; Surprenant, A.

    1993-01-01

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

  9. Progressive Hemorrhage and Myotoxicity Induced by Echis carinatus Venom in Murine Model: Neutralization by Inhibitor Cocktail of N,N,N',N'-Tetrakis (2-Pyridylmethyl) Ethane-1,2-Diamine and Silymarin

    PubMed Central

    Nanjaraj Urs, Ankanahalli N.; Ramakrishnan, Chandrasekaran; Joshi, Vikram; Suvilesh, Kanve Nagaraj; Veerabasappa Gowda, Teregowda; Velmurugan, Devadasan; Vishwanath, Bannikuppe Sannanaik

    2015-01-01

    Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn2+ metalloproteases (Zn2+MPs), phospholipase A2s (PLA2s) and hyaluronidases (HYs) in the onset and progression of local toxicity from the bitten site. As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N’,N’-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn2+MPs of ECV (IC50: 6.7 μM). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA2s (IC50: 12.5 μM) and HYs (IC50: 8 μM) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min (for hemorrhage alone) of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity. PMID:26274501

  10. 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.

  11. Plasma Lipoprotein-Associated Phospholipase A2 Level Is an Independent Predictor of High Thrombus Burden in Patients With Acute ST-segment Elevation Myocardial Infarction.

    PubMed

    Wu, Xiangqi; Zhang, Yingqiang; Wu, Zhiming; You, Wei; Liang, Fengshuo; Ye, Fei; Chen, Shaoliang

    2016-12-02

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an independent risk factor for plaque rupture and atherothrombotic events. However, the associations between serum Lp-PLA2 level and thrombus burden in ST-segment elevation myocardial infarction (STEMI) patients remain unknown.We consecutively enrolled 351 STEMI patients who underwent primary percutaneous coronary intervention (pPCI). Patients were assigned to a high thrombus burden (HTB) group (n = 230) and a low thrombus burden (LTB) group (n = 121). Baseline data were recorded during hospital admission. Plasma Lp-PLA2 concentration, coronary angiography results, and in-hospital mortality were measured. Plasma Lp-PLA2 level had a high correlation with thrombus burden score (TBS) before pPCI and it was found to be a significant independent predictor of HTB in STEMI patients (P < 0.05). Moreover, TBS, corrected thrombolysis in myocardial infarction (TIMI) frame count (cTFC), and plasma Lp-PLA2 level after pPCI in patients with HTB were significantly higher than those in patients with LTB (P < 0.05). Meanwhile, TIMI flow grade (TFG) and TIMI myocardial perfusion grade (TMPG) of HTB patients were markedly lower than those of LTB patients (P < 0.05). Additionally, correlations of plasma Lp-PLA2 level before pPCI with TFG before pPCI and TBS, cTFC, and TMPG after pPCI were modest (P < 0.05). However, the associations of plasma Lp-PLA2 level after pPCI with TFG, TBS, cTFC and TMPG were low (P < 0.05).These results demonstrated that the plasma Lp-PLA2 level before pPCI is an independent predictor of HTB in STEMI patients, resulting in modestly predicting blood flow and myocardial perfusion of the culprit artery.

  12. AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs

    PubMed Central

    El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

    2015-01-01

    Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs. PMID:26162096

  13. Circulating n-3 fatty acids and trans-fatty acids, PLA2G2A gene variation and sudden cardiac arrest.

    PubMed

    Lemaitre, Rozenn N; Bartz, Traci M; King, Irena B; Brody, Jennifer A; McKnight, Barbara; Sotoodehnia, Nona; Rea, Thomas D; Johnson, Catherine O; Mozaffarian, Dariush; Hesselson, Stephanie; Kwok, Pui-Yan; Siscovick, David S

    2016-01-01

    Whether genetic factors influence the associations of fatty acids with the risk of sudden cardiac arrest (SCA) is largely unknown. To investigate possible gene-fatty acid interactions on SCA risk, we used a case-only approach and measured fatty acids in erythrocyte samples from 1869 SCA cases in a population-based repository with genetic data. We selected 191 SNP in ENCODE-identified regulatory regions of fifty-five candidate genes in fatty acid metabolic pathways. Using linear regression and additive genetic models, we investigated the association of the selected SNP with erythrocyte levels of fatty acids, including DHA, EPA and trans-fatty acids among the SCA cases. The assumption of no association in non-cases was supported by analysis of publicly available datasets containing over 8000 samples. None of the SNP-fatty acid associations tested among the cases reached statistical significance after correction for multiple comparisons. One SNP, rs4654990 near PLA2G2A, with an allele frequency of 0·33, was nominally associated with lower levels of DHA and EPA and higher levels of trans-fatty acids. The strongest association was with DHA levels (exponentiated coefficient for one unit (1 % of total fatty acids), 0·90, 95 % CI 0·85, 0·97; P = 0·003), indicating that for subjects with a coded allele, the OR of SCA associated with one unit higher DHA is about 90 % what it is for subjects with one fewer coded allele. These findings suggest that the associations of circulating n-3 and trans-fatty acids with SCA risk may be more pronounced in carriers of the rs4654990 G allele.

  14. Expression analysis of the group IIA secretory phospholipase A(2) in mice with differential susceptibility to azoxymethane-induced colon tumorigenesis.

    PubMed

    Papanikolaou, A; Wang, Q S; Mulherkar, R; Bolt, A; Rosenberg, D W

    2000-02-01

    The murine non-pancreatic secretory phospholipase A(2) (sPLA(2)) has been proposed as a tumor modifier of multiple intestinal neoplasia (Min). A genetic polymorphism in the mouse gene that causes a disruption in exon 3 results in loss of functional protein. Mouse strains with a disrupted sPLA(2) gene are susceptible to the Min phenotype and develop numerous intestinal polyps, whereas mice with normal sPLA(2) develop only a limited number of polyps. The following study was undertaken to test the hypothesis that sPLA(2) plays an equivalent role in murine susceptibility to the colon carcinogen azoxymethane (AOM). sPLA(2) status was confirmed by sequencing in mice that are highly susceptible (A/J), susceptible (SWR/J) and resistant (AKR/J) to AOM-induced tumorigenesis. Constitutive expression of sPLA(2) mRNA was compared in small intestine and colon of untreated mice using semi-quantitative RT-PCR. Whereas mRNA expression was nearly absent in A/J mice, AKR/J mice exhibited extensive expression throughout the intestine. Despite the wild-type sPLA(2) gene, colonic mRNA expression in SWR/J mice was significantly lower relative to AKR/J. Immunohistochemical analysis of sPLA(2) protein confirmed the mRNA data. The effect of AOM on colonic sPLA(2) expression was also examined. Twenty-four weeks after the last of six weekly injections of AOM (10 mg/kg i.p.), RT-PCR analysis of distal colons revealed a significant increase in mRNA in normal-appearing epithelium and tumor tissue from AOM-treated mice relative to controls. However, there was no corresponding increase in protein expression in A/J mice. The absence of sPLA(2) expression within control colons of tumor-susceptible A/J mice together with low expression in SWR/J colons is consistent with its potential role as an intestinal tumor modifier, but the carcinogen-induced increase in expression raises doubts as to the significance of sPLA(2) in inhibiting carcinogenesis.

  15. Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

    PubMed

    Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H; Boilard, Eric

    2015-01-01

    Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

  16. Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

    PubMed Central

    Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A.; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H.; Boilard, Eric

    2015-01-01

    Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes. PMID:25587983

  17. Role of Ca2+-independent phospholipase A2 and store-operated pathway in urocortin-induced vasodilatation of rat coronary artery.

    PubMed

    Smani, Tarik; Domínguez-Rodríguez, Alejandro; Hmadcha, Abdelkrim; Calderón-Sánchez, Eva; Horrillo-Ledesma, Angélica; Ordóñez, Antonio

    2007-11-26

    Urocortin has been shown to produce vasodilatation in several arteries, but the precise mechanism of its action is still poorly understood. Here we demonstrate the role of store operated Ca2+ entry (SOCE) regulated by Ca2+-independent phospholipase A2 (iPLA2) in phenylephrine hydrochloride (PE)-induced vasoconstriction, and we present the first evidence that urocortin induces relaxation by the modulation of SOCE and iPLA2 in rat coronary artery. Urocortin produces an endothelium independent relaxation, and its effect is concentration-dependent (IC50 approximately = 4.5 nmol/L). We show in coronary smooth muscle cells (SMCs) that urocortin inhibits iPLA2 activation, a crucial step for SOC channel activation, and prevents Ca2+ influx evoked by the emptying of the stores via a cAMP and protein kinase A (PKA)-dependent mechanism. Lysophophatidylcholine and lysophosphatidylinositol, products of iPLA2, exactly mimic the effect of the depletion of the stores in presence of urocortin. Furthermore, we report that long treatment with urocortin downregulates iPLA2 mRNA and proteins expression in rat coronary smooth muscle cells. In summary, we propose a new mechanism of vasodilatation by urocortin which involves the regulation of iPLA2 and SOCE via the stimulation of a cAMP/PKA-dependent signal transduction cascade in rat coronary artery.

  18. Complete amino acid sequence of an acidic, cardiotoxic phospholipase A2 from the venom of Ophiophagus hannah (King Cobra): a novel cobra venom enzyme with "pancreatic loop".

    PubMed

    Huang, M Z; Gopalakrishnakone, P; Chung, M C; Kini, R M

    1997-02-15

    A phospholipase A2 (OHV A-PLA2) from the venom of Ophiophagus hannah (King cobra) is an acidic protein exhibiting cardiotoxicity, myotoxicity, and antiplatelet activity. The complete amino acid sequence of OHV A-PLA2 has been determined using a combination of Edman degradation and mass spectrometric techniques. OHV A-PLA2 is composed of a single chain of 124 amino acid residues with 14 cysteines and a calculated molecular weight of 13719 Da. It contains the loop of residues (62-66) found in pancreatic PLA2s and hence belongs to class IB enzymes. This pancreatic loop is between two proline residues (Pro 59 and Pro 68) and contains several hydrophilic amino acids (Ser and Asp). This region has high degree of conformational flexibility and is on the surface of the molecule, and hence it may be a potential protein-protein interaction site. A relatively low sequence homology is found between OHV A-PLA2 and other known cardiotoxic PLA2s, and hence a contiguous segment could not be identified as a site responsible for the cardiotoxic activity.

  19. Protein interactions between the C-terminus of Aβ-peptide and phospholipase A2--a structure biology based approach to identify novel Alzheimer's therapeutics.

    PubMed

    Mirza, Zeenat; Pillai, Vikram G; Kamal, Mohammad A

    2014-01-01

    Amyloid β (Aβ) polypeptide plays a key role in determining the state of protein aggregation in Alzheimer's disease. The hydrophobic C-terminal part of the Aβ peptide is critical in triggering the transformation from α-helical to β- sheet structure. We hypothesized that phospholipase A2 (PLA2) may inhibit the aggregation of Aβ peptide by interacting with the peptide and keeping the two peptide chains apart. In order to examine the nature of interactions between PLA2 and Aβ peptide, we prepared and crystallized complex of Naja naja sagittifera PLA2 with the C-terminal hepta-peptide Val-Gly-Gly-Val-Val-Ile-Ala. The X-ray intensity data were collected to 2.04 A resolution and the structure was determined by molecular replacement and refined to the crystallographic R factor of 0.186. The structural analysis revealed that the peptide binds to PLA2 at the hydrophobic substrate binding cavity forming at least eight hydrogen bonds and approximately a two dozen Van der Waals interactions. The number and nature of interactions indicate that the affinity between PLA2 and the hepta-peptide is greater than the affinity between two Aβ peptide chains. Therefore, PLA2 is proposed as a probable ligand to prevent the aggregation of Aβ peptides.

  20. Specific nonpeptide inhibitors of puromycin-sensitive aminopeptidase with a 2,4(1H,3H)-quinazolinedione skeleton.

    PubMed

    Kakuta, Hiroki; Tanatani, Aya; Nagasawa, Kazuo; Hashimoto, Yuichi

    2003-11-01

    Potent, specific, chemically stable and non-peptide/small-molecular inhibitors of puromycin-sensitive aminopeptidase, such as 3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (PAQ-22, 5), were prepared by the structural development of a potent PSA inhibitor, 2-(2,6-diethylphenyl)-1,2,3,4-tetrahydroisoquinoline-1,3-dione (PIQ-22, 4). The design was carried out partly by applying electrostatic potential field information obtained from PIQ-22 (4) and its derivatives based on thalidomide (2). This information revealed that a positive electrostatic potential field around the benzylic methylene in the tetrahydroisoquinoline ring is necessary for potent activity. Lineweaver-Burk plot analysis showed that PAQ-22 (5) and its derivatives inhibit puromycin-sensitive aminopeptidase (PSA) in a non-competitive manner. These potent and specific PSA inhibitors showed dose-dependent cell invasion-inhibitory activity in a Matrigel assay using mouse melanoma B16F10/L5 cells, in spite of their low cell toxicity.

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2014-11-07

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

  3. Mechanistic characterization of a 2-thioxanthine myeloperoxidase inhibitor and selectivity assessment utilizing click chemistry--activity-based protein profiling.

    PubMed

    Ward, Jessica; Spath, Samantha N; Pabst, Brandon; Carpino, Philip A; Ruggeri, Roger B; Xing, Gang; Speers, Anna E; Cravatt, Benjamin F; Ahn, Kay

    2013-12-23

    Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Despite a high level of interest in MPO as a therapeutic target, there have been limited reports about MPO inhibitors that are suitable for evaluating MPO in pharmacological studies. 2-Thioxanthine, 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (A), has recently been reported to inhibit MPO by covalently modifying the heme prosthetic group. Here we report a detailed mechanistic characterization demonstrating that A possesses all the distinguishing features of a mechanism-based inactivator. A is a time-dependent MPO inhibitor and displays saturable inactivation kinetics consistent with a two-step mechanism of inactivation and a potency (k(inact)/K(I) ratio) of 8450 ± 780 M⁻¹ s⁻¹. MPO inactivation by A is dependent on MPO catalysis and is protected by substrate. A reduces MPO compound I to compound II with a second-order rate constant of (0.801 ± 0.056) × 10⁶ M⁻¹ s⁻¹, and its irreversible inactivation of MPO occurs prior to release of the activated inhibitory species. Despite its relatively high selectivity against a broad panel of more than 100 individual targets, including enzymes, receptors, transporters, and ion channels, we demonstrate that A labels multiple other protein targets in the presence of MPO. By synthesizing an alkyne analogue of A and utilizing click chemistry-activity-based protein profiling, we present that the MPO-activated inhibitory species can diffuse away to covalently modify other proteins, as reflected by the relatively high partition ratio of A, which we determined to be 15.6. This study highlights critical methods that can guide the discovery and development of next-generation MPO inhibitors.

  4. Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells.

    PubMed

    Yao, Mu; Xie, Chanlu; Kiang, Mei-Yee; Teng, Ying; Harman, David; Tiffen, Jessamy; Wang, Qian; Sved, Paul; Bao, Shisan; Witting, Paul; Holst, Jeff; Dong, Qihan

    2015-10-27

    Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A2α (cPLA2α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA2α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA2α with a phosphorylation at Ser505 was lower compared to their proliferating state. Conversely, the phospho-cPLA2α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA2α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G0/G1 and low percentage of cells in S and G2/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA2α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA2α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.

  5. Group IVA phospholipase A2-associated production of MMP-9 in macrophages and formation of atherosclerotic lesions.

    PubMed

    Ii, Hiromi; Hontani, Naoya; Toshida, Issei; Oka, Mayuko; Sato, Takashi; Akiba, Satoshi

    2008-03-01

    Matrix metalloproteinase-9 (MMP-9) is involved in atherogenesis, and the production of MMP-9 in macrophages is considered to be mediated by the arachidonic acid cascade. The present study examined the possible involvement of group IVA phospholipase A2 (IVA-PLA2), a key enzyme in the arachidonic acid cascade, in the production of MMP-9 induced by oxidized low-density lipoprotein (oxLDL) in macrophages and high-fat diet-induced formation of atherosclerotic lesions using IVA-PLA2-deficient mice (C57BL/6 background). In wild-type mouse peritoneal macrophages, oxLDL induced an increase in MMP-9 in the culture medium. The oxLDL-promoted production of MMP-9 was markedly reduced in IVA-PLA2-deficient macrophages compared to wild-type macrophages. Feeding of wild-type mice with a high-fat diet caused the formation of early atherosclerotic lesions in the aortic root with increases in MMP-9 and macrophages in the lesions and with higher serum levels of total cholesterol. Such lesions were apparently less severe in IVA-PLA2-deficient mice fed a high-fat diet, despite higher total cholesterol levels. Under the conditions, a high-fat diet reduced the serum levels of high-density lipoprotein-cholesterol (HDL-C) in wild-type mice. However, IVA-PLA2-deficient mice fed a high-fat diet were protected against the decrease in HDL-C levels. The present results suggest that IVA-PLA2 is involved in the oxLDL-induced production of MMP-9 in macrophages and the high-fat diet-induced formation of early atherosclerotic lesions. The protection against the lesions in IVA-PLA2-deficient mice may be ascribable, in part, to the impaired production of MMP-9 and/or the maintained levels of HDL-C.

  6. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    PubMed

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-07

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  7. Biochemical and enzymatic characterization of two basic Asp49 phospholipase A2 isoforms from Lachesis muta muta (Surucucu) venom.

    PubMed

    Damico, Daniela C S; Lilla, Sérgio; de Nucci, Gilberto; Ponce-Soto, Luis A; Winck, Flávia V; Novello, José Camillo; Marangoni, Sérgio

    2005-10-30

    Two basic phospholipase A2 (PLA2) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 mu-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg65 (LmTX-I)-->Pro65 (LmTX-II). The amino acid sequence showed a high degree of homology between PLA2 isoforms from Lachesis muta muta and other PLA2 snake venoms. LmTX-I and LmTX-II had PLA2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35-45 degrees C. Full PLA2 activity required Ca2+ and was respectively inhibited by Cu2+ and Zn2+ in the presence and absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P<0.05) the enzymatic activity of LmTX-I, suggesting that the binding site for crotapotin in this PLA2 was similar to another in the basic PLA2 of the crotoxin complex from C. durissus cascavella venom.

  8. Phospholipase A2 receptor positive membranous nephropathy long after living donor kidney transplantation between identical twins.

    PubMed

    Saito, Hisako; Hamasaki, Yoshifumi; Tojo, Akihiro; Shintani, Yukako; Shimizu, Akira; Nangaku, Masaomi

    2015-07-01

    Although membranous nephropathy (MN) is a commonly observed cause of post-transplant glomerulonephritis, distinguishing de novo from recurrent MN in kidney allograft is often difficult. Phospholipase A2 receptor (PLA2R) staining is useful for diagnosing recurrent MN in allografts similarly to idiopathic MN in native kidney. No specific treatment strategy has been established for MN, especially when accompanied with HCV infection in kidney transplant recipients. This report describes a 66-year-old man who was diagnosed as having PLA2R positive membranous nephropathy accompanied with already-known IgA nephropathy and HCV infection 26 years after kidney transplantation conducted between identical twins. PLA2R was detected along capillary loops, implying that this patient is affected by the same pathogenic mechanism as idiopathic MN, not secondary MN associated with other disorders such as HCV infection. The patient successfully achieved clinical remission after steroid therapy.

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

    PubMed

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

    2002-06-01

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

  10. 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

  11. Identification of Alpha 2 Macroglobulin (A2M) as a master inhibitor of cartilage degrading factors that attenuates post-traumatic osteoarthritis progression

    PubMed Central

    Wang, Shaowei; Wei, Xiaochun; Zhou, Jingming; Zhang, Jing; Li, Kai; Chen, Qian; Terek, Richard; Fleming, Braden C.; Goldring, Mary B.; Ehrlich, Michael G.; Zhang, Ge; Wei, Lei

    2014-01-01

    Objective To determine if supplemental intra-articular alpha-2 macroglobulin (A2M) has a chondroprotective effect in a rat OA model. Methods A2M was identified as a potential therapeutic agent by comparing A2M concentrations in serum, synovial fluid (SF), and cartilage from normal and osteoarthritic (OA) patients by Western blotting, mass spectrometry, ELISA, and immunohistochemistry (IHC). The effects of A2M on IL-1-induced cartilage catabolic enzymes were evaluated by Luminex and ELISA in cultured chondrocytes. In vivo effects on cartilage degeneration and MMP-13 concentration were evaluated in male rats (N=120) randomized to four treatments: (1) CLT+saline, (2) ACLT+A2M (1IU/kg), (3) ACLT+A2M (2IU/kg) or (4) sham surgery+saline. Intra-articular injections were given for 6 weeks. The concentration of MMP-13 in SF lavages was measured using ELISA. OA-related gene expression was quantified by RT-qPCR. Histology was performed to grade OA. Results In both normal and OA patients, the levels of A2M were lower in SF compared to serum, and MMP-13 was higher in SF than serum of OA patients. In vitro, A2M inhibited the induction of MMP-13 by IL-1 in a dose-dependent manner in human chondrocytes. In the rat ACLT OA model, supplemental intra-articular injection of A2M reduced the concentration of MMP-13 in SF, had a favorable effect on OA-related gene expression, and attenuated OA progression. Conclusion A2M is a plasma protease inhibitor that is not present in sufficient concentrations to inactivate the high concentrations of catabolic factors found in OA SF. Our findings suggest that supplemental intra-articular A2M provides chondral protection for post traumatic OA. PMID:24578232

  12. No dose adjustment on coadministration of the PDE4 inhibitor roflumilast with a weak CYP3A, CYP1A2, and CYP2C19 inhibitor: an investigation using cimetidine.

    PubMed

    Böhmer, Gabriele M; Gleiter, Christoph H; Mörike, Klaus; Nassr, Nassr; Walz, Antje; Lahu, Gezim

    2011-04-01

    This nonrandomized, fixed-sequence, 2-period crossover study investigated potential pharmacokinetic interactions between the phosphodiesterase 4 inhibitor roflumilast, currently in clinical development for the treatment of chronic obstructive pulmonary disease, and the histamine 2 agonist cimetidine. Participants received roflumilast, 500 µg once daily, on days 1 and 13. Cimetidine, 400 mg twice daily, was administered from days 6 to 16. Pharmacokinetic analysis of roflumilast and its active metabolite roflumilast N-oxide was performed, and the ratio of geometric means for roflumilast alone and concomitantly with steady-state cimetidine was calculated. The effect of cimetidine on the total PDE4 inhibitory activity (tPDE4i; total exposure to roflumilast and roflumilast N-oxide) was also calculated. Coadministration of steady-state cimetidine increased mean tPDE4i of roflumilast and roflumilast N-oxide by about 47%. The maximum plasma concentration (C(max)) of roflumilast increased by about 46%, with no effect on C(max) of roflumilast N-oxide. The increase in tPDE4i of roflumilast and roflumilast N-oxide following coadministration with cimetidine was mainly due to the inhibitory effect of cimetidine on cytochrome P450 (CYP) isoenzymes CYP1A2, CYP3A, and CYP2C19. These moderate changes indicate that dose adjustment of roflumilast is not required when coadministered with a weak inhibitor of CYP1A2, CYP3A, and CYP2C19, such as cimetidine.

  13. Comparison of Lipoprotein-Associated Phospholipase A2 and High Sensitive C-Reactive Protein as Determinants of Metabolic Syndrome in Subjects without Coronary Heart Disease: In Search of the Best Predictor

    PubMed Central

    Acevedo, Mónica; Kramer, Verónica; Adasme, Marcela; Briones, Luisa

    2015-01-01

    High sensitivity C-reactive protein (hsCRP) is a marker of metabolic syndrome (MS) and cardiovascular (CV) disease. Lipoprotein-associated phospholipase A2 (Lp-PLA2) also predicts CV disease. There are no reports comparing these markers as predictors of MS. Methods. Cross-sectional study comparing Lp-PLA2 and hsCRP as predictors of MS in asymptomatic subjects was carried out; 152 subjects without known atherosclerosis participated. Data were collected on demographics, cardiovascular risk factors, anthropometric and biochemical measurements, and hsCRP and Lp-PLA2 activity levels. A logistic regression analysis was performed with each biomarker and receiver operating characteristic (ROC) curves were constructed for MS. Results. Mean age was 46 ± 11 years, and 38% of the subjects had MS. Mean Lp-PLA2 activity was 185 ± 48 nmol/mL/min, and mean hsCRP was 2.1 ± 2.2 mg/L. Subjects with MS had significantly higher levels of Lp-PLA2 (P = 0.03) and hsCRP (P < 0.0001) than those without MS. ROC curves showed that both markers predicted MS. Conclusion. Lp-PLA2 and hsCRP are elevated in subjects with MS. Both biomarkers were independent and significant predictors for MS, emphasizing the role of inflammation in MS. Further research is necessary to determine if inflammation predicts a higher risk for CV events in MS subjects. PMID:26089902

  14. Comparison of Lipoprotein-Associated Phospholipase A2 and High Sensitive C-Reactive Protein as Determinants of Metabolic Syndrome in Subjects without Coronary Heart Disease: In Search of the Best Predictor.

    PubMed

    Acevedo, Mónica; Varleta, Paola; Kramer, Verónica; Valentino, Giovanna; Quiroga, Teresa; Prieto, Carolina; Parada, Jacqueline; Adasme, Marcela; Briones, Luisa; Navarrete, Carlos

    2015-01-01

    High sensitivity C-reactive protein (hsCRP) is a marker of metabolic syndrome (MS) and cardiovascular (CV) disease. Lipoprotein-associated phospholipase A2 (Lp-PLA2) also predicts CV disease. There are no reports comparing these markers as predictors of MS. Methods. Cross-sectional study comparing Lp-PLA2 and hsCRP as predictors of MS in asymptomatic subjects was carried out; 152 subjects without known atherosclerosis participated. Data were collected on demographics, cardiovascular risk factors, anthropometric and biochemical measurements, and hsCRP and Lp-PLA2 activity levels. A logistic regression analysis was performed with each biomarker and receiver operating characteristic (ROC) curves were constructed for MS. Results. Mean age was 46 ± 11 years, and 38% of the subjects had MS. Mean Lp-PLA2 activity was 185 ± 48 nmol/mL/min, and mean hsCRP was 2.1 ± 2.2 mg/L. Subjects with MS had significantly higher levels of Lp-PLA2 (P = 0.03) and hsCRP (P < 0.0001) than those without MS. ROC curves showed that both markers predicted MS. Conclusion. Lp-PLA2 and hsCRP are elevated in subjects with MS. Both biomarkers were independent and significant predictors for MS, emphasizing the role of inflammation in MS. Further research is necessary to determine if inflammation predicts a higher risk for CV events in MS subjects.

  15. Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

    PubMed

    Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

    2014-03-10

    Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overa